clÁudia isabel rodrigues martins lisados de plaquetas … · 2020. 1. 17. · figure 2.4:...

Universidade de Aveiro

2017 Departamento de Química

CLÁUDIA ISABEL RODRIGUES MARTINS

Micropartículas de laminarina com incorporação de lisados de plaquetas para adesão e expansão celular Platelet lysates loaded laminarin microparticles for cell attachment and expansion

Universidade de Aveiro

2017 Departamento de Química

CLÁUDIA ISABEL RODRIGUES MARTINS

Micropartículas de laminarina com incorporação de lisados de plaquetas para adesão e expansão celular Platelet lysates loaded laminarin microparticles for cell attachment and expansion Dissertação apresentada à Universidade de Aveiro para cumprimento dos

requisitos necessários à obtenção do grau de Mestre em Biotecnologia

Industrial e Ambiental, realizada sob a orientação científica da Doutora Catarina

Custódio, Investigadora Pós-Doutorada de Departamento de Química da

Universidade de Aveiro, e do Professor Doutor João Mano, Professor

Catedrático do Departamento de Química da Universidade de Aveiro.

o júri

Presidente

vogal – Arguente principal

vogal - orientador

Doutora Mara Guadalupe Freire Martins Equiparado a investigador Coordenador do Departamento de Química da Universidade

de Aveiro

Professora Doutora Cláudia Alexandra Martins Lobato da Silva Professor Auxiliar do Instituto Superior Técnico de Lisboa

Doutora Catarina de Almeida Custódio Bolseiro de pós-doutoramento de Departamento de Química d Universidade de Aveiro

agradecimentos

A última parte da tese e com isto a última parte deste percurso.

Em primeiro lugar gostaria de agradecer aos meus pais, irmão e avós por

estarem sempre presentes e por todo o apoio. Por aturarem o mau feitio,

por me fazerem sorrir sempre que o trabalho não corria da melhor forma

e por me fazerem acreditar que era capaz.

De seguida, não posso deixar de agradecer aos meus amigos, a todos

aqueles que a vida académica me deu e a todos aqueles que estão na minha

vida desde antes. Sem o apoio deste nada seria possível. Aqui não posso

esquecer os meus colegas de laboratório. Durante este ano partilhámos

muitas histórias, muitas aventuras. Sem dúvida que sem eles foram muito

importantes durante este ano de tese. “Aqueles que passam por nós não

vão sós, não nos deixam sós. Deixam um pouco de si, levam um pouco de

nós”. Alguém me disse esta frase este ano e sem dúvida agora que chega

o fim faz todo o sentido. Todas as pessoas do grupo COMPASS com quem

me cruzei neste ano de alguma forma marcaram este ano. E duas pessoas

que marcaram de forma única foram os meus orientadores tiveram sem

dúvida um papel central durante este ano. Ainda me lembro da primeira

reunião em que eu mal sabia o que era um hidrogel… Agora que chegou

ao fim, vejo que foi um ano de muita aprendizagem, uma área nova, um

laboratório novo e uma bagagem sem dúvida bem mais vaste de

conhecimentos.

“Escolhe um trabalho de que gostes, e não terás que trabalhar nem um dia

da tua vida”.

palavras-chave

Engenaria de tecidos, microgeis, bottom-up, microfluídica, microcarriers

resumo

A engenharia de tecidos tem estudado a combinação de vários materiais com células com o intuito de solucionar alguns problemas que as atuais terapias não conseguem dar resposta. Umas das respostas passa pelo desenvolvimento de pequenas estruturas, microgéis, que mimetizam a matriz extracelular e podem interagir de várias formas para criar uma estrutura tridimensional complexa. Este trabalho resume as principais técnicas de microfabricação que têm sido usadas no desenvolvimento de microgéis assim como as principais aplicações destas na engenharia de tecidos. Uma dessas técnicas, a microfluídica foi neste trabalho usada para produzir micropartículas de laminarina. Este polímero foi quimicamente modificado, adicionando grupos metacrilato que permitiram a reticulação das gotículas formadas pela microfluídica quando expostas a luz ultravioleta. Esta tecnologia permite encapsular lisados de plaquetas humanos durante a produção das micropartículas. As micropartículas apresentam elevado grau de monodispersão e uma forma esférica com diâmetro médio de 326.6 μm nas micropartículas com elevado grau de modificação e 303.3 µm nas micropartículas com baixo grau de modificação. Estudos de libertação demonstraram que diferentes graus de modificação da laminarina permite obter diferentes perfis de libertação das proteínas encapsuladas. Ao fim de 14 dias as micropartículas com alto grau de modificação tinham libertado 56.89 ± 1.03% da proteína encapsulada enquanto as micropartículas com baixo nível de modificação libertaram 35.31 ± 2.73%. As partículas funcionais foram então testadas como suporte para cultura de células. Os resultados mostraram que as partículas funcionalizadas com RGD e lisados encapsulados permitem uma eficiente adesão e expansão celular. As células cultivadas na superfície dos microgéis promovem ainda agregação dos mesmos em estruturas tridimensionais. Para aplicações clínicas, hidrogéis injetáveis/moldáveis são muitas vezes necessários para preencher defeitos de geometrias irregulares. Estes microgéis de elevada versatilidade podem vir a traduzir-se no desenvolvimento de sistemas de elevado desempenho para a produção de sistemas injetáveis para aplicações em engenharia de tecidos.

keywords

Tissue engineering, microgels, bottom-up, microfluidic, microcarries

abstract

Tissue engineering has studied the combination of diverse biomaterials with cells in order to overcome some limitations of traditional therapies. One of solutions is related with the development of microstructures, microgels, that mimic the extracellular matrix and can assemble to create a complex three-dimensional structure. This work reviews the microfabrication techniques that been used to fabricate these microstructures as well as the main application of these in tissue engineering. One of this technique, microfluidic flow-focusing has been used to produce laminarin microparticles. This polymer was chemical modified, by adding methacrylated groups that allowed the photopolymerization of laminarin droplets when exposed to UV light. This technology allows to encapsulate human platelet lysates during fabrication of microgels by microfluidic. The microparticles shows high monodisperse and spherical shape with an average diameter around 326.6 μm in the microparticles with high degree of modification and 303.3 µm in the microparticles with low degree of modifications. The release studies demonstrate that different levels of modification of the laminarin microparticles allow to obtain different encapsulated proteins release profiles. At the end of 14 days the high methacrylated laminarin released 56.89 ± 1.03% of total amount of protein while low methacrylated laminarin released 35.31 ± 2.73%. The results showed that RGD functionalized particles with encapsulated platelet lysates allow an efficient cell adhesion and expansion. The cells cultured on the surface of microgels promote the assembly in three dimensional structures. For clinical applications, injectable/moldable hydrogels are several times necessaries to fill defects of irregular geometries. This highly versatility microgels can be translated in the development of the high-performance systems to production of injectable systems for applications in tissue engineering

i

Contents

Chapter 1 – Motivation…………………………………………………………………… .1

Chapter 2 - Recent trends on microfabrication of hydrogels ............................................... 4

1. Introduction .............................................................................................................. 4

2. Microfabrication of hydrogel…………………………………………………………4

2.1. Micromolding ............................................................................................... 6

2.2. Bioprinting ................................................................................................... 7

2.3. Microfluidics ................................................................................................ 9

2.4. Spray based technologies ............................................................................ 11

2.5. Superhydrophobic-based platforms for microgel

production……………………………………………….…………………………13

3. Applications of microfabricated hydrogels .............................................................. 14

3.1. Cell laden microgels as building blocks for 3D constructs ........................... 14

3.2. Cell microcarriers ....................................................................................... 16

3.3. High-throughput screening .......................................................................... 17

3.4. Drug Delivery ............................................................................................. 18

3.5. Assembly of microgels ............................................................................... 19

3.5.1. Surface Tension .......................................................................................... 20

3.5.2. Acoustic assembly ...................................................................................... 20

3.5.3. Magnetic assembly ..................................................................................... 21

3.5.4. Biomolecular recognition ............................................................................ 21

3.5.5. Cellular controlled assembly ....................................................................... 23

4. Conclusions ............................................................................................................ 23

5. References: ............................................................................................................. 23

ii

Chapter 3 – Materials and Methods .................................................................................. 32

1. Laminarin ............................................................................................................... 32

2. Platelet Lysates ....................................................................................................... 32

3. Methods .................................................................................................................. 32

3.1. Synthesis and characterization of methacrylated laminarin .......................... 32

3.2. Fabrication of MeLam microparticles ......................................................... 33

3.3. RGD functionalization of MeLam microparticles ........................................ 34

3.4. Scanning electron microscopy (SEM) ......................................................... 35

3.5. Studies on the release of PL ........................................................................ 35

3.6. Cell culture on MeLam microparticles ........................................................ 35

3.7. Cell morphology analysis ............................................................................ 36

3.7.1. DNA quantification..................................................................................... 37

3.7.2 MTS Viability Assay .................................................................................. 37

3.8. Statistical analysis ....................................................................................... 37

4. References .............................................................................................................. 37

Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and expansion ...... 39

1. Introduction ............................................................................................................ 39

2. Materials and Methods ............................................................................................ 40

2.1. Synthesis and characterization of methacrylated laminarin .......................... 40

2.2. Fabrication of MeLam microparticles ......................................................... 41

2.3. RGD functionalization of MeLam microparticles ........................................ 42

2.4. Scanning electron microscopy (SEM) ......................................................... 43

2.5. Studies on the release of PL ........................................................................ 43

2.6. Cell culture on MeLam microparticles ........................................................ 43

2.7. Cell morphology analysis ............................................................................ 44

iii

2.8. DNA quantification..................................................................................... 45

2.9. MTS Viability Assay .................................................................................. 45

2.10. Statistical analysis ....................................................................................... 45

3. Results and Discussion............................................................................................ 46

3.1. Synthesis and Characterization of Methacrylated Laminarin ....................... 46

3.2. Fabrication of Methacrylated Laminarin microparticles .............................. 47

3.3. Cumulative release of platelet lysates from MeLam microparticles ............. 49

3.4. RGD functionalization of MeLam microparticles ........................................... 51

3.5. L929 fibroblast capture and expansion on MeLam microparticles .................. 52

4. Conclusions ............................................................................................................ 54

5. References: ............................................................................................................. 54

Chapter 5 – General Conclusions ..................................................................................... 59

v

List of figures

Figure 2.1: Schematic representation of different techniques to produce microgels and their

applications. .......................................................................................................................6

Figure 2.2: Micromolding-based fabrication of hydrogel microstructures. (A) Schematic

representation of the surface tension-induced droplet formation.52 (B) Schematic

representation of the fabrication process of Janus microstrips and optical microscope images

of self-bent Janus microstrips in response to changes in the pH. The scale bar in each figure

indicates 300 μm.53 ............................................................................................................7

Figure 2.3: Bioprinting techniques. (A) Inkjet bioprinting: the drops are ejected by thermal

or acoustic forces. (B) Laser-assisted bioprinting constituted by a pulse laser and a ribbon.

(C) Extrusion bioprinting: the application of a continuous force can print bioink droplets and

fibers.69 ..............................................................................................................................9

Figure 2.4: Schematic representation of microfluidic technologies to fabricate hydrogels

microstructures. (A) Schematic representation of microfluidic technology used to produce

protein microgel based in water-in-oil emulsion.80 (B) The capillarity microfluidic device

with a three-barrel injection capillary and three parallel inserted spindlier capillaries for the

generation of microfibers and the biomimetic vessels.82 ................................................... 11

Figure 2.5: Schematic representation of the apparatus used to produce microparticles. (A)

Representation of the dropwise UV curing apparatus developed for the processing PEG-

fibrinogen cell-laden microparticles.90 .............................................................................. 13

Figure 2.6: Schematic representation of droplet microarray platform. Crosslinking of

alginate droplets by performing parallel addition of CaCl2 solutions into the individual

droplets via the sandwiching method. By changing the position of the slide 1 (bottom vs top)

containing CaCl2 droplets, it is possible to form either an array of fixed hydrogel particles

(Step 2a) or detach hydrogel particles to form freefloating hydrogel particles (Step 2b).50 14

vi

Figure 2.7: Schematic representation of the open porous microgels. After cell seeding,

spheroid cells were maintained in the open porous Nutrients could be diffused very well. 30

mg/ml of microgel – human adipose stem cells (hASCs) dispersion were injected to evaluate

the formation of adipose tissue after 14 days.100 ............................................................... 15

Figure 2.8: Chitosan microparticles are functionalized with antibodies and used for specific

cell isolation/separation from an cell heterogenous population and for further cell

expansion.45 ..................................................................................................................... 17

Figure 2.9: Schematic representation of the fabrication process showing the deposition of

cell-laden pre-polymers.118 ............................................................................................... 18

Figure 2.10: (A) Schematic of magnetic directed assembly of microgels. Microgels are

assembled to fabricate three-layer spheroids through the application of external magnetic

fields.139 (B) Assembly of magnetic freestanding hydrogel particles and representative

images of (i) brightfield, (ii) fluorescent, and (iii) overlay of coculture hydrogels. HeLa cells

expressing GFP cells (green) were immobilized in circle-shaped freestanding hydrogels and

MLTy-mCherry cells (expressing fluorescent red) were immobilized in squared-shaped

freestanding hydrogels. Scale bars: 1 mm.50 ..................................................................... 21

Figure 2.11: a. Scheme illustrating microgel formation using a microfluidic water-in-oil

emulsion system. A pre-gel and crosslinker solution are segmented into monodisperse

droplets followed by in-droplet mixing and crosslinking via Michael addition. b. microgels

are purified into an aqueous solution and annealed using FXIIIa into a microporous scaffold,

either in the presence of cells or as a pure scaffold. c. Fluorescent images showing purified

microgel building blocks (left) and a subsequent cell-laden MAP scaffold (right).22 ......... 22

Figure 3.1: Schematic representation of microfluidic system used to produce MeLam

microparticles. (A) Schematic representation of PDMS microchip with T-junction shape. (B)

Droplets formation into the microchip. (C) UV crosslinked MeLam microparticles .......... 34

vii

Figure 3.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD

with MeLam microparticles. After modification, microparticles were cultured with L929

cells. This scheme also represents cell attachment on the microparticles and subsequent

assembly of the structures. ............................................................................................... 36

Figure 4.1: Schematic representation of microfluidic system used to produce MeLam

microparticles. (A) Schematic representation of PDMS microchip with T-junction shape. (B)

Droplets formation into the microchip. (C) UV crosslinked MeLam microparticles .......... 42

Figure 4.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD

with MeLam microparticles. After modification, microparticles were cultured with L929

cells. This scheme also represents cell attachment on the microparticles and subsequent

assembly of the structures. ............................................................................................... 44

Figure 4.3: (A) Schematic illustration of the methacrylation reaction of laminarin with

glycidyl methacrylate. (B) 1H NMR spectrum of laminarin before modification. (C) Low

methacrylated laminarin (D) High methacrylated laminarin. ............................................ 47

Figure 4.4: Images of the high MeLam microparticles in oil (A) in PBS (B) obtained by

optical microscopy and respective histogram of the distribution (C) of microparticles (n =

74) after washed with PBS. Images of the low MeLam microparticles in oil (D) in PBS (E)

obtained by optical microscopy and respective histogram of the distribution (F) of

microparticles size (n = 74) after washed with PBS. SEM image of monodisperse high (G)

and low (H) MeLam microparticles .................................................................................. 48

Figure 4.5: (A) Cumulative protein profile release from low and high methacrylate

laminarin microparticles by incubation in PBS solution up to 14 days quantified by micro-

BCA assay. ELISA assay performance to quantify specific growth factors release from

microparticles. (B) Concentration of VEGF and TGF-β1 presented in PL sample. VEFG

release profiled (C) and TGF-β1 release profile (D) up to 14 days from low and high

methacrylate laminarin. Error bars represent standard deviation (n = 3). ........................... 51

viii

Figure 4.6: Fluorescence imagens showing the functionalization of MeLam microparticles.

Images of MeLam microparticles with biotin-PEG-SH (A) and MeLam microparticles

without biotin (control) (B) after incubation with fluorescently labeled SaV..................... 52

Figure 4.7: Fluorescence images of the high MeLam microparticles with encapsulated PL

(A-D) culture with L929 cells up to 14 days. These images demonstrate the ability to cell

attach, cytoskeleton was stained with phalloidin (red) and nuclei was stained with DAPI

(blue). (E) Cell viability by MTS assay was determined at 3, 7 and 11 days. (F) DNA

quantification of all formulations tested up to 11 days of culture. Results are present as mean

± standard error of the mean (n = 3). ................................................................................ 54

Figure S4.1: Images of MeLam microparticles with biotin-PEG-SH bioconjugate with pure

SaV (A) and without bioconjugation with pure SaV (control) (B) after incubation with

fluorescence biotin ........................................................................................................... 57

Figure S4.2: Images assessed by optical microscopy with encapsulated PL (A and B) and

without PL (C and D) for 24 hours and 7 days. ................................................................. 58

Figure S4.3: Fluorescence images of the high MeLam microparticles without encapsulated

PL (A-C) culture with L929 cells up to 14 days. These images demonstrate the ability to cell

attach, cytoskeleton was stained with phalloidin (red) and nuclei was stained with DAPI

(blue). .............................................................................................................................. 58

ix

Abbreviations

2D – Bidimensional

3D – Three dimensional

DMAP – 4-(N,N-dimethylamino)pyridine

DMSO – Dimethyl Sulfoxide

DS – Degree of Substituion

ECM – Extracellular Matrix

FBS – Fetal Bovine Serum

FEP – Fluorinated Ethylene Propylene

GFs – Growth Factors

HTS – High-throughput screening

MCS – Maleic Anhydride-modified Chitosan

MeLam – Methacrylated Laminarin

MSCs – Mesenchymal Stem Cells

PBS – Phosphate Buffered Saline

PDMS – Polydimethylsiloxane

PEG – Poly(ethylene glycol)

PDGF – Platelet Derived Growth Factor

PL – Platelet Lysates

PLGA – Poly(DL-lactic-co-glycolic acid)

PRP – Platelet Rich Plasma

RT – Room Temperature

SaV – Streptavidin

TE – Tissue Engineering

TGF-β1 – Transforming Growth Factor

UV – Ultraviolet

VEGF – Vascular Epithelial Growth Factor

CHAPTER 1

Motivation

1

Chapter 1 - Motivation


Tissue Engineering (TE) is a multidisciplinary field of research that applies the principles

of engineering, biology and materials science to either enhance or replace biological

tissues.1,2 Conventional TE strategies typically employ a “top-down” approach, in which

cells are seeded onto a biodegradable polymeric scaffold with specific biochemical and

physical features (e.g. porosity, pore architecture, chemical composition) to act as a

temporary support matrix in which cells are cultured.3–5 This approach has been shown

promising results towards the development of the scaffolds for the regeneration of

tissues.6,7 Nevertheless, the fabrication of complex and fully functional tissues is still a

challenge due to the limited diffusion of nutrients and oxygen and lack of vascularization

systems.8 For reconstructing hierarchically organized and complex 3D tissues that more

accurately mimic organs, the bottom-up tissue engineering approach has emerged as an

alternative strategy. This strategy employs the use of small units as building blocks with

a specific microarchitecture, that assembly to form complex 3D tissue constructs. Several

methods have been proposed for the fabrication of these tissue building blocks, including

generation and assembly of cell sheets, direct printing of cells and fabrication of cell-

laden microscale hydrogels.

Hydrogels are hydrophilic polymer networks with high water content, which allows high

biocompatibility and similar structure to the ECM.4 The recent convergence of nano and

microtechnologies, has resulted in the emergence of microscale hydrogels (microgels),

synthesized through different microfabrication techniques. For instance, micromolding,

bioprinting, microfluidics, electrospraying and superhydrophobic platforms for microgels

production are some of these techniques, which are describe in detail in chapter 2 of this

thesis. Moreover, these chapter also goes through the materials that have been explored

in the microfabrication of hydrogels as well their main applications.

Laminarin has recently been proposed as an ideal polymer to produce cell laden microgels

while retaining high cell viability.9 Laminarin is a β-1,3-glucan with β-1,6-branchings

extracted from brown algae. The low molecular weight of this polymer associated with

the intrinsic low viscosity and high solubility in aqueous or organic solvents makes this

polymer particularly appealing for the microfabrication of hydrogels.9,10 A main goal in

this project is the fabrication of cell microcarriers that in addition to serve as a support

for cell culture and expansion may be also used as an injectable system for tissue

regeneration purposes. In this project, microfluidics was used to produce laminarin

2


microparticles with well controlled dimension and high monodispersed. Two parallel

studies were conducted, fabrication of laminarin derived microparticles that were

explored as cell microcarriers for cell expansion. In this strategy, human platelet lysates

(PLs) were used as a source of growth factors. PL are a whole-blood derivative with high

concentration of growth factors that are being established as a safe and efficient cell

culture supplement. Besides the influence of PLs in the cultured cells, the release profile

from the laminarin microparticles was also studied. The results suggest that microfluidics

is an effective technique for the production of laminarin microparticles. The size of the

particles is easily controlled by adjusting parameters such as flow rates and polymer

concentrations. The laminarin microparticles allowed a slow release of PL for about 14

days. Cell culture studies demonstrated the positive effect of PL release on the culture

cells. Only particles containing PL were able to support cell expansion.

References:

1. Boos, A. M. et al. The potential role of telocytes in Tissue Engineering and Regenerative

Medicine. Semin. Cell Dev. Biol. 55, 70–78 (2016).

2. Bhowmick, S. et al. Biomimetic electrospun scaffolds from main extracellular matrix components

for skin tissue engineering application – The role of chondroitin sulfate and sulfated hyaluronan.

Mater. Sci. Eng. C 79, 15–22 (2017).

3. Bajaj, P., Schweller, R. M., Khademhosseini, A., West, J. L. & Bashir, R. 3D Biofabrication

Strategies for Tissue Engineering and Regenerative Medicine. Annu Rev Biomed Eng 16, 247–

276 (2014).

4. Slaughter, B. B. V et al. Hydrogels in Regenerative Medicine. Adv. Mater. 21, 3307–3329 (2009).

5. Pang, Y. et al. Novel integrative methodology for engineering large liver tissue equivalents based

on three-dimensional scaffold fabrication and cellular aggregate assembly. Biofabrication 8, 1–16

(2016).

6. Shen, X. et al. Sequential and sustained release of SDF-1 and BMP-2 from silk fibroin-

nanohydroxyapatite scaffold for the enhancement of bone regeneration. Biomaterials 106, 205–

216 (2016).

7. Huang, B. J., Hu, J. C. & Athanasiou, K. A. Cell-based tissue engineering strategies used in the

clinical repair of articular cartilage. Biomaterials 98, 1–22 (2016).

8. Causa, F., Netti, P. A. & Ambrosio, L. A multi-functional scaffold for tissue regeneration: The

need to engineer a tissue analogue. Biomaterials 28, 5093–5099 (2007).

9. Custódio, C. A., Reis, R. L. & Mano, J. F. Photo-Cross-Linked Laminarin-Based Hydrogels for

Biomedical Applications. Biomacromolecules 17, 1602–1609 (2016).

10. Wang, D. et al. The first bacterial β-1,6-endoglucanase from Saccharophagus degradans 2-40T

for the hydrolysis of pustulan and laminarin. Appl. Microbiol. Biotechnol. 101, 197–204 (2017).

CHAPTER 2 Recent trends on microfabrication of

hydrogels

4

Chapter 2 – Recent trends on microfabrication of hydrogels

Chapter 2 - Recent trends on microfabrication of hydrogels

Martins CR1, Custódio CA1, Mano JF1

1-Department of Chemistry, CICECO, Aveiro Institute of Materials, University of Aveiro Campus

Universitário de Santiago, 3810-193 Aveiro - Portugal

Abstract

Hydrogels are polymeric 3D networks with high water content that have become very

popular in multiple biomedical applications due to their unique properties such as,

biocompatibility and versatile fabrication. Microfabricated hydrogels (microgels) are

presently under intense investigation for application as platforms for controlled release of

drugs and bioactive molecules, cell encapsulation and cell expansion. More recently,

microgels have become popular in the design of 3D hierarchically organized constructs

for tissue engineering applications. Microgels serve as building blocks, where living cells

can be cultured and arranged in complex 3D structures that resemble in vivo complexity

of tissues and organs.

This review highlights the efforts developed in last years regarding microfabrication of

hydrogels, their applications, strategies for microgels assembly and future perspectives in

the field.

Key words: Microfabrication, Hydrogels, Bottom-up, Tissue Engineering, Microgels

1. Introduction

Hydrogels are three dimensional (3D) hydrophilic polymeric networks that partially

resemble the physical characteristics of the native extracellular matrix (ECM).1–6 Their

resemblance to living tissues opens up many opportunities for applications in biomedical

areas. The main areas of hydrogel applications are contact lenses, wound dressings,

controlled release of bioactive molecules and 3D cell culture.6–9 In particular, 3D

biomimetic structures have widespread applications in tissue engineering (TE) and have

been used for cell culture in substitution of flat 2D supports.

Methods for the preparation of 3D structures for applications in TE comprise two large

categories: top-down and bottom-up approaches.10,11 The traditional top-down approach

5


involves seeding cells into full sized porous scaffolds to form tissue constructs. These 3D

matrices can be then cultured in static conditions or inserted in bioreactors for dynamic

cell culture. Commonly used techniques to fabricate top-down scaffolds include freeze-

drying,12 solvent-casting,13 electrospinning14 and supercritical fluids.16 Scaffolds

prepared by top-down strategies have been widely explored for engineering tissues such

as skin,17 bone,18,19 or cartilage.20,21 Still, top-down approaches often have difficulty in

recreating the intricate microstructural and hierarchical features of native tissues.

Recently, bottom-up strategies have been proposed, by culturing living cells in small units

and arranging them into 3D large constructs.22 This method is based in the use of small

units as building blocks with a specific microarchitecture and the assembly of these units

to engineer large and well organized constructs.23 The bottom-up approach allows for the

creation of structures with controlled porosity and organization, multiple cell types and

high cellular densities.24 Different structures have been produced using bottom-up

approaches such as, stacked cell sheets,25 cell aggregation to form spheroids26 and

complex and organized assembly of microgels.27,28

Microgels are small soft hydrogel units formed by a crosslinked polymeric structure using

microfabrication methods. Physical and chemical crosslinking methods of microgels

preparation will be discussed in this review. Some examples of hydrogel microfabrication

techniques are micromolding,29 bioprinting,30 microfluidics,31 electrospray32 and spray

drying33 and droplet microarrays34 that will be overviewed. The assembly of microgel

units has an excellent potential for the reconstruction of 3D complex structures for TE

applications.

2. Microfabrication of hydrogels

Traditionally, hydrogels have been classified based on the method of crosslinking as

either chemically (covalently) or physically crosslinked networks. Chemical crosslinking,

includes radical polymerization,35,36 enzyme-catalyzed crosslinking37 and click

chemistry.38 A common method to promote radical polymerization is the use of ultraviolet

(UV), visible or infrared light along with a photoinitiator that allows the formation of a

radical group. The free radical reacts with specific functional groups on the polymer

backbone allowing the polymerization.39–41 The use of click chemistry to produce

hydrogels has been increasing during the last years due to the high reactivity and

selectivity and mild reaction conditions.35 In particular, recent development of radical

6


mediated thiol-ene chemistry, Diels-Alder reaction, tetrazole-alkene photo-click

chemistry, and oxime renders it possible to form hydrogels.40,42,43

Physical crosslinking includes ionic interaction,44,45 hydrogen bonds interactions,46

polymer self-aggregation via non-covalent interactions and electrostatic interactions.47,48

Different stimulus can promote these reactions, such as temperature and pH variations.

The physical crosslinked hydrogels can be classified in thermosensitive,49

stereocomplexed30 and ionically50 depending on the crosslinking response.

The previous listed methods have also been used associated with microfabrication

techniques to form a variety of microgels (figure 2.1). Such microfabrication strategies

will be described in the following topics.

Figure 2.1: Schematic representation of different techniques to produce microgels and their applications.

2.1.Micromolding

Micromolding is a versatile technique, with associated low cost and simplicity that can

be used to mold a variety of different materials in multiple geometries and sizes.29 This

technique is based on printing, molding and embossing that allow for the production of

microgels.51 A prepolymer solution is first casted into a mold featuring well designed

arrays of wells, typically fabricated from elastomeric materials such as

polydimethylsiloxane (PDMS). After crosslinking, the microgels are taken out from the

mold. For example, Jung and co-works developed a micromolding-based approach to

synthesize poly(ethylene glycol)-based microspheres with controlled macroporous

structures.52 For that an aqueous prepolymer solutions was used to fill the wells of

7


micropatterned PDMS molds (figure 2.2 A) Next, hydrophobic wetting fluid consisting

of hexadecane and a photoinitiator is pipetted onto the molds to allow surface tension-

induced droplet formation of the prepolymer solution. More recently, Oh and co-workers

have explored micromolding to produce a pH-responsive self-bending of bilayered Janus

hydrogel microstrips (figure 2.2 B).53 The prepolymer solution was deposited in PDMS

mold and polymerized under UV radiation. This technique allowed to define the

dimensions and shape of Janus microstrips on demand and precisely tune the chemical

properties.

The versatility of micromolding allows the fabrication of microgels with different

morphologies and capable to encapsulate cells, proteins, to create responsive systems and

vascularized structures.4,54 This ability to produce microgels molded in any shape enables

great control over the assembly process into 3D structures.4,53

Figure 2.2: Micromolding-based fabrication of hydrogel microstructures. (A) Schematic representation of

the surface tension-induced droplet formation.52 (B) Schematic representation of the fabrication process of

Janus microstrips and optical microscope images of self-bent Janus microstrips in response to changes in

the pH. The scale bar in each figure indicates 300 μm.53

2.2.Bioprinting

3D bioprinting allows for the rapid prototyping of complex 3D constructs and it has been

used as a powerful tool to engineer tissues and organs. This technology allows for the

specific placement of cells and tissues, enabling the production of complex and highly

organized 3D constructs.57–60 In TE bioprinting techniques can be divided into inkjet,

laser-assisted, and extrusion bioprinting.

A B

8


Inkjet printing technique allows to handle and print biological materials at high

resolution, precision and speed.61 In this technique electrostatic, thermal62 or acoustic63

forces can be applied to eject droplets from the nozzle (figure 2.3 - A). This system offers

some advantages such as low cost when compared with other similar techniques, high

resolution and high speed and compatibility with many biological materials. Beyond this,

inkjet printing has the potential to introduce concentration gradients of cells, materials or

growth factors throughout the 3D structure by altering drop densities or sizes.64,65 Still,

inkjet printing has some disadvantages, such as the risk of exposing cells and materials

to thermal and mechanical stress, low droplet directionality, non-uniform droplet size,

frequent clogging of nozzle and unreliable cell encapsulation.61 Iwanaga and co-workers

have used inkjet printing to produce alginate microparticles.65 They tested distinct

alginate solutions to analyse the influence on particle size, which exhibit a linear

correlation with the concentration of alginate. Zhu and co-workers developed a gold

nanorod-incorporated gelatin methacryloyl-based bioink for printing 3D functional

cardiac tissue constructs.66 The authors demonstrated that the developed bioink with low

viscosity, allows for the easy integration of cells at high densities and improved cell

adhesion and organization.

Laser-assisted is another bioprinting technique used to engineer 3D constructs. The

essential part of this technique is a donor layer that responds to laser stimulation. This

system is composed by three main components constituted by a pulse laser source beam,

a ribbon that prints the structures and a substrate that collects the printed materials (figure

2.3 – B).61,67 Due to its unprecedented cell printing resolution and precision and ability to

print viscous materials, is an attractive tool for the in printing of tissues and organs

substitutes.67 Keriquel and co-workers reported the use of laser-assisted bioprinting to test

different cell printing geometries. The authors demonstrated for the first time that printed

cells remain viable and proliferate, both in vitro and in vivo, independent of the geometry

used.68 Nevertheless, lasers with high resolution and intensity are expensive compared to

other nozzle-based printing methods, and this could represent a limitation for laser

assisted printing techniques.64,69

An alternative bioprinting technique is extrusion, a modification of inkjet printing.69

Generally, extrusion bioprinting functions by the robotically controlled extrusion of a

material, which is deposited onto a substrate.61 This system can be used to deposit

materials with high viscosity. It is applied to the system a continuous force, that allow to

print uninterrupted cylindrical lines rather than a single bioink droplet (figure 2.3 - C).

9


Some concerns about cell viability have been reported using extrusion bioprinting, and

this may be a limitation of this technique. A main advantage of this technique is the

compatibility with a high range of materials such as hydrogels, copolymers and cell

spheroids.61,64 An additional advantage is its good compatibility with photo, chemical and

thermal crosslinking.69 In a recent study, Zhu and co-workers developed a gold nanorod-

incorporated gelatin methacryloyl based bioink for printing 3D functional cardiac tissues.

They used extrusion bioprinting for the printing of cell-laden fibers at a high resolution

and high cell viability.66

Figure 2.3: Bioprinting techniques. (A) Inkjet bioprinting: the drops are ejected by thermal or acoustic

forces. (B) Laser-assisted bioprinting constituted by a pulse laser and a ribbon. (C) Extrusion bioprinting:

the application of a continuous force can print bioink droplets and fibers.69

2.3.Microfluidics

Microfluidics have recently become a highly attractive tool to overcome limitations of

the traditional bulk emulsion methods to generate monodisperse microgels in a reliable

manner. Microfluidic technologies have also been used to produce microplatforms to

study new drugs and to produce microparticles that can be used to building 3D constructs.

The principle of microfluidics is the creation of a stream of polymer solution in a

microchannel (dispersed phase) and the induction of a periodic break-up by flow focusing

with a second immiscible fluid (continuous phase). The two fluids cross each other on a

chip which can have different shapes, such as T-junction, flow Focusing and co-flow.

When these two fluids meet, droplets form in a “drop-by-drop” fashion due to a balance

of interfacial tension and the shear of the continuous phase acting on the dispersed phase

.31,68 The disperse phase may contain a crosslinking agent, cells or biomolecules which

will be encapsulated in the droplet/microparticle.72 An important advantage of this

technology is the ability to produce droplets with highly controlled morphology with the

added benefit of providing droplet manipulation (merging, sorting, deformation, single-

10


cell loading, etc.) due to the facility in tuning different flow rates solutions.73 Another

advantage of microfluidics is the possibility to rapidly produce monodispersed

microparticles with encapsulated living cells,74,75 drugs76 or other bioactive compounds

in a unique step. One of the most widely used systems in droplet generation using

microfluidics, is based in water-in-oil emulsifications.77–79 Mao and co-workers reported

a microfluidic-based method for encapsulating single cells within a thin hydrogel layer

using a one-step method.72 The results of this study show that cell viability was

maintained over a three-day period and that the microgels are mechanically tractable. In

another study, Shimanovich and co-workers have described an approach for generating

microgels composed of amyloid fibril networks using a microfluidic droplet maker

device.80 They have demonstrated that these microgels enable the local release of

encapsulated molecules resulting in enhanced antimicrobial action (figure 2.4 - A). An

interesting conclusion from this work was that they were able to generate dynamic

materials that enable the internal content of nanofibrils to be changed at the post-synthetic

stage in response to exposure to monomeric protein molecules in solution. Importantly,

the protein nanofibril microgels demonstrated to be nontoxic to a human cell line and

have the potential for effective drug encapsulation.

Krutkramelis and co-workers have described the photopolymerization of aqueous

hydrogel forming solutions within microscale emulsion droplets.81 They studied the

inhibitory effect of oxygen in photopolymerization. After understanding this limitation,

it was possible to fabricate a continuous PEG-DA microgel on a chip, a nitrogen-jacketed

microfluidic device for in situ oxygen purging has been developed. The works previously

reported confirm precise control offered by microfluidic technology under the structures

produced. This demonstrate the great importance of the technology can offer to create

tissue constructs.

The microgels may be formed in situ by the action of crosslinking reactions occurring

along the time (the crosslinking agent already exists in the aqueous solution), through

high irradiation or by a temperature change. The droplets can also gellify at the exit of

the tube using spatial precipitation co-axial baths.

Cheng and co-workers used microfluidics to fabricate bioactive microfibers for creating

different tissue constructs.82 By employing a multiple laminar flow they were able to

fabricate a series of cell-laden hydrogel microgels with tunable morphological and

structural features from designed multiple injection flows (figure 2.4 - B). They proposed

11


this technology to generate complex fiber-shaped cellular building-blocks continuously

in a one-step microfluidic spinning process.

Figure 2.4: Schematic representation of microfluidic technologies to fabricate hydrogels microstructures.

(A) Schematic representation of microfluidic technology used to produce protein microgel based in water-

in-oil emulsion.80 (B) The capillarity microfluidic device with a three-barrel injection capillary and three

parallel inserted spindlier capillaries for the generation of microfibers and the biomimetic vessels.82

2.4.Spray based technologies

Electrospray is a broadly used technique to generate submicrometric particles with high

yields. In electrospraying, the solution is forced through a nozzle spray machine or

syringe needle and a Taylor cone is created due to the applied electric field forming

droplets that are further crosslinked.56,83,84 Electrospraying can be used to produce small,

nearly monodisperse particles when a colloidal suspension of solid nanoparticles or a

solution of a material is sprayed. Although this technique allows to produce monodisperse

particles, microfluidic technique allows to improve the monodispersity of the

microparticles. Different parameters can be manipulated to control morphological

features of the microgels in terms of size and distribution, shape, surface roughness and

porosity.85 The droplets size can be controlled by the liquid flow rate and by adjusting the

voltage applied to the nozzle.86 Electrospraying allows the encapsulation of living cells,

growth factors or drugs in a single step procedure and this could be an advantage when

12


compared with other time consuming techniques.87,88 Kim and co-workers described the

fabrication of RGD-alginate microgels formed by electrospraying for the encapsulation

of endothelial cells and growth factors.87 They have proved that encapsulated endothelial

cells maintain viability and are able to proliferate within the microgels. In addition, the

RGD-alginate microgels demonstrated the long-term release of the encapsulated growth

factors in vitro. In a similar study, Yao and co-workers developed a fibrin nanofiber

hydrogel loaded with drug encapsulated poly(DL-lactic-co-glycolic acid) (PLGA)

microspheres prepared via electrospray and electrospinning.89 Electrospray allowed the

production of PLGA microspheres with high drug efficiency and convenient drug release

control. After, by electrostretching process microspheres was encapsulated into

microfibers hydrogel. This system showed a good release behaviour comparing to the

PLGA microspheres and hUMSCs showed a better adhesion demonstrating the great

potential in the neural regeneration to the system. Another method based on droplet

formation and subsequent conversion into particles is co-axial air-flow. Here, the solution

is forced through a nozzle and injected air led the solution to break up into a spray at the

outlet of nozzle.27 Oliveira and co-workers developed a system to fabricate PEG-

fibrinogen cell laden particles.90 The polymer solution was exposed to UV light prior to

its entry into the jet-in-air nozzle that promote the increases the viscosity of the solution

(figure 2.5).

Spray-drying involves the use of a spray drier, mainly consisting of an atomizer and

drying chamber. The solvent is atomized in droplets induced by a stream of hot air that

promotes solvent evaporation in the spray-drying chamber, resulting in the formation of

microparticles, which are then separated from the air through a cyclone or a filter bag.91,92

Spray drying technique offers many advantages, including efficiency, reproducibility,

simplicity, scale-up and the ability to produce homogenous powders.93 These advantages,

led to the use of this system in the production of microgels, coatings and powders.33 Zhao

and co-workers reported the synthesis of stimuli-responsive hemicellulose microgels via

a single-step crosslinking chemistry using spray drying.33 The crosslinking reaction

occurred during spray drying, and the functional hemicellulose microgels were made

responsive to different external stimuli such as pH, electroactivity, magnetic field, and

dual-stimuli (pH and electric field). The simultaneous use of the both methods offers the

potential for fabrication of microgels with rapid stimuli response and good

biocompatibility. Some disadvantages of spray-drying include the possible degradation

of the heat sensitive fine particles, inefficient yields and heterogeneous size

13


distribution.92,94 Take into account this, spray dry method can be used to produce

hydrogels microparticles, although it is not better option, being more used in

pharmaceutical industry to produce powders.

Figure 2.5: Schematic representation of the apparatus used to produce microparticles. (A) Representation

of the dropwise UV curing apparatus developed for the processing PEG-fibrinogen cell-laden

microparticles.90

2.5.Superhydrophobic-based platforms for microgel production

In most of the techniques previously reported, the hydrogels were produced in liquid

environment. The superhydrophobic-based platforms are an alternative microparticle

processing method. The droplets are formed using a superhydrophobic surface that allows

to change the contact angle between the aqueous solution and the surface. When the liquid

is suspended on the surface, a sphere is formed involving a basic liquid-air interface.

Superhydrophobic-based platforms are potentially attractive to produce multiple

microgels with encapsulated proteins or other biomolecules and living cells.95 Song and

co-workers were pioneers in the processing of biomaterials at the microscale using

superhydrophobic platforms.95 Such technology was later on adapted to produce

multilayered particles96 or liquified particles.97 However, the proposed strategy,

encompasses multiple pipetting and subsequent crosslinking, resulting in the production

of particles not smaller than 1 mm in diameter, hampering in consequence their tentative

use for cell encapsulation. In order to overcome these limitations, Costa and co-workers

have proposed a new method to produce droplet microarrays that allows the production

of microgels with sizes under 500m.98 The deposition of droplets over fully covered

14


superhydrophobic surfaces enables the production of sphere-like objects. Recently, Neto

and co-workers developed a versatile platform (figure 2.6) that allows to create a thousand

microdroplets of specific geometry and volume, based on the use of superhydrophobic

surfaces patterned with a wettable superhydrophilic domains.50 This approach enables the

dispensing of aqueous solutions into thousands of droplets without the need for manual

pipetting or robotic devices. This is very promising due to its ability to easily create and

manipulate thousands of microgels of controlled size and geometry.

Figure 2.6: Schematic representation of droplet microarray platform. Crosslinking of alginate droplets by

performing parallel addition of CaCl2 solutions into the individual droplets via the sandwiching method.

By changing the position of the slide 1 (bottom vs top) containing CaCl2 droplets, it is possible to form

either an array of fixed hydrogel particles (Step 2a) or detach hydrogel particles to form freefloating

hydrogel particles (Step 2b).50

3. Applications of microfabricated hydrogels

Microfabricated hydrogels have found multiple applications as cell encapsulation, cell

expansion used as building units to engineer complex and hierarchal 3D constructs, drug

delivery or even as microreactors. In the next topics, recent developments in microgel

applications are presented and discussed.

3.1.Cell laden microgels as building blocks for 3D constructs

Most living tissues are composed of repeating microunits, which are combinations of

multiple cell types with well-defined 3D microarchitectures and tissue-specific functional

properties. A major challenge in TE is to engineer biomimetic tissues that contain

appropriate cell microenvironment and the multicellular architectural features found in

vivo. Taking this into account, microgels became attractive entities for TE applications,

15


namely for the fabrication of complex and hierarchal 3D structures. Du and co-workers

were pioneers in the fabrication of biomimetic 3D tissue constructs using cell laden PEG

microgels opening the paradigm for directing the assembly of mesoscale materials.25

Yuebi and co-workers demonstrated the self-assembly of alginate microgels modified

with combinations of binding pair molecules.99 These molecules bind to their

complements rapidly and under physiological conditions. The modified alginate

microgels were shown to support viable cell encapsulation. The modified microgels can

be induced to self-assemble either in suspension or following a brief centrifugation step

forming complex 3D constructs that can combine multiple cell types. In a more recent

work, Xia and co-workers developed a strategy to fabricate open porous microgels with

high hydrophobicity and great injectability (figure 2.7).100 The microgels were based on

double bonded poly-(L-glutamic acid)-g-2-Hydroxylethyl methacrylate (PLGA-g-

HEMA) and maleic anhydride-modified chitosan (MCS). The high hydrophobicity of the

system, made the stem cell shape spheroid to favour adipogenic differentiation. The

porous structure promoted the nutrients diffusion that promotes the high stem cell

viability. Lienemann and co-workers developed a strategy based on microfluidic to create

monodisperse pre-microgel droplets.101 This work presented a strategy to bypass Poisson

encapsulation statistics in synthetic microniches by selection of only cell-laden microgels.

They demonstrated of the versatility of this work, developing a strategy that allows

efficient encapsulation of individual cells that can be used for different cell types.

Cell-laden microgels can be applied in various applications as shown the works

previously reported. Encapsulation of single cells can be useful to study biological

responses a specific cellular type.

Figure 2.7: Schematic representation of the open porous microgels. After cell seeding, spheroid cells were

maintained in the open porous Nutrients could be diffused very well. 30 mg/ml of microgel – human adipose

stem cells (hASCs) dispersion were injected to evaluate the formation of adipose tissue after 14 days.100

16


3.2.Cell microcarriers

Microcarriers have been widely used in biotechnology industry for the large-scale

production of proteins or viruses.102,103 Cell microcarriers are also a promising culture

system for producing great quantities of anchorage-dependent cells for biomedical and

biochemical applications.104–106 In this case, cells are usually cultured over the surface of

microparticles and are expanded in dynamic conditions using bioreactors. Expanded cells

are recovered from the microcarriers by tripsinization. The coating of microparticles

using smart polymers could be also envisaged to use temperature or other mild stimuli

for cell recovery.107 From cell microcarriers used up to date, Cytodex 3 is probably the

mostly used. This commercial microcarrier consists of a thin layer of denatured collagen

chemically coupled to a matrix of crosslinked dextran. For instance, Nie and co-workers

have used Cytodex to expand and differentiate human embryonic stem cells (ESCs) into

the three germ layers.108 Gelatin based microcarriers (CultiSpher-S) have also been

broadly used for cell expansion. Bender and co-workers have shown the potential of these

gel microcarriers in enhanced cortical neuron adhesion, viability and neurite extension.109

In a more recent work, Soure and co-workers developed a xenogeneic-free microcarrier-

based system for the effective expansion of umbilical cord matrix derived from

mesenchymal stem cells (MSCs) under dynamic conditions.110 In this work, they used a

bioreactor-based manufacturing of MSCs cultured in medium supplemented with human

platelet lysates.

These principals were extrapolated to TE strategies where these systems might be used

both as a strategy for cell expansion and simultaneously injectable systems to be used in

vivo using minimally invasive procedures.111 For example, Custódio and co-workers

reported the use of chitosan microparticles to capture and expand a specific cell type that

can also be regarded as an injectable biomaterial (figure 2.8).45 The microparticles were

modified with specific antibodies enabling the particles to select specific cell types from

mixed cell populations and further cell expansion. The authors suggest that this system

may be used in vivo for tissue regeneration using minimally invasive procedures. Kim

and co-workers also developed an injectable multifunctional microparticle system.87

However, while the work of the Custódio and co-workers promotes cell adhesion on the

surface of microparticle, in Kim´s work cells and growth factors were encapsulated in

microparticles. Cells encapsulated within the microgel exhibited a time-dependent

proliferation with enhanced cell viability, and the size-controlled microgels resulted in

17


sustained release of growth factors for enhanced new vessel formation. This method

shows a decrease in the host immune response, but also may be a promising strategy to

improve the therapeutic efficacy for the delivery of therapeutic cells and angiogenic

proteins. Such kind of assembled particles mediated by multiple cell attachment may be

compartmentalized in liquified environments to produce microtissues that can be used in

therapies or as disease models.112,113

Figure 2.8: Chitosan microparticles are functionalized with antibodies and used for specific cell

isolation/separation from an cell heterogenous population and for further cell expansion.45

3.3.High-throughput screening

Engineering tissues combines the use of different biomaterials with proteins and/or cells.

Reactions and interactions between the cells and biomaterials need be studied to evaluated

the cell biology and cytotoxicity of the material. This led to the emerging of high-

throughput screening (HTS) approaches. This powerful technology allows to evaluate a

large combination of structural, biophysical and biochemical parameters and screening

the individual and combined effects of multivariate biological cues.50,114,115

The HTS is developed under the principles: sufficiently sensitive to measure a relevant

cellular response, easy to automate and reproducible.114,116 Basically, this system consists

of a library of distinct material or molecular entities, displayed on a single substrate at

specific addressed locations, so that comparative cell response to each test entity can be

assessed.117 The main advantages of this technology are the significant decrease in cost

and time for screening a biological target. Based on these principles, Guermani and co-

workers developed a microprinting approach to print cellular structures capable to mimic

native tissues architecture.118 The goal of their work, was the synthesis of a high-

18


throughput platform to understand the effects of structural, cellular and

microenvironmental heterogeneity on human mesenchymal stem cells differentiation.

(figure 2.9). In a similar approach, superhydrophobic surface patterned with wettable

spots have been used to dispense and fix small volumes of aqueous based solutions that

can give rise to patterned microgels.119 Such technology permits the distribution of a large

amount of controlled combinations of materials and cells in the hydrogels that can be

analysed directly in the chip using image analysis. Besides the biological behaviour of

the cells other properties of the individual microgels can be assessed. Oliveira and co-

workers developed an on-chip microarray platform based on this type of surfaces to test

the mechanical and viscoelastic properties of miniaturized hydrogels and their effect in

cell function.120

Figure 2.9: Schematic representation of the fabrication process showing the deposition of cell-laden pre-

polymers.118

19


3.4.Drug Delivery

The polymer network can easily be tuned by controlling the density of crosslinks in the

gel matrix and the affinity of the hydrogels for the aqueous environment in which they

are swollen. The properties of the hydrogel at the macromolecular scale will influence

diffusion and interfacial properties. Due to such versatility, microgel beads have been

widely recognized as a promising material for controlled drug delivery.121,122 The

presence of macromolecular chains in the hydrogels that react with external stimuli

permits to develop microgels able to control the release profile of encapsulated drugs

depending on parameters such as pH, temperature or the presence of specific

molecules.107,123,124 Lee and co-workers demonstrated the possibility of ascorbic acid

delivery in response to different pH stimulus using P(MAA-co-EGMA) hydrogel

microparticles.125 The swelling of the microparticles also showed different responses to

pH changes. This system allows to control the ascorbic acid delivery to control pH of the

environment. Sivakumaran and co-workers explored the application of soft

nanocomposite injectable hydrogels containing entrapped microgels for drug delivery.126

Copolymer microgels based on N-isopropylacrylamide and acrylic acid were synthesized

that exhibited both ionic and hydrophobic affinity for binding to bupivacaine. Microgels

were subsequently immobilized within an in situ-gelling hydrogel network to achieve

longer and more constant drug release kinetics than can be achieved with microgels alone.

In a more recent work, Lai and co-workers developed an approach based of electrospray

technique to produce core-shell hydrogel microspheres.127 They used

carboxymethylcellulose (CMC) and alginate polymers to produce microparticles with

multi cores with distinct properties. This system allows a better control in drug delivery

and a great potential for multi-drug therapy, due to the possibility to separate drugs in

different cores. Different materials can be used to produce hydrogels that enables the

encapsulation of different drugs, proteins or other biomolecules. Controlling the porosity,

the shape and the layers of hydrogels are possible to control the drug delivery.

3.5.Assembly of microgels

In TE, there has been significant interest in using assembly of small building blocks to

form biological tissues in a bottom-up engineering strategy. To date, bottom-up assembly

of microgels has been achieved by a variety of techniques. Such methods can be divided

in two main groups: self-assembly or directed assembly. Self-assembly is the process by

which an organized structure spontaneously forms from individual components into

20


patterns or structures.11,128 Self-assembly properties have been exploited for the

preparation of 3D nanostructured bioactive scaffolds, by using specific complementary

molecular interactions. Microgels have been conjugated with ionic-complementary self-

assembling peptides, oligosaccharides, binding pair molecules and nucleic acids allowing

the self-organization of the microgels.99,129,130

Directed assembly is the use of specific driving forces to assemble microgels in a directed

manner. Directed assembly approaches include programmable molecular recognition and

binding scheme, hydrophilic-hydrophobic interactions, surface template, microfluidic

and magnetic assembly are promising technologies to assemble microgels.131

3.5.1. Surface Tension

Whitesides and co-workers were pioneers in the microfabrication of 3D constructs that

assemble by surface tension. Using this strategy, hydrophilic microgels can be assembled

in millimetre-scale objects into well-defined 3D structures through minimization of the

interfacial free energy of the liquid–liquid interface.132,133 Parameters such as external

energy input, surface tension, and microgel dimensions can be controlled and influence

the assembly process. Du and co-workers also reported about shape-controlled microgels

that direct assemble within multiphase reactor systems into predetermined geometric

configurations.25,134 By increasing the hydrophilicity of the microgels and reducing the

surface tension of the surrounding solution they were able to improve the assembly

process. In addition, by combining this directed assembly process with a second

crosslinking they were able produce 3D structures with increased stability. The increase

of hydrophobicity at the surface of hydrogels can lead to floatable microbjects that can

move over the surface of aqueous media with minimum friction.135

3.5.2. Acoustic assembly

Acoustics has been also used in the biomedical field to manipulate droplets, cells and

biomolecules. The application of the surface waves has showed some advantages like as

non-invasive, simple and inexpensive approach towards the precise and rapid patterning

of microparticles and cells.136 For example, Feng Xu and co-workers reported the use of

acoustic fields to direct the assembly of PEG-methacrylate microgels (figure 2.11).131 By

applying an acoustic field to droplet encapsulating microgels of different shapes and sizes

the particles are agitated and self-assemble. They also investigated the effect of acoustic

frequency and amplitude on the assembly process, and the effect of acoustic excitation

21


on the viability of cells encapsulated in the microgels. The results indicated that

crosslinking and acoustic excitation did not have significant effect on cell viability.

Despite the very promising results in acoustic assembly, a main limitation of this method

is the uni-directional patterning, that limited the creation of line patterns.

3.5.3. Magnetic assembly

Magnetic fields have different applications in TE such as cellular manipulation, cell

sorting and isolation, 3D cell culture and clinical in vivo imaging.137 Recently, magnetic

hydrogels have emerged as a novel biocomposite for their active response properties and

extended applications.138 The potential of the magnetic hydrogels for the fabrication of

modular 3D scaffolds have been also explored to promote directed assembly between

structures. Xu and co-workers reported about the microfabrication of gelatin methacrylate

(GelMA) and PEG microgels incorporating magnetic nanoparticles that were easily

assembled into microscopic hydrogels (figure 2.10 - A).139 These microgels were used as

building blocks to create 3D complex multilayer constructs using external magnetic

fields. More recently, Neto and co-workers reported about the fabrication of freestanding

hydrogel particles with defined geometries and sizes containing magnetic particles and

cells (figure 2.10 - B).50 The full control of the magnetic fields is still a challenge to truly

be able to build organized and predefined 3D structures using such magnetic-responsive

building blocks.

Figure 2.10: (A) Schematic of magnetic directed assembly of microgels. Microgels are assembled to

fabricate three-layer spheroids through the application of external magnetic fields.139 (B) Assembly of

magnetic freestanding hydrogel particles and representative images of (i) brightfield, (ii) fluorescent, and

(iii) overlay of coculture hydrogels. HeLa cells expressing GFP cells (green) were immobilized in circle-

shaped freestanding hydrogels and MLTy-mCherry cells (expressing fluorescent red) were immobilized in

squared-shaped freestanding hydrogels. Scale bars: 1 mm.50

22


3.5.4. Biomolecular recognition

Biomolecular recognition has been also explored to assemble microgels into 3D

structures. For example, Griffin and co-workers synthesized microgel building blocks

composed of multi-armed poly(ethylene) glycol-vinyl sulphone backbones decorated

with a cell adhesive peptide and two transglutaminase peptide substrates (K and Q).

(figure 2.11).22 The microgel building blocks were then assembled through Michael-type

addition between the K and Q peptides mediated by activated Factor XIII (FXIIIa), a

naturally occurring enzyme responsible for stabilizing blood clots. This enzyme-mediated

annealing process allowed incorporation of living cells into a dynamically forming MAP

scaffold that contained interconnected microporous networks.

In a different strategy, using DNA nucleotide bases as programmable, sequence-specific

‘glues’, shape-controlled hydrogel units have been self-assembled into complex 3D

structures.140 Li and co-workers modified the surface of cell-laden PEG microgels with

complementary DNA to form DNA-templated assembly of PEG microtissues. The

recognition capabilities of DNA are the key to achieve rapid assembly of multiple

building blocks. In a similar approach Qi and co-workers have developed a strategy to

use complementary DNA molecules as glue to direct the self-assembly of hydrogel

cubes.141 Using hydrogel cubes that display giant DNA on multiple designated faces, they

were able to construct linear chain structures and net-like structures.99 The use more than

one pair of complementary DNA chains (on other kind of biochemical recognition) would

enable the independent assembly of different points and to produce more controlled and

complex structures.

Figure 2.11: a. Scheme illustrating microgel formation using a microfluidic water-in-oil emulsion system.

A pre-gel and crosslinker solution are segmented into monodisperse droplets followed by in-droplet mixing

and crosslinking via Michael addition. b. microgels are purified into an aqueous solution and annealed using

23


FXIIIa into a microporous scaffold, either in the presence of cells or as a pure scaffold. c. Fluorescent

images showing purified microgel building blocks (left) and a subsequent cell-laden MAP scaffold (right).22

3.5.5. Cellular controlled assembly

A number of works have been focused on the development of materials that self-assembly

through the presence of cells. Cruz and co-workers have shown that chitosan microgels

support cell adhesion and proliferation.142 When cells spread actively on a number of

microgels they trigger the assembly of the microstructures, forming a 3D scaffold.

Oliveira and co-workers have reported similar outcomes using protein based microgels to

form cell-induced aggregation 3D constructs.143 More recently, Custódio and co-workers

developed a refined strategy using functional microgels capable of binding to receptors

on the cell surface.27 They demonstrated that microgels when combined with cells, are

able to establish interconnected networks and lead to the formation of stable and robust

3D structures. They has also reported that chitosan microgels bioconjugated with specific

antibodies provide suitable surfaces to capture a target cell type, subsequent expansion of

the captured cells and assembly in a complex 3D structure.45

4. Conclusions

Microgels are microfabricated hydrophilic polymeric networks in the range of several

micrometers down to nanometers. In the past decades, multiple methods and technologies

have been developed to microfabricate hydrogels. Microfabrication has been used to

produce microgels for cell encapsulation with high cell viability due to the increased

diffusion of oxygen and nutrients. Microfabrication techniques have also been used for

encapsulation of drugs and other biomolecules without compromising their bioactivity.

Biological systems are highly hierarchical in structure. To mimic such organization

microfabrication tools that can generate and manipulate multi-scale building units are

needed. Is in this context microgels are particularly attractive as building blocks to

engineer complex and organized tissues.

Still, producing functional constructs is challenged by the limitations in the assembly

processes and the effective fabrication of tissue constructs with relevant length scales.

5. References:

1. Geckil, H., Xu, F., Zhang, X., Moon, S. & Demirci, U. Engineering hydrogels as extracellular

24


matrix mimics. Nanomedicine 5, 469–84 (2010).

2. Shapiro, J. M. & Oyen, M. L. Hydrogel composite materials for tissue engineering scaffolds. Jom

65, 505–516 (2013).

3. Zhu, J. & Marchant, R. E. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev.

Med. Devices 8, 607–626 (2011).

4. Yeh, J. et al. Micromolding of shape-controlled, harvestable cell-laden hydrogels. Biomaterials

27, 5391–5398 (2006).

5. Ahmed, E. M. Hydrogel: Preparation, characterization, and applications: A review. J. Adv. Res. 6,

105–121 (2015).

6. Slaughter, B. B. V et al. Hydrogels in Regenerative Medicine. Adv. Mater. 21, 3307–3329 (2009).

7. Wu, J. et al. Fabrication and characterization of monodisperse PLGA-alginate core-shell

microspheres with monodisperse size and homogeneous shells for controlled drug release. Acta

Biomater. 9, 7410–7419 (2013).

8. Riederer, M. S., Requist, B. D., Payne, K. A., Way, J. D. & Krebs, M. D. Injectable and

microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels.

Carbohydr. Polym. 152, 792–801 (2016).

9. Caló, E. & Khutoryanskiy, V. V. Biomedical applications of hydrogels: A review of patents and

commercial products. Eur. Polym. J. 65, 252–267 (2015).

10. Oliveira, S. M., Reis, R. L. & Mano, J. F. Towards the design of 3D multiscale instructive tissue

engineering constructs: Current approaches and trends. Biotechnol. Adv. 33, 842–855 (2015).

11. Lu, T., Yuhui, L. & Chen, T. Techniques for fabrication and construction of three-dimensional

scaffolds for tissue engineering. Int. J. Nanomedicine 8, 337–350 (2013).

12. Correia, C. R. et al. Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering.

Tissue Eng. Part C. Methods 17, 717–730 (2011).

13. Kong, J., Hwang, I.-W. & Lee, K. Top-Down Approach for Nanophase Reconstruction in Bulk

Heterojunction Solar Cells. Adv. Mater. 26, 6275–6283 (2014).

14. Jayakumar, R., Prabaharan, M., Nair, S. V. & Tamura, H. Novel chitin and chitosan nanofibers in

biomedical applications. Biotechnol. Adv. 28, 142–150 (2010).

15. Wijesena, R. N. et al. A method for top down preparation of chitosan nanoparticles and

nanofibers. Carbohydr. Polym. 117, 731–738 (2015).

16. Santo, V. E. et al. Enhancement of osteogenic differentiation of human adipose derived stem cells

by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid

foaming. J. Control. Release 162, 19–27 (2012).

17. Bhowmick, S. et al. Biomimetic electrospun scaffolds from main extracellular matrix components

for skin tissue engineering application – The role of chondroitin sulfate and sulfated hyaluronan.

Mater. Sci. Eng. C 79, 15–22 (2017).

18. Sun, X. et al. Modeling vascularized bone regeneration within a porous biodegradable CaP

scaffold loaded with growth factors. Biomaterials 34, 4971–4981 (2013).

19. Shen, X. et al. Sequential and sustained release of SDF-1 and BMP-2 from silk fibroin-

nanohydroxyapatite scaffold for the enhancement of bone regeneration. Biomaterials 106, 205–

25


216 (2016).

20. Parmar, P. A. et al. Collagen-mimetic peptide-modifiable hydrogels for articular cartilage

regeneration. Biomaterials 54, 213–225 (2015).

21. Huang, B. J., Hu, J. C. & Athanasiou, K. A. Cell-based tissue engineering strategies used in the

clinical repair of articular cartilage. Biomaterials 98, 1–22 (2016).

22. Griffin, D. R., Weaver, W. M., Scumpia, P. O., Di Carlo, D. & Segura, T. Accelerated wound

healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat

Mater 14, 737–744 (2015).

23. Causa, F., Netti, P. A. & Ambrosio, L. A multi-functional scaffold for tissue regeneration: The

need to engineer a tissue analogue. Biomaterials 28, 5093–5099 (2007).

24. Khan, O. F., Voice, D. N., Leung, B. M. & Sefton, M. V. A novel high-speed production process

to create modular components for the bottom-up assembly of large-scale tissue-engineered

constructs. Adv. Healthc. Mater. 4, 113–120 (2015).

25. Du, Y., Lo, E., Ali, S. & Khademhosseini, A. Directed assembly of cell-laden microgels for

fabrication of 3D tissue constructs. Proc. Natl. Acad. Sci. 105, 9522–9527 (2008).

26. Cheng, N. C., Wang, S. & Young, T. H. The influence of spheroid formation of human adipose-

derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials 33,

1748–1758 (2012).

27. Custódio, C. A. et al. Functionalized microparticles producing scaffolds in combination with

cells. Adv. Funct. Mater. 24, 1391–1400 (2014).

28. Chan, H. F. et al. Rapid formation of multicellular spheroids in double-emulsion droplets with

controllable microenvironment. Sci. Rep. 3, 3462 (2013).

29. Tekin, H. et al. Responsive micromolds for sequential patterning of hydrogel microstructures. J.

Am. Chem. Soc. 133, 12944–12947 (2011).

30. Poldervaart, M. T. et al. Prolonged presence of VEGF promotes vascularization in 3D bioprinted

scaffolds with de fi ned architecture. J. Control. Release 184, 58–66 (2014).

31. Seiffert, S. & Weitz, D. A. Microfluidic fabrication of smart microgels from macromolecular

precursors. Polymer (Guildf). 51, 5883–5889 (2010).

32. Correia, D. M. et al. Electrosprayed poly(vinylidene fluoride) microparticles for tissue

engineering applications. RSC Adv. 4, 33013–33021 (2014).

33. Zhao, W. et al. In situ cross-linking of stimuli-responsive hemicellulose microgels during spray

drying. ACS Appl. Mater. Interfaces 7, 4202–4215 (2015).

34. Berthuy, O. I. et al. Multiplex cell microarrays for high-throughput screening. Lab Chip 16,

4248–4262 (2016).

35. Jiang, Y., Chen, J., Deng, C., Suuronen, E. J. & Zhong, Z. Click hydrogels, microgels and

nanogels: Emerging platforms for drug delivery and tissue engineering. Biomaterials 35, 4969–

4985 (2014).

36. Fraser, A. K., Ki, C. S. & Lin, C. C. PEG-based microgels formed by visible-light-mediated thiol-

ene photo-click reactions. Macromol. Chem. Phys. 215, 507–515 (2014).

37. Henke, S. et al. Enzymatic Crosslinking of Polymer Conjugates is Superior over Ionic or UV

26


Crosslinking for the On-Chip Production of Cell-Laden Microgels. Macromol. Biosci. 1524–1532

(2016). doi:10.1002/mabi.201600174

38. El-Sagheer, A. H. & Brown, T. Click chemistry with DNA. Chem. Soc. Rev. 39, 1388–1405

(2010).

39. Wang, J. & Wei, J. Hydrogel brushes grafted from stainless steel via surface-initiated atom

transfer radical polymerization for marine antifouling. Appl. Surf. Sci. 382, 202–216 (2016).



41. Nguyen, K. T. & West, J. L. Photopolymerizable hydrogels for tissue engineering applications.

Biomaterials 23, 4307–4314 (2002).

42. Malkoch, M. et al. Synthesis of well-defined hydrogel networks using Click chemistry. Chem.

Commun. 2774–2776 (2006).

43. Hein, C. D., Liu, X.-M. & Wang, D. Click chemistry, a powerful tool for pharmaceutical

sciences. Pharm. Res. 25, 2216–2230 (2008).

44. Zhou, Y. et al. Chitosan microspheres with an extracellular matrix-mimicking nanofibrous

structure as cell-carrier building blocks for bottom-up cartilage tissue engineering. R. Soc. Chem.

8, 309–317 (2016).

45. Custódio, C. A., Cerqueira, M. T., Marques, A. P., Reis, R. L. & Mano, J. F. Cell selective

chitosan microparticles as injectable cell carriers for tissue regeneration. Biomaterials 43, 23–31

(2015).

46. Wang, M. & Kim, J. C. Microgels of poly(hydroxyethyl acrylate-co-coumaryl acrylate-co-

octadecyl acrylate): Photo-responsive release. Colloid Polym. Sci. 291, 2319–2327 (2013).

47. Ye, X. et al. Self-healing pH-sensitive cytosine- and guanosine-modified hyaluronic acid

hydrogels via hydrogen bonding. Polymer (Guildf). 108, 348–360 (2017).

48. Berger, J. et al. Structure and interactions in covalently and ionically crosslinked chitosan

hydrogels for biomedical applications. European Journal of Pharmaceutics and

Biopharmaceutics 57, 19–34 (2004).

49. Laurenti, M. et al. Synthesis of thermosensitive microgels with a tunable magnetic core. Am.

Chem. Soc. 27, 10484–10491 (2011).

50. Neto, A. I. et al. Fabrication of Hydrogel Particles of Defined Shapes Using Superhydrophobic-

Hydrophilic Micropatterns. Adv. Mater. 28, 7613–7619 (2016).

51. Tran, K. T. M. & Nguyen, T. D. Lithography-based methods to manufacture biomaterials at small

scales. J. Sci. Adv. Mater. Devices 2, 1–14 (2017).

52. Jung, S. & Yi, H. Facile Micromolding-Based Fabrication of Biopolymeric - Synthetic Hydrogel

Microspheres with Controlled Structures for Improved Protein Conjugation. Chem. Mater. 27,

3988–3998 (2015).

53. Oh, M. S. et al. Control of Reversible Self-Bending Behavior in Responsive Janus Microstrips.

ACS Appl. Mater. Interfaces 8, 8782–8788 (2016).

54. Heath, D. E. et al. Regenerating the cell resistance of micromolded PEG hydrogels. Lab Chip 15,

2073–2089 (2015).

27


55. Oh, J. K., Drumright, R., Siegwart, D. J. & Matyjaszewski, K. The development of

microgels/nanogels for drug delivery applications. Prog. Polym. Sci. 33, 448–477 (2008).

56. Farjami, T. & Madadlou, A. Fabrication methods of biopolymeric microgels and microgel-based

hydrogels. Food Hydrocoll. 62, 262–272 (2017).

57. Demirci, U. & Montesano, G. Single cell epitaxy by acoustic picolitre droplets. Lab Chip 7,

1139–1145 (2007).

58. Tasoglu, S. & Demirci, U. Bioprinting for stem cell research. Trends in Biotechnology 31, 10–19

(2013).

59. Wu, Z. et al. Bioprinting three-dimensional cell-laden tissue constructs with controllable

degradation. Sci. Rep. 6, 1–10 (2016).

60. Yanagawa, F., Sugiura, S. & Kanamori, T. Hydrogel microfabrication technology toward three

dimensional tissue engineering. Regen. Ther. 3, 45–57 (2016).

61. Murphy, S. V & Atala, A. 3D bioprinting of tissues and organs. Nat. Biotechnol. 32, 773–785

(2014).

62. Udey, R. N., Jones, a D. & Farquar, G. R. Aerosol and Microparticle Generation Using a

Commercial Inkjet Printer. Aerosol Sci. Technol. 47, 361–372 (2013).

63. Gao, Q., He, Y., Fu, J. Z., Qiu, J. J. & Jin, Y. an. Fabrication of shape controllable alginate

microparticles based on drop-on-demand jetting. J. Sol-Gel Sci. Technol. 77, 610–619 (2016).

64. Zhu, W. et al. 3D printing of functional biomaterials for tissue engineering. Current Opinion in

Biotechnology 40, 103–112 (2016).

65. Iwanaga, S., Saito, N., Sanae, H. & Nakamura, M. Facile fabrication of uniform size-controlled

microparticles and potentiality for tandem drug delivery system of micro/nanoparticles. Colloids

Surfaces B Biointerfaces 109, 301–306 (2013).

66. Zhu, K. et al. Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs. Adv. Funct.

Mater. 27, 1–12 (2017).

67. Li, J., Chen, M., Fan, X. & Zhou, H. Recent advances in bioprinting techniques: approaches,

applications and future prospects. J. Transl. Med. 14, 1–15 (2016).

68. Keriquel, V. et al. In situ printing of mesenchymal stromal cells , by laser-assisted bioprinting ,

for in vivo bone regeneration applications. Sci. Rep. 7, 1–10 (2017).

69. Mandrycky, C., Wang, Z., Kim, K. & Kim, D. H. 3D bioprinting for engineering complex tissues.

Biotechnol. Adv. 34, 422–434 (2016).

70. Huang, R., Wang, Y., Qi, W., Su, R. & He, Z. Chemical catalysis triggered self-assembly for the

bottom-up fabrication of peptide nanofibers and hydrogels. Mater. Lett. 128, 216–219 (2014).

71. Wang, Q., Liu, S., Wang, H., Zhu, J. & Yang, Y. Alginate droplets pre-crosslinked in

microchannels to prepare monodispersed spherical microgels. Colloids Surfaces A Physicochem.

Eng. Asp. 482, 371–377 (2015).

72. Mao, A. S. et al. Deterministic encapsulation of single cells in thin tunable microgels for niche

modelling and therapeutic delivery. Nat. Mater. 1, 1–10 (2016).

73. Hu, Y., Azadi, G. & Ardekani, A. M. Microfluidic fabrication of shape-tunable alginate

microgels: Effect of size and impact velocity. Carbohydr. Polym. 120, 38–45 (2015).

28


74. Konry, T., Dominguez-Villar, M., Baecher-Allan, C., Hafler, D. A. & Yarmush, M. L. Droplet-

based microfluidic platforms for single T cell secretion analysis of IL-10 cytokine. Biosens.

Bioelectron. 26, 2707–2710 (2011).

75. Shi, Y. et al. High throughput generation and trapping of individual agarose microgel using

microfluidic approach. Microfluid. Nanofluidics 15, 467–474 (2013).

76. Dashtimoghadam, E., Mirzadeh, H., Taromi, F. A. & Nyström, B. Microfluidic self-assembly of

polymeric nanoparticles with tunable compactness for controlled drug delivery. Polymer (Guildf).

54, 4972–4979 (2013).

77. Bawazer, L. A. et al. Combinatorial microfluidic droplet engineering for biomimetic material

synthesis. Sci. Adv. 2, 1–12 (2016).

78. Shui, L., Eijkel, J. C. T. & Berg, A. van den. Multiphase flow in microfluidic systems – Control

and applications of droplets and interfaces. Adv. Colloid Interface Sci. 133, 35–49 (2007).

79. Yang, C. H. et al. Microfluidic-assisted synthesis of hemispherical and discoidal chitosan

microparticles at an oil/water interface. Electrophoresis 33, 3173–3180 (2012).

80. Shimanovich, U. et al. Protein microgels from amyloid fibril networks. ACS Nano 9, 43–51

(2015).

81. Krutkramelis, K., Xia, B. & Oakey, J. Monodisperse polyethylene glycol diacrylate hydrogel

microsphere formation by oxygen-controlled photopolymerization in a microfluidic device. Lab

Chip 16, 1457–1465 (2016).

82. Cheng, Y. et al. Controlled Fabrication of Bioactive Microfibers for Creating Tissue Constructs

Using Microfluidic Techniques. ACS Appl. Mater. Interfaces 8, 1080–1086 (2016).

83. Bock, N., Woodruff, M. A., Hutmacher, D. W. & Dargaville, T. R. Electrospraying, a

reproducible method for production of polymeric microspheres for biomedical applications.

Polymers (Basel). 3, 131–149 (2011).

84. Guarino, V., Khartini, W., Abdul, W. & Ambrosio, L. Biodegradable microparticles and

nanoparticles by electrospraying techniques. J. Appl. Biomater. Funct. Mater. 10, 191–196

(2012).

85. Altobelli, R., Guarino, V. & Ambrosio, L. Micro- and nanocarriers by electrofludodynamic

technologies for cell and molecular therapies. Process Biochem. 51, 2143–2145 (2016).

86. Jaworek, A. et al. Electrospinning and electrospraying techniques for nanocomposite non-woven

fabric production. Fibres Text. East. Eur. 17, 77–81 (2009).

87. Kim, P. H. et al. Injectable multifunctional microgel encapsulating outgrowth endothelial cells

and growth factors for enhanced neovascularization. J. Control. Release 187, 1–13 (2014).

88. Di, J., Kim, J., Hu, Q., Jiang, X. & Gu, Z. Spatiotemporal drug delivery using laser-generated-

focused ultrasound system. J. Control. Release 220, 592–599 (2015).

89. Yao, S., Yang, Y., Wang, X. & Wang, L. Fabrication and characterization of aligned fibrin

nanofiber hydrogel loaded with PLGA microspheres. Macromol. Res. 25, 528–533 (2017).

90. Oliveira, M. B., Kossover, O., Mano, J. F. & Seliktar, D. Injectable PEGylated fibrinogen cell-

laden microparticles made with a continuous solvent- and oil-free preparation method. Acta

Biomater. 13, 78–87 (2015).

29


91. Oh, J. K., Lee, D. I. & Park, J. M. Biopolymer-based microgels/nanogels for drug delivery

applications. Prog. Polym. Sci. 34, 1261–1282 (2009).

92. Silva, A. S., Tavares, M. T. & Aguiar-Ricardo, A. Sustainable strategies for nano-in-micro

particle engineering for pulmonary delivery. J. Nanoparticle Res. 16, 1–17 (2014).

93. Sollohub, K. & Cal, K. Spray drying technique: II. Current applications in pharmaceutical

technology. Journal of Pharmaceutical Sciences 99, 587–597 (2010).

94. Beck-Broichsitter, M., Strehlow, B. & Kissel, T. Direct fractionation of spray-dried polymeric

microparticles by inertial impaction. Powder Technol. 286, 311–317 (2015).

95. Song, W., Lima, A. C. & Mano, J. F. Bioinspired methodology to fabricate hydrogel spheres for

multi-applications using superhydrophobic substrates. Soft Matter 6, 5868–5871 (2010).

96. Lima, A. C., Custódio, C. A., Alvarez-Lorenzo, C. & Mano, J. F. Biomimetic methodology to

produce polymeric multilayered particles for biotechnological and biomedical applications. Small

9, 2487–2492 (2013).

97. Costa, A. M. S. & Mano, J. F. Solvent-free strategy yields size and shape-uniform capsules. J.

Am. Chem. Soc. 139, 1057–1060 (2017).

98. Costa, A. M. S., Alatorre-Meda, M., Oliveira, N. M. & Mano, J. F. Biocompatible polymeric

microparticles produced by a simple biomimetic approach. Langmuir 30, 4535–4539 (2014).

99. Hu, Y. et al. Controlled self-assembly of alginate microgels by rapidly binding molecule pairs.

Lab Chip 17, 2481–2490 (2017).

100. Xia, P. et al. Injectable Stem Cell Laden Open Porous Microgels That Favor Adipogenesis: In

Vitro and in Vivo Evaluation. ACS Appl. Mater. Interfaces 9, 34751–34761 (2017).

101. Lienemann, P. S. et al. Single cell-laden protease-sensitive microniches for long-term culture in

3D. Lab Chip 17, 727–737 (2017).

102. Alfred, R. et al. Efficient suspension bioreactor expansion of murine embryonic stem cells on

microcarriers in serum-free medium. Biotechnol. Prog. 27, 811–823 (2011).

103. Van Wezel, A. L. Growth of cell-strains and primary cells on micro-carriers in homogeneous

culture. Nature 216, 64–65 (1967).

104. Zhang, Z. Injectable Biomaterials for Stem Cell Delivery and Tissue Regeneration. Expert Opin.

Biol. Ther. 17, 49–62 (2017).

105. Oliveira, M. B. & Mano, J. F. Polymer-based microparticles in tissue engineering and

regenerative medicine. Biotechnol. Prog. 27, 897–912 (2011).

106. Li, B. et al. Past, present, and future of microcarrier-based tissue engineering. Journal of

Orthopaedic Translation 3, 51–57 (2015).

107. Mano, F. J. Stimuli-Responsive Polymeric Systems for Biomedical Applications. Adv. Eng.

Mater. 10, 515–527 (2008).

108. Nie, Y., Bergendahl, V., Hei, D. J., Jones, J. M. & Palecek, S. P. Scalable Culture and

Cryopreservation of Human Embryonic Stem Cells on Microcarriers. Biotechnol. Prog. 25, 20–31

(2009).

109. Bender, M. D., Bennett, J. M., Waddell, R. L., Doctor, J. S. & Marra, K. G. Multi-channeled

biodegradable polymer/CultiSpher composite nerve guides. Biomaterials 25, 1269–1278 (2004).

30


110. de Soure, A. M. et al. Integrated culture platform based on a human platelet lysate supplement for

the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal

stem/stromal cells. J. Tissue Eng. Regen. Med. 11, 1630–1640 (2017).

111. Mano, J. F. et al. Natural origin biodegradable systems in tissue engineering and regenerative

medicine: present status and some moving trends. J. R. Soc. Interface 4, 999–1030 (2007).

112. Correia, C. R. et al. Semipermeable capsules wrapping a multifunctional and self-regulated co-

culture microenvironment for osteogenic differentiation. Sci. Rep. 6, 1–12 (2016).

113. Correia, C. R. et al. In vivo osteogenic differentiation of stem cells inside compartmentalized

capsules loaded with co-cultured endothelial cells. Acta Biomater. 53, 483–494 (2017).

114. Oliveira, M. B. & Mano, J. F. High-throughput screening for integrative biomaterials design:

Exploring advances and new trends. Trends Biotechnol. 32, 627–636 (2014).

115. Oliveira, M. B. et al. Superhydrophobic chips for cell spheroids high-throughput generation and

drug screening. ACS Appl. Mater. Interfaces 6, 9488–9495 (2014).

116. Mohanraj, B. et al. A high throughput mechanical screening device for cartilage tissue

engineering. J. Biomech. 47, 2130–2136 (2014).

117. Rasi Ghaemi, S., Harding, F. J., Delalat, B., Gronthos, S. & Voelcker, N. H. Exploring the

mesenchymal stem cell niche using high throughput screening. Biomaterials 34, 7601–7615

(2013).

118. Guermani, E. et al. Engineering complex tissue-like microgel arrays for evaluating stem cell

differentiation. Sci. Rep. 6, 1–8 (2016).

119. Salgado, C. L., Oliveira, M. B. & Mano, J. F. Combinatorial cell–3D biomaterials

cytocompatibility screening for tissue engineering using bioinspired superhydrophobic substrates.

Integr. Biol. 4, 318 (2012).

120. Oliveira, M. B., Luz, G. M. & Mano, J. F. A combinatorial study of nanocomposite hydrogels:

on-chip mechanical/viscoelastic and pre-osteoblast interaction characterization. J. Mater. Chem.

B 2, 5627–5638 (2014).

121. Lyon, L. A. & Serpe, M. J. Hydrogel Micro and Nanoparticles. Wiley (2012).

doi:10.1002/9783527646425

122. Smeets, N. M. B. & Hoare, T. Designing responsive microgels for drug delivery applications. J.

Polym. Sci. Part A Polym. Chem. 51, 3027–3043 (2013).

123. Rodríguez-Velázquez, E., Alatorre-Meda, M. & Mano, J. F. Polysaccharide-Based

Nanobiomaterials as Controlled Release Systems for Tissue Engineering Applications. Curr.

Pharm. Des. 21, 4837–4850 (2015).

124. Sood, N., Bhardwaj, A., Mehta, S. & Mehta, A. Stimuli-responsive hydrogels in drug delivery

and tissue engineering. Drug Deliv. 23, 748–770 (2016).

125. Lee, E. et al. Development of smart delivery system for ascorbic acid using pH-responsive P (

MAA-co-EGMA ) hydrogel microparticles. Drug Deliv. 17, 573–580 (2010).

126. Sivakumaran, D., Maitland, D. & Hoare, T. Injectable microgel-hydrogel composites for

prolonged small-molecule drug delivery. Biomacromolecules 12, 4112–4120 (2011).

127. Lai, W.-F. et al. Electrospray-mediated preparation of compositionally homogeneous core–shell

31


hydrogel microspheres for sustained drug release. RSC Adv. 7, 44482–44491 (2017).

128. Rahman, A., Majewski, P. W., Doerk, G., Black, C. T. & Yager, K. G. Non-native three-

dimensional block copolymer morphologies. Nat. Commun. 7, 1–8 (2016).

129. Guven, S. et al. Multiscale assembly for tissue engineering and regenerative medicine. Trends

Biotechnol. 33, 269–279 (2015).

130. Franchi, S. et al. Self-assembling peptide hydrogels immobilized on silicon surfaces. Mater. Sci.

Eng. C 69, 200–207 (2016).

131. Xu, F. et al. The assembly of cell-encapsulating microscale hydrogels using acoustic waves.

Biomaterials 32, 7847–7855 (2011).

132. Breen, T. L., Tien, J., Oliver, S. R. J., Hadzic, T. & Whitesides, G. M. Design and Self-Assembly

of Open, Regular, 3D Mesostructures. Science (80-. ). 284, 948–951 (1999).

133. Choi, I. S., Bowden, N. & Whitesides, G. M. Macroscopic, hierarchical, two-dimensional self-

assembly. Angew. Chemie - Int. Ed. 38, 3078–3081 (1999).

134. Du, Y. et al. Surface-directed assembly of cell-laden microgels. Biotechnol. Bioeng. 105, 655–

662 (2010).

135. Oliveira, N. M. et al. Hydrophobic Hydrogels: Toward Construction of Floating

(Bio)microdevices. Chem. Mater. 28, 3641–3648 (2016).

136. Naseer, S. M. et al. Surface acoustic waves induced micropatterning of cells in gelatin

methacryloyl ( GelMA ) hydrogels Surface acoustic waves induced micropatterning of cells in

gelatin methacryloyl ( GelMA ) hydrogels. Biofabrication 9, 1–11 (2017).

137. Li, Y. et al. Magnetic hydrogels and their potential biomedical applications. Adv. Funct. Mater.

23, 660–672 (2013).

138. Gil, S. & Mano, J. F. Magnetic composite biomaterials for tissue engineering. Biomater. Sci. 2,

812–818 (2014).

139. Xu, F. et al. Three-dimensional magnetic assembly of microscale hydrogels. Adv. Mater. 23,

4254–4260 (2011).

140. Li, C. Y., Wodd, D. K., Hsu, C. M. & Bhatia, S. N. DNA-templated assembly of droplet-derived

PEG microtissues. Lab Chip 11, 2967–2975 (2011).

141. Qi, H. et al. DNA-directed self-assembly of shape-controlled hydrogels. Nat. Commun. 4, 1–10

(2013).

142. Cruz, D. M. G. et al. Chitosan microparticles as injectable scaffolds for tissue engineering. J.

Tissue Eng. Regen. Med. 2, 378–380 (2008).

143. Oliveira, M. B. et al. Development of an injectable system based on elastin-like recombinamer

particles for tissue engineering applications. Soft Matter 7, 6426–6434 (2011).

CHAPTER 3

Materials and methods

32

Chapter 3 – Materials and Methods


1. Laminarin

Laminarin is a storage glucan found in many macro-algae and most phytoplankton, and

is one of the most abundant carbon sources in the marine ecosystem. It is a glucan wherein

units of glucose are bonded through β-(1,3) repeating units, with sporadic β-(1,6)

branches, easily soluble in aqueous and organic solvents forming clear and stable

solutions.12 Laminarin has been shown to stimulate immunity, and to have antitumor

effects and antibacterial activity.1,3 An attractive feature of this particular polymer is its

inherent low viscosity and high water solubility over a temperature range of 4ºC to 40ºC

that facilitates processing. This is especially useful for microfabrication protocols such

microfluidics processing.3

2. Platelet Lysates

Platelet derived products include Platelet Lysates (PLs) and Platelet Rich Plasma (PRP)

and have been studied and used since the 1970s.4 It is now well established that platelets

are an important source of autologous GFs that can modulate stem cell proliferation and

differentiation. Human PLs can be generated through a simple freeze-thaw procedure of

platelet units. In this work, PLs were purchased from STEMCELL Technologies.

Multiple donor units are pooled during manufacturing to minimize lot-to-lot variability

and multiple lots were used during the experiments.

3. Methods

3.1.Synthesis and characterization of methacrylated laminarin

Methacrylated laminarin (MeLam) was modified by a common chemical reaction

following the protocol previously described.3 Briefly, MeLam was synthetized by

reacting laminarin (6kDa) (Carbosynth, U.K.) with glycidyl methacrylate (Acros

Organics, Germany). Laminarin (1g) and 4-(N,N-dimethylamino)pyridine (DMAP) (167

mg) (Acros Organic, Germany) were dissolved in 10 mL of dimethyl sulfoxide (DMSO)

(Sigma-Aldrich, Germany) under nitrogen atmosphere. Varying the amount of glycidyl

methacrylate added is possible to manipulate the degree of modification; low degree of

modification was obtained by adding 2.9 × 10-3 mol of glycidyl methacrylate to the

33


previously prepared solution and high degree of modification by adding 5.1 × 10-3 mol of

glycidyl methacrylate. The mixture was stirred at room temperature (RT) for 48 hours

protected from light, being stopped by adding HCl solution (37%) (Sigma-Aldrich, USA)

to neutralize DMAP. Subsequently, the solution was purified by dialysis using a

benzoylated membrane (2000 MWCO) (Sigma-Aldrich, USA) for at least 7 days in

distilled water. The final product was freeze-dried and stored at RT until further use.

Degree of substitution (DS, fraction of modified hydroxyl groups per repeating unit) was

calculated by 1H NMR (Bruker Avance III (300 MHz)) by integrating the peak

correspondent to the acetyl group of the methacrylate centered at ∼2 ppm (IAc) against

the polymer backbone region ∼3 –5.5 ppm (ILam). The following formula (Eq. 1) was

used to calculate the DS value:

𝐷𝑆 =𝐼𝐴𝐶

𝐼𝐿𝑎𝑚 Equation 1

3.2.Fabrication of MeLam microparticles

For the aqueous phase, MeLam was dissolved in phosphate buffered saline (PBS, pH 7.4)

(Sigma, USA) at a concentration of 15% (w/v) with 0.5% (w/v) 2-hydroxy-4-(2-

hydroxyethoxy)-2-methylpropiophenone (Sigma, USA). Moreover, biotin-PEG-thiol

(Polypure AS, Norway) (0.5 mg/mL) was also added to the previously prepared solution.

Biotinylated microparticles are then formed by a Michael-type addition between thiol

group of biotin and alkene group of MeLam. The previously prepared solution was loaded

into plastic syringes (BD Luer-Lok syringe) and connected to the inlets with Fluorinated

Ethylene Propylene (FEP) tubing. For the continuous phase, mineral oil (Fisher) was

loaded into the same type of syringes. Syringe pumps (Harvard Apparatus, PhD Ultra,

USA) were used to inject fluids at controlled flow rates into the microfluidic chip (figure

3.1 A). For these experiments, a T-junction hydrophobic microfluidic chip with header

(190µm etch depth) (Dolomite, UK) was used. This chip allows for the fabrication of

water-in-oil droplets in the size range Ø100 – 300µm (figure 3.1 B). Upon formation in

the flow-focusing device, droplets were photopolymerized with UV light (Omnicure

S2000, Canada) to form microparticles. The outlet tubing (0.5 mm of diameter) was

coiled to make a spiral microchannel ensuring that the microdroplets are kept for at least

60 seconds under UV light (6.12 W/cm2) for the efficient crosslinking of microparticles

(figure 3.1 C). For the experiments, flow rates are set by the syringe pumps to be 8 μl/min

34


for the aqueous phase (QAq) and 160 μl/min for the continuous phase (QC). The MeLam

microparticles were collected to a falcon (15 mL) and the mineral oil removed after

centrifugation.

To produce microparticles with encapsulated platelet lysates (PL) the protocol was

slightly changed by including PL in the aqueous phase. Briefly, PL (25% w/v) was mixed

with the MeLam solution previously dissolved in PBS and loaded into the plastic

syringes.

Figure 3.1: Schematic representation of microfluidic system used to produce MeLam microparticles. (A)

Schematic representation of PDMS microchip with T-junction shape. (B) Droplets formation into the

microchip. (C) UV crosslinked MeLam microparticles

3.3.RGD functionalization of MeLam microparticles

To access the functionalization with biotin, modified particles were incubated with

DyLight 488 Streptavidin (BioLegend) (10μg/ml), washed with PBS and observed under

fluorescence microscopy (Zeiss, Axio Imager M2). Polymeric microparticles with

covalently attached biotin are proposed as versatile targeting vehicles for multiple

biomolecules, in this work the particles were further functionalized with the tripeptide

Arg-Gly-Asp (RGD) to promote cell adhesiveness.

Biotinylated microparticles were incubated with purified streptavidin (Promega)

(25μg/ml) in PBS under constant stirring for 15min at room temperature. A washing step

was then performed to remove unbound streptavidin. Finally, the particles were incubated

with biotinylated RGD (25 μg/mL) (AnaSpec, USA) in PBS under constant stirring for

15min at room temperature (figure 3.2). The microparticles were washed with PBS

solution to remove all unbound RGD biotinylated.

35


3.4.Scanning electron microscopy (SEM)

SEM was performed as a means to evaluate the morphology and porosity of the

microparticles produced by microfluidics. For SEM analysis, particles were dried at 25ºC

for 3 days), sputtered with gold and evaluated by SEM (Hitachi SU-70, Japan).

3.5.Studies on the release of PL

Aliquots of the microparticles (triplicate samples from each batch) were suspended in 5

mL phosphate buffered saline (PBS, pH 7.4); samples were gently shaked at 60 rpm in a

water bath at 37ºC. At defined time intervals, 500 μL of PBS were removed and replaced

with 500 μl fresh PBS. The removed supernatants were stored frozen until required and

were then assayed for total protein content using the Micro BCA assay kit. Briefly, 50 μL

of the collected samples were diluted in 100 μL of PBS solution and mixed with 150 μL

of the working solution and incubated for 2 hours at 37ºC. Afterwards, the quantity of

protein was measured by absorbance at a wavelength 592 nm in a Synergy HTX multi-

mode reader (Biotek Instruments, Inc, USA).

The total protein release was calculated following equation described by Hailong5

equation (2):

𝐶𝑢𝑚𝑢𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝐿 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 (%) = 𝑉𝑒 ∑ 𝐶𝑖+ 𝑉0𝐶𝑛

𝑛−1𝑖

𝑚𝑑𝑟𝑢𝑔 ×100 Equation 2

ELISA assay (ThermoFisherScientific, USA) was also performed to evaluate the release

of transforming growth factor (TGF-β1) and vascular epithelial growth factor (VEGF)

from the microparticles. The assay was performed according to the manufacturer’s

standard protocols. The optical density values were measured using a Synergy HTX

multi-mode reader (Biotek Instruments, Inc, USA) set at 450 nm.

3.6.Cell culture on MeLam microparticles

For the cell culture tests, microparticles prepared with high degree of substitution were

used. Mouse fibroblast (L929) were used to verify the potential of methacrylated

laminarin microparticles as cell carriers (figure 3.2). L929 were cultured in Dulbecco’s

modified Eagle’s medium DMEM (Thermo Scientific, USA) supplemented with 10% of

fetal bovine serum (FBS) and 1% antibiotic/antimycotic at 37ºC under 5% CO2. The high

MeLam 15% (w/v) microparticles were seeded with cells in non-adherent 48 well

microplates in portion 200 microparticles per 1×104 cells under gently shaking (150 rpm)

36


and at cell culture conditions (5% CO2, 37ºC). The cultured microparticles were

maintained in culture for 11 days. At pre-determined time points, cell morphology, cell

viability and proliferation were assessed. The assembly process was followed and imaged

at day 11. Constructs were evaluated for cell proliferation.

Figure 3.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD with MeLam

microparticles. After modification, microparticles were cultured with L929 cells. This scheme also

represents cell attachment on the microparticles and subsequent assembly of the structures.

3.7.Cell morphology analysis

Phalloidin and DAPI staining were used to visualize actin cytoskeleton and to label the

DNA, respectively. The assay was conducted as outlined by the supplier’s protocol

(Sigma, Germany). Briefly, cultured microparticles were washed with PBS, fixed in 4%

formaldehyde/PBS (v/v) for 1h at room temperature (RT) and washed extensively in PBS

to remove all traces of the fixative. Cells were then stained with 50 μg/ml fluorescent

phalloidin-conjugate solution in PBS for 45 min at room temperature. DAPI labeling

solution 0.5 μg/ml was incubated for 5 min at room temperature. The microparticles were

washed in PBS to remove remaining staining solutions and imaged using a fluorescent

microscope Zeiss Imager M2 (Carl Zeiss SMT Inc).

37


3.7.1. DNA quantification

Cell proliferation on laminarin microparticles was determined by DNA quantification

using a fluorimetric dsDNA quantification kit (PicoGreen, Invitrogen). This assay allows

measurement of the fluorescence produced when PicoGreen dye is excited by UV light

while bounded to dsDNA. After each time-point, cells were lysed by osmotic and thermal

shock and the supernatant used for double-stranded DNA (dsDNA) content analysis.

Briefly, samples collected after each time point were washed with PBS and immersed in

1 ml of ultrapure water, frozen for at −80 °C, thawed at room temperature, and sonicated

for 30 min. Fluorescence was measured on a microplate reader (Synergy HTX multi-

mode reader). The DNA amount was calculated from a standard curve

3.7.2 MTS Viability Assay

The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2Htetrazolium) assay (Promega) was performed to evaluate cell viability.

Briefly, after each time period, culture media was removed and cells were washed with

PBS solution. The MTS solution 1:10 ratio to PBS was added to the cells and incubated

for 4 h (37ºC, 5% CO2). After the incubation period, the optical density (OD) was read

at 490 nm in a microplate reader (Synergy HTX multi-mode reader).

3.8.Statistical analysis

All results were subjected a statistical analysis and results were presented as mean ±

standard deviation. Statistical analysis of results was performed using Student’s t-test,

with a significant level of 95% (p < 0.05).

4. References

1. Anjugam, M. et al. A study on β-glucan binding protein (β-GBP) and its involvement in

phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus. Mol. Immunol. 92, 1–11

(2017).





4. Soomekh, D. J. Current Concepts for the Use of Platelet-Rich Plasma in the Foot and Ankle. Clin.

Podiatr. Med. Surg. 28, 155–170 (2011).

38


5. Che, H. et al. CO 2 -switchable drug release from magneto-polymeric nanohybrids. Polym. Chem.

6, 2319–2326 (2015).

CHAPTER 4

Multifunctional laminarin microparticles

for cell adhesion and expansion

39

Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and

expansion

Chapter 4 - Multifunctional laminarin microparticles for cell adhesion

and expansion

Martins CR,1 Custódio CA,1 Mano JF1



Abstract

Microfabrication technologies have been explored to produce microgels that can be

assembled in functional constructs for tissue engineering applications. Here, we propose

microfluidics coupled to a source of UV light to produce monodisperse multifunctional

laminarin microparticles by photopolymerization. In an attempt to enhance cell adhesion

and proliferation, the microparticles were loaded with platelet lysates and further

conjugated with an adhesive peptide. The modified microparticles were cultured with

L929 cells, the results showed a high cellular adhesion on microparticles with

encapsulated platelet lysates. The multifunctional laminarin microparticles provide an

effective support for cell attachment and cell expansion, moreover the microparticles tend

to aggregate in robust 3D structures. This showed the potential for using the

microplatforms to rapidly produce large tissue engineered constructs.

Key words: Microfluidic, microcarrier, microgels, platelet lysates

1. Introduction

Living tissues are hierarchically organized three-dimensional (3D) structures composed

of multiple types of cells and extracellular matrix (ECM).1 Thus, effective strategies to

engineer living constructs that mimic native tissues requires the development of structures

with well-defined spatial distributions of different cells embedded in ECM.

Current strategies for tissue and organ development include “bottom-up” tissue

engineering, that consists in the self-assembly of smaller units to build a 3D construct or

“top-down” approaches, involves scaffold-based cell-seeding.

Bottom-up approaches for the directed or random assembly of microunits, which mimic

the living tissue architecture from repeating functional units, have been gaining increasing

40


expansion

attention in tissue engineering applications.2–5 In the last years, different microfabrication

strategies have been explored for the production of miniaturized structures for cell culture

and cell encapsulation.5–7 Given the advantages including high continuity, reproducibility

and scalability, microfluidics have been widely used to fabricate microparticles with

controlled sizes and structures.8,9

Laminarin is a natural polymer obtained from brown algae with low molecular weight

and low viscosity.10 These properties make this polymer particularly appealing to be used

in microfabrication techniques. Laminarin hydrogels have recently been proposed as a

platform for cell encapsulation and good cell viability has been demonstrated.11 Addition

of methacrylate groups to the hydroxyl-containing groups of laminarin was performed to

make it light polymerizable into a hydrogel.

In this work, we report a simple and efficient microfluidic approach to produce

monodisperse laminarin microgels with encapsulated PL. The methacrylate groups act

also as anchoring sites for the immobilization of thiol-biotin molecules and streptavidin,

which subsequently form complexes with biotin-RGD. Cell adhesion to the microgels

was enhanced by the conjugation of the microparticles with an adhesive peptide (RGD).

The use of streptavidin–biotin should be applicable to a broad range of proteins,

expanding the options available for microparticles modification. By using a

biocompatible and biodegradable polymer as support for cell culture, we provide

simultaneously a support for cell culture and expansion that can be also used to fill tissue

defects in tissue engineering strategies.

Additionally, encapsulation of platelet lysates (PL) within the microparticles showed

improved cell attachment and expansion in the laminarin microgels. PL, offer much

potential owing to its autogenous nature and high content of proteins and growth factors

(GFs). PL have been used as a natural source of GFs for tissue regeneration purposes and

as a substitute of animal derived components for in vitro cell culture.12

2. Materials and Methods

2.1.Synthesis and characterization of methacrylated laminarin

Methacrylated laminarin (MeLam) was modified by a common chemical reaction

following the protocol previously described.11 Briefly, MeLam was synthetized by

reacting laminarin (6kDa) (Carbosynth, U.K.) with glycidyl methacrylate (Acros

Organics, Germany). Laminarin (1g) and 4-(N,N-dimethylamino)pyridine (DMAP) (167

41


expansion

mg) (Acros Organic, Germany) were dissolved in 10 mL of dimethyl sulfoxide (DMSO)

(Sigma-Aldrich, Germany) under nitrogen atmosphere. Varying the amount of glycidyl

methacrylate added is possible to manipulate the degree of modification; low degree of

modification was obtained by adding 2.9 × 10-3 mol of glycidyl methacrylate to the

previously prepared solution and high degree of modification by adding 5.1 × 10-3 mol of

glycidyl methacrylate. The mixture was stirred at room temperature for 48 hours protected

from light, being stopped by adding HCl solution (37%) (Sigma-Aldrich, USA) to

neutralize DMAP. Subsequently, the solution was purified by dialysis using a

benzoylated membrane (2000 MWCO) (Sigma-Aldrich, USA) for at least 7 days in

distilled water. The final product was freeze-dried and stored at room temperature until

further use.

Degree of substitution (DS, fraction of modified hydroxyl groups per repeating unit) was

calculated by 1H NMR (Bruker Avance III (300 MHz)) by integrating the peak

correspondent to the acetyl group of the methacrylate centered at ∼2 ppm (IAc) against

the polymer backbone region ∼3 –5.5 ppm (ILam). The following formula (Eq. 1) was

used to calculate the DS value:

𝐷𝑆 =𝐼𝐴𝐶

𝐼𝐿𝑎𝑚 Equation 1

2.2.Fabrication of MeLam microparticles

For the aqueous phase, MeLam was dissolved in phosphate buffered saline (PBS, pH 7.4)

(Sigma, USA) at a concentration of 15% (w/v) with 0.5% (w/v) 2-hydroxy-4-(2-

hydroxyethoxy)-2-methylpropiophenone (Sigma, USA). Moreover, biotin-PEG-thiol

(Polypure AS, Norway) (0.5 mg/mL) was also added to the previously prepared solution.

Biotinylated microparticles are then formed by a Michael-type addition between thiol

group of biotin and alkene group of MeLam. The previously prepared solution was loaded

into plastic syringes (BD Luer-Lok syringe) and connected to the inlets with Fluorinated

Ethylene Propylene (FEP) tubing. For the continuous phase, mineral oil (Fisher) was

loaded into the same type of syringes. Syringe pumps (Harvard Apparatus, PhD Ultra,

USA) were used to inject fluids at controlled flow rates into the microfluidic chip (figure

4.1 A). For these experiments, a T-junction hydrophobic microfluidic chip with header

(190µm etch depth) (Dolomite, UK) was used. This chip allows for the fabrication of

water-in-oil droplets in the size range Ø 100 – 300µm (figure 4.1 B). Upon formation in

42


expansion

the flow-focusing device, droplets were photopolymerized with UV light (Omnicure

S2000, Canada) to form microparticles. The outlet tubing (0.5 mm of diameter) was

coiled to make a spiral microchannel ensuring that the microdroplets are kept for at least

60 seconds under UV light (6.12 W/cm2) for the efficient crosslinking of microparticles

(figure 4.1 C). For the experiments, flow rates are set by the syringe pumps to be 8 μl/min

for the aqueous phase and 160 μl/min for the continuous phase. The MeLam

microparticles were collected to a falcon (15 mL) and the mineral oil removed after

centrifugation.

To produce microparticles with encapsulated platelet lysates (PL) the protocol was

slightly changed by including PL in the aqueous phase. Briefly, PL (25% w/v) was mixed

with the MeLam solution previously dissolved in PBS and loaded into the plastic

syringes.

Figure 4.1: Schematic representation of microfluidic system used to produce MeLam microparticles. (A)

Schematic representation of PDMS microchip with T-junction shape. (B) Droplets formation into the

microchip. (C) UV crosslinked MeLam microparticles

2.3.RGD functionalization of MeLam microparticles

To access the functionalization with biotin, modified particles were incubated with

DyLight 488 Streptavidin (BioLegend) (10μg/ml), washed with PBS and observed under

fluorescence microscopy (Zeiss, Axio Imager M2). Polymeric microparticles with

covalently attached biotin are proposed as versatile targeting vehicles for multiple

biomolecules, in this work the particles were further functionalized with the tripeptide

Arg-Gly-Asp (RGD) to promote cell adhesiveness.

Biotinylated microparticles were incubated with purified streptavidin (Promega)

(25μg/ml) in PBS under constant stirring for 15min at room temperature. A washing step

was then performed to remove unbound streptavidin. Finally, the particles were incubated

43


expansion

with biotinylated RGD (25 μg/mL) (AnaSpec, USA) in PBS under constant stirring for

15min at room temperature. The microparticles were washed with PBS solution to

remove all unbound RGD biotinylated.

2.4.Scanning electron microscopy (SEM)

SEM was performed as a means to evaluate the morphology and porosity of the

microparticles produced by microfluidics. For SEM analysis, particles were dried at 25ºC

for 3 days), sputtered with gold and evaluated by SEM (Hitachi SU-70, Japan).

2.5.Studies on the release of PL

Aliquots of the microparticles (triplicate samples from each batch) were suspended in 5

mL phosphate buffered saline (PBS, pH 7.4), samples were gently shaked at 60 rpm in a

water bath at 37ºC. At defined time intervals, 500 μL of PBS were removed and replaced

with 500 μL fresh PBS. The removed supernatants were stored frozen until required and

were then assayed for total protein content using the Micro BCA assay kit. Briefly, 50 μL

of the collected samples were diluted in 100 μL of PBS solution and mixed with 150 μL

of the working solution and incubated for 2 hours at 37ºC. Afterwards, the quantity of

protein was measured by absorbance at a wavelength 592 nm in a Synergy HTX multi-

mode reader (Biotek Instruments, Inc, USA).

The total protein release was calculated following equation described by Hailong13

equation (2):

𝐶𝑢𝑚𝑢𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝐿 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 (%) = 𝑉𝑒 ∑ 𝐶𝑖+ 𝑉0𝐶𝑛

𝑛−1𝑖

𝑚𝑑𝑟𝑢𝑔 ×100 Equation 2

ELISA assay (ThermoFisherScientific, USA) was also performed to evaluate the release

of transforming growth factor (TGF-β1) and vascular epithelial growth factor (VEGF)

from the microparticles. The assay was performed according to the manufacturer’s

standard protocols. The optical density values were measured using a Synergy HTX

multi-mode reader (Biotek Instruments, Inc, USA) set at 450 nm.

2.6.Cell culture on MeLam microparticles

For the cell culture tests, microparticles prepared with high degree of substitution were

used. Mouse fibroblast (L929) were used to verify the potential of methacrylated

44


expansion

laminarin microparticles as cell carriers (figure 4.2). L929 were cultured in Dulbecco’s

modified Eagle’s medium DMEM (Thermo Scientific, USA) supplemented with 10% of

fetal bovine serum (FBS) and 1% antibiotic/antimycotic at 37ºC under 5% CO2. The high

MeLam 15% (w/v) microparticles were seeded with cells in non-adherent 48 well

microplates in portion 200 microparticles per 1×104 cells under gently shaking (150 rpm)

and at cell culture conditions (5% CO2, 37ºC). The cultured microparticles were

maintained in culture for 11 days. At pre-determined time points, cell morphology, cell

viability and proliferation were assessed. The assembly process was followed and imaged

at day 11. Constructs were evaluated for cell proliferation.

Figure 4.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD with MeLam

microparticles. After modification, microparticles were cultured with L929 cells. This scheme also

represents cell attachment on the microparticles and subsequent assembly of the structures.

2.7.Cell morphology analysis

Phalloidin and DAPI staining were used to visualize actin cytoskeleton and to label the

DNA, respectively. The assay was conducted as outlined by the supplier’s protocol

(Sigma, Germany). Briefly, cultured microparticles were washed with PBS, fixed in 4%

45


expansion

formaldehyde/PBS (v/v) for 1h at room temperature (RT) and washed extensively in PBS

to remove all traces of the fixative. Cells were then stained with 50 μg/ml fluorescent

phalloidin-conjugate solution in PBS for 45 min at room temperature. DAPI labeling

solution 0.5 μg/ml was incubated for 5 min at room temperature. The microparticles were

washed in PBS to remove remaining staining solutions and imaged using a fluorescent

microscope Zeiss Imager M2 (Carl Zeiss SMT Inc).

2.8.DNA quantification

Cell proliferation on laminarin microparticles was determined by DNA quantification

using a fluorimetric dsDNA quantification kit (PicoGreen, Invitrogen). This assay allows

measurement of the fluorescence produced when PicoGreen dye is excited by UV light

while bounded to dsDNA. After each time-point, cells were lysed by osmotic and thermal

shock and the supernatant used for double-stranded DNA (dsDNA) content analysis.

Briefly, samples collected after each time point were washed with PBS and immersed in

1 mL of ultrapure water, frozen for at −80 °C, thawed at room temperature, and sonicated

for 30 min. Fluorescence was measured on a microplate reader (Synergy HTX multi-

mode reader). The DNA amount was calculated from a standard curve.

2.9.MTS Viability Assay

The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2Htetrazolium) assay (Promega) was performed to evaluate cell viability.

Briefly, after each time period, culture media was removed and cells were washed with

PBS solution. The MTS solution 1:10 ratio to PBS was added to the cells and incubated

for 4 h (37ºC, 5% CO2). After the incubation period, the optical density (OD) was read

at 490 nm in a microplate reader (Synergy HTX multi-mode reader).

2.10. Statistical analysis

All results were subjected a statistical analysis and results were presented as mean ±

standard deviation. Statistical analysis of results was performed using Student’s t-test,

with a significant level of 95% (p < 0.05).

46


expansion

3. Results and Discussion

3.1.Synthesis and Characterization of Methacrylated Laminarin

Laminarin is a low-molecular-weight polysaccharide and bioactive compound present in

brown algae.10 The abundance of hydroxyl groups in the laminarin structure may be used

to insert a polymerizable moiety or to chemically bind a bioactive agent. To synthesize

laminarin with UV crosslinking ability via grafting acrylate units, the modification of

laminarin using glycidyl methacrylate has recently been proposed.11 Briefly,

methacrylated laminarin was synthesized as a hydrogel precursor by taking advantage of

the functionality of the hydroxyl groups in laminarin as well as the reactivity of the epoxy

group in glycidyl methacrylate. (figure 4.3 A). In this work, the functionalization protocol

was followed as previously described and two different degree of modification were

obtained. The chemical modification and degree of substitution of laminarin was assessed

by 1H NMR. Comparing the original 1H NMR spectrum of laminarin (figure 4.3 B) with

the 1H NMR spectrum (figure 4.3 C and D) after modification it was possible to observe

the appearance of two new peaks. The peaks at δ = 5.7 ppm and δ = 6.1 ppm corresponds

of vinylic protons (C=CH2) and the peak at δ = 1.9 ppm corresponds of the protons of

methyl group (CH3). Integration and normalization of the methyl group peak in the

methacrylate segment in relation to the hydrogen peaks of the laminarin backbone (3 ∼

5.5 ppm) provides consistent means for calculating the degree of substitution of hydroxyl

groups in laminarin by the methacrylate group. The average degree substitution increased

from 30% (Low - MeLam) to 60% (High -MeLam), by adding 7% (400 μl) and 14%

(700 μl) (v/v) glycidyl methacrylate to laminarin during the synthesis.11 This value is

relative to the total amount of hydrogens per monomer of laminarin. The equation 1 give

the value of the substitutions taking account the total of hydrogens.

47


expansion

Figure 4.3: (A) Schematic illustration of the methacrylation reaction of laminarin with glycidyl

methacrylate. (B) 1H NMR spectrum of laminarin before modification. (C) Low methacrylated laminarin

(D) High methacrylated laminarin.

3.2.Fabrication of Methacrylated Laminarin microparticles

Based on the promising results of laminarin hydrogels as cell culture platforms,11 and on

the intrinsic low viscosity of this polymer (6 KDa) we hypothesized that microfabricated

laminarin hydrogels could provide effective microplatforms for cell culture. MeLam was

processed in a microfluidic flow-focusing device. Aqueous MeLam droplets were formed

using a water-in-oil emulsion. The continuous stream of laminarin is broken into droplets

by the shear forces exerted from the oil phase using a microchip with a T-junction (figure

4.1). Microfluidics technique allowed to efficiently produce high monodisperse

microparticles with spherical shape.14,15 The microparticles size and shape can be tuned

to a desired value by adjusting the flow rates of the aqueous and dispersed phase or by

adjusting the diameter of the microfluidic channel. In the present work, different flow

rates were tested being noticeable that the increase of the ratio aqueous phase/continuous

phase (QAq)/QC decreases the size of microparticles. The flow rates were optimized to

prepared microparticles with average diameter of 100 µm. A continuous phase at 160

µL/min and aqueous phase of 8 µL/min were found to be the ideal flow rates (figure 4.4

A and D). The formed droplets go through a FEP tube under UV light with intensity of

6.12w/cm2 for 60 seconds that allowed the crosslinking of MeLam microspheres. The

formed MeLam microparticles were collected immersed in mineral oil. The excess of

48


expansion

mineral oil was cleared and the microparticles were washed with PBS solution to remove

the remaining oil. Some previous works have reported the use of surfactants or organic

solvents (e.g. ethanol 70%) for a complete removal of the mineral oil.14 In this particular

work this was not possible, as such solvents may denature the encapsulated proteins. The

fabricated particles exhibit an increase in size after the removal of oil and incubation of

PBS, due to the swelling of the microgels. The average diameter of the microparticles

was 326.6 µm to high degree of methacrylation (figure 4.4 B and C) and 303.3 µm to low

degree of methacrylation (figure 4.4 E and F) and they exhibit a smooth surface. The

homogenous and spherical shape and smooth surface of microparticles is notable in the

SEM images in both degrees of modification microparticles (figure 4.4 G and H).

Figure 4.4: Images of the high MeLam microparticles in oil (A) in PBS (B) obtained by optical microscopy

and respective histogram of the distribution (C) of microparticles (n = 74) after washed with PBS. Images

of the low MeLam microparticles in oil (D) in PBS (E) obtained by optical microscopy and respective

histogram of the distribution (F) of microparticles size (n = 74) after washed with PBS. SEM image of

monodisperse high (G) and low (H) MeLam microparticles

49


expansion

3.3.Cumulative release of platelet lysates from MeLam microparticles

Hydrogels microparticles have demonstrated great potential as drug delivery systems due

to facile incorporation and finely tuned release of biomolecules. The MeLam

microparticles are highly porous and exhibit water uptake, that was confirmed by the

swelling degree when in exposure to the PBS solution. In this work, platelet lysates were

used as a source of growth factors to improve cell expansion. The lysates were mixed

with the laminarin solution and this mixture was processed as previously described, using

the microfluidic device. To further assess their potential for the controlled release of

proteins, these particles were incubated in PBS at 37ºC and the protein content in the

supernatant was evaluated after pre-determined time-points. The cumulative release

profile of the total protein content released from the MeLam microparticles is show in

figure 4.5 A. The release profile of encapsulated PL in high and low MeLam

microparticles was followed up to 14 days. High MeLam microparticles exhibit a burst

release of 40.31 ± 0.27% after 12 hours while the low MeLam exhibit a burst release of

22.54 ± 1.13% after 12 hours. At the end of 14 days 56.89 ± 1.03% of the total protein

was released from the high MeLam microparticles, while only 35.31 ± 2.73% of the total

encapsulated protein was released from the low MeLam microparticles. Protein release

rate depends on the solubility, diffusion and biodegradation of the matrix of

encapsulation. The difference in release profile of the microparticles can be related to the

microparticle crosslinking level. According to the previously characterization of the

microparticles, it was expected a high release from the gels with low degree of

substitution due to larger pores than the ones with high degree of substitution.11 However,

our results demonstrated that the high MeLam microparticles had a better release profile.

Due to different levels of substitution of the microparticles, the chemical interactions

between them and proteins will be different, which may explain the different release

profiles. Ngyyen and co-workers studied the influence of different degrees of

methacrylation and the ability to bind and release GFs.16 They demonstrated that

decreasing of the degree of methacrylation increases GFs binding. This corroborates the

results obtained once the release profile is higher for high MeLam microparticles. This

can be explained by covalent or electrostatic interactions between the matrix of the

microgel and proteins. Some authors defend that sometimes nucleophilic double bonds,

including vinylsulfone, methacrylate and maleimide, can be react with the protein amine

groups under physiological conditions. Yu and co-workers demonstrated that in your

works.17 Here, after polymerization some methacrylate groups continuous available to

50


expansion

react with amine groups of proteins. Also, the kinetics of GFs release in vitro has been

controlled by the types of non-covalent interactions between the microgels and GFs. The

low MeLam microparticles possess more hydroxyl groups available to link the proteins

by hydrogen bonds.18 PLs are rich in several chemokines and growth factors (GFs) such

as platelet derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming

growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), vascular endothelial

growth factor (VEGF) and bone morphogenetic protein 2, -4 and -6 (BMP-2, -4, -6).19

Considering the PL composition and the response of the PL in wound healing, several

works have been exploring PLs as a possible substitute to animal derived serum (FBS) in

cell culture. FBS is rich in growth factors like PL, however this shows high risk of

contamination and a potential to promote xenogeneic immune reactions, which make PL

an excellent alternative.20,21 The release of specific GFs, namely VEGF and TGF-β1 was

confirmed by ELISA assay (figure 4.5 C and D). The total amount of these GFs in PL

was quantified and results show 9.78 ± 0.07 pg for VEGF and 73.94 ± 4.44 pg for TGF-

β1 (figure 4.5 B). In this study, we have demonstrated the capacity to produce

microparticles with encapsulated PL capable to release GFs present in PL. The

comparison between different studies using PL is not easy, once the composition of PL is

variable depending of the donor. The release results of this specific growth factors showed

a similar behavior to total protein amount.22 Comparing the total amount of VEGF and

TGF-β1 encapsulated into the microparticles can conclude which the VEGF is practically

all released into the high MeLam. In its turn, the release profile of TGF-β1 confirms the

higher release from the high MeLam microparticles. But instead of VEGF, here the

microparticles release 6.62 ± 1.68 pg of total amount encapsulated in high MeLam

microparticles while the release of TGF-β1 was 45.78 ± 1.20 pg (figure 4.5 C and D).

High MeLam microparticles revealed an increased release of proteins and were chosen to

evaluate the ability this system to support cell expansion.

51


expansion

Figure 4.5: (A) Cumulative protein profile release from low and high methacrylate laminarin

microparticles by incubation in PBS solution up to 14 days quantified by micro-BCA assay. ELISA assay

performance to quantify specific growth factors release from microparticles. (B) Concentration of VEGF

and TGF-β1 presented in PL sample. VEFG release profiled (C) and TGF-β1 release profile (D) up to 14

days from low and high methacrylate laminarin. Error bars represent standard deviation (n = 3).

3.4. RGD functionalization of MeLam microparticles

One of the most common strategies used to conjugate biomolecules on the surface of

biomaterials is through the use of specific interaction between streptavidin (SaV) and

biotin.5,23,24 Biotin is a small molecule with high affinity to SaV. Due to the high stability

and specificity, this complex has been a very powerful tool in the study of biological

systems being used for chemical conjugation of biomolecules (e.g. antibodies, peptides

sequences and GFs.25 Taking advantage of the SaV-biotin pair, the focus in this work was

the immobilization of biotinylated RGD to promote cell attachment to the laminarin

microgels. The MeLam microparticles were first modified with biotin-PEG-SH by

reaction with the alkene groups presents in MeLam microparticles via Michael type

reaction. A second modification step was performed to create a coating of SaV in the

microgels, followed by an incubation with biotin-RGD. One the biggest advantage of the

use of this system, is the possibility to modify different surfaces with a large number of

different biotinylated molecules. The effective modification of the laminarin

52


expansion

microparticles was assessed by florescence microscopy using fluorescent-labeled SaV

and the unmodified microparticles were used as control. The fluorescence images

demonstrate the efficient conjugation with the microparticles with SaV, confirming the

effective modification of the microparticles with biotin. (figure 4.6 A and B). The last

step of the microparticles functionalization was the addition of the RGD-biotinylated.

RGD is a peptide sequence (Arg-Gly-Asp) that constitute a major recognition system for

cell adhesion (figure S4.1).26 The goal of this work is to use the microparticles as a support

for cell expansion and prove the PL improve the cell adhesion. Taking into account the

results from PL release, the high MeLam showed a better profile release, being expected

which that shows better results in cell culture.

Figure 4.6: Fluorescence imagens showing the functionalization of MeLam microparticles. Images of

MeLam microparticles with biotin-PEG-SH (A) and MeLam microparticles without biotin (control) (B)

after incubation with fluorescently labeled SaV.

3.5. L929 fibroblast capture and expansion on MeLam microparticles

Microcarrier beads of different materials, have been widely used to culture anchorage-

dependent cells. Microcarriers have innumerous advantageous when compared with the

conventional cell culture systems. The low cost and great surface-to-volume ratio allow

the culture of high cell numbers, eliminating multiple trypsinization steps.27 Recently,

Soure and co-workers studied the effect of PL on the expansion of umbilical cord matrix

derived from mesenchymal stem cells (MSCs) in plastic microcarriers under dynamic

conditions.28 Their results demonstrated the advantages of the use of PL for the effective

expansion of MSCs in a xenogeneic-free microcarrier-based system. In this work, we

proposed the fabrication of MeLam microparticles with encapsulated PL to be used as

microplatforms for cell culture. Here, we not use PL to supplement the culture media, we

propose the encapsulation of PL to improve cell adhesion and expansion on the cell

53


expansion

microcarriers. L929 cells were used to evaluate our hypothesis. L929 cells were seeded

on the RGD functionalized microparticles, with encapsulated PL and cultured for 11 days.

In order to study the influence of encapsulated PL in cell adhesion, RGD functionalized

microparticles without encapsulated PL were used as a control. Cell adhesion was

monitored by optical microscopy (figure S 4.2). The images shown an increased in cell

attachment in microparticles with encapsulated PL. This may be due to the initial burst

release of protein from the microcarriers that stimulate cell attachment. Also, phalloidin

and DAPI staining were performed to evaluate cell morphology on the surface of

microparticles at 3, 7 and 11 days (figure 4.7 A to D and figure S 4.3). The dependence

of cell adhesion and morphology from the encapsulated PLs was evident. The L929 cells

adopt an elongated, spreading morphology on the microparticles containing PL. After 11

days, is possible to observe the surface of microparticles completely covered with cells

and a cell matrix connecting the microparticles. In the absence of PLs a few cells adhered

on the surface. Nevertheless, this may be justified by the presence of the RGD moieties

as plain particles did not show any cell attachment.

The cell viability of L929 was assessed at day 3, 7 and 11 (figure 4.7 E). In the first-time

points, were no significant differences between the sample and control were observed.

Only after 11 days of culture we could observe a significant difference of cell viability in

the two conditions. The microparticles with encapsulated PL showed increase cell

viability. Lastly, DNA assay quantification was performed to evaluated the cell

proliferation. Results corroborate the hypothesis that PL have a positive influence in cell

expansion. (figure 4.7 F).

54


expansion

Figure 4.7: Fluorescence images of the high MeLam microparticles with encapsulated PL (A-D) culture

with L929 cells up to 14 days. These images demonstrate the ability to cell attach, cytoskeleton was stained

with phalloidin (red) and nuclei was stained with DAPI (blue). (E) Cell viability by MTS assay was

determined at 3, 7 and 11 days. (F) DNA quantification of all formulations tested up to 11 days of culture.

Results are present as mean ± standard error of the mean (n = 3).

4. Conclusions

Here, we demonstrated an efficient one-step method to generate monodisperse MeLam

microparticles incorporating PL using a microfluidics device coupled to a source of UV

light. The pendant acrylate groups of MeLam allowed also the conjugation of thiolated

biotin via thiol-Michael addition and further conjugation with RGD peptides.

The size of microparticles was easily controlled by the adjustment of the flow rates of the

aqueous phase and continuous phase. The multifunctional MeLam microparticles were

seeded with L929 cells and the results demonstrate their potential to support cell adhesion

and expansion. MeLam microgels offer a high degree of tunability over both structural

and chemical properties, and can be used to recapitulate highly varied tissue

environments.

Cultured microgels could self-assemble to form structures with packing densities,

suggesting potential applications in tissue engineering and regenerative medicine.

5. References:

1. Discher, D. E., Mooney, D. J. & Zandstra, P. W. Growth Factors, Matrices, and Forces Combine

and Control Stem Cells. Science (80 ). 324, 1673–1677 (2009).

2. Hu, Y. et al. Controlled self-assembly of alginate microgels by rapidly binding molecule pairs.

Lab Chip 17, 2481–2490 (2017).

3. Kim, P. H. et al. Injectable multifunctional microgel encapsulating outgrowth endothelial cells

and growth factors for enhanced neovascularization. J. Control. Release 187, 1–13 (2014).

55


expansion

4. Cavalieri, F., Postma, A., Lee, L. & Caruso, F. Assembly and Functionalization of DNA -

Polymer Microcapsules. ACS NanoNano 3, 234–240 (2009).

5. Custódio, C. A., Cerqueira, M. T., Marques, A. P., Reis, R. L. & Mano, J. F. Cell selective

chitosan microparticles as injectable cell carriers for tissue regeneration. Biomaterials 43, 23–31

(2015).

6. Mao, A. S. et al. Deterministic encapsulation of single cells in thin tunable microgels for niche

modelling and therapeutic delivery. Nat. Mater. 1, 1–10 (2016).

7. Neto, A. I. et al. Fabrication of Hydrogel Particles of Defined Shapes Using Superhydrophobic-

Hydrophilic Micropatterns. Adv. Mater. 28, 7613–7619 (2016).

8. Wang, Q., Liu, S., Wang, H., Zhu, J. & Yang, Y. Alginate droplets pre-crosslinked in

microchannels to prepare monodispersed spherical microgels. Colloids Surfaces A Physicochem.

Eng. Asp. 482, 371–377 (2015).

9. Hu, Y., Azadi, G. & Ardekani, A. M. Microfluidic fabrication of shape-tunable alginate

microgels: Effect of size and impact velocity. Carbohydr. Polym. 120, 38–45 (2015).





12. Juhl, M. et al. Comparison of clinical grade human platelet lysates for cultivation of

mesenchymal stromal cells from bone marrow and adipose tissue. Scand. J. Clin. Lab. Invest. 76,

93–104 (2016).

13. Che, H. et al. CO 2 -switchable drug release from magneto-polymeric nanohybrids. Polym. Chem.

6, 2319–2326 (2015).

14. Cha, C. et al. Microfluidics-Assisted Fabrication of Gelatin-Silica Core − Shell Microgels for

Injectable Tissue Constructs. Biomacromolecules 15, 283–290 (2014).

15. Zhao, X. et al. Injectable Stem Cell-Laden Photocrosslinkable Microspheres Fabricated Using

Microfluidics for Rapid Generation of Osteogenic Tissue Constructs. Adv. Funct. Mater. 26,

2809–2819 (2016).

16. Nguyen, A. H., McKinney, J., Miller, T., Bongiorno, T. & McDevitt, T. C. Gelatin Methacrylate

Microspheres for Growth Factor Controlled Release. Acta Biomater. 13, 101–110 (2015).

17. Yu, Y. & Chau, Y. Formulation of in situ chemically cross-linked hydrogel depots for protein

release: From the blob model perspective. Biomacromolecules 16, 56–65 (2015).

18. King, W. J. & Krebsbach, P. H. Growth factor delivery: How surface interactions modulate

release in vitro and in vivo. Adv. Drug Deliv. Rev. 64, 1239–1256 (2012).

19. Burnouf, T., Strunk, D., Koh, M. B. C. & Schallmoser, K. Human platelet lysate: Replacing fetal

bovine serum as a gold standard for human cell propagation? Biomaterials 76, 371–387 (2016).

20. Bieback, K. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell

cultures. Transfus. Med. Hemotherapy 40, 326–335 (2013).

21. Turner, P. A., Thiele, J. S. & Stegemann, J. P. Growth factor sequestration and enzyme-mediated

release from genipin-crosslinked gelatin microspheres. J. Biomater. Sci. Polym. Ed. 28, 1826–

56


expansion

1846 (2017).

22. Santo, V. E. et al. Enhancement of osteogenic differentiation of human adipose derived stem cells

by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid

foaming. J. Control. Release 162, 19–27 (2012).

23. Li, C. Y., Wood, D. K., Hsu, C. M. & Bhatia, S. N. DNA-templated assembly of droplet-derived

PEG microtissues. Lab Chip 11, 2967 (2011).

24. Riccardi, C. et al. Fluorescent Thrombin Binding Aptamer-Tagged Nanoparticles for an Efficient

and Reversible Control of Thrombin Activity. ACS Appl. Mater. Interfaces 9, 35574–35587

(2017).

25. Chivers, C. E., Koner, A. L., Lowe, E. D. & Howarth, M. How the biotin–streptavidin interaction

was made even stronger: investigation via crystallography and a chimaeric tetramer. Biochem. J.

435, 55–63 (2011).

26. Ruoslahti, E. RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12,

697–715 (1996).

27. Sun, L. et al. Novel konjac glucomannan microcarriers for anchorage-dependent animal cell

culture. Biochem. Eng. J. 96, 46–54 (2015).

28. de Soure, A. M. et al. Integrated culture platform based on a human platelet lysate supplement for

the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal

stem/stromal cells. J. Tissue Eng. Regen. Med. 11, 1630–1640 (2017).

57

Supporting Information

Multifunctional laminarin microparticles for cell adhesion and expansion

Martins CR,1 Custódio CA,1 Mano JF1



Figure S4.1: Images of MeLam microparticles with biotin-PEG-SH bioconjugate with pure SaV (A) and

without bioconjugation with pure SaV (control) (B) after incubation with fluorescence biotin

58

Figure S4.2: Images assessed by optical microscopy with encapsulated PL (A and B) and without PL (C

and D) for 24 hours and 7 days.

Figure S4.3: Fluorescence images of the high MeLam microparticles without encapsulated PL (A-C)

culture with L929 cells up to 14 days. These images demonstrate the ability to cell attach, cytoskeleton was

stained with phalloidin (red) and nuclei was stained with DAPI (blue)

CHAPTER 5

General Conclusions

59

Chapter 5 – General Conclusions


Tissue engineering (TE) combines mainly three different areas: biology, chemistry and

engineering to mimic or replace the native architecture of tissues. TE uses two approaches

to recreate a native tissue: the top-down approach in which cells are seeded in a scaffold

and the bottom-up approach in which small microunits are used to construct a complex

3D structure. Hydrogels are a versatile class of polymeric networks that have been used

for a wide variety of cell culture and TE applications. Additionally, hydrogels can be

selectively functionalized to present specific chemical or mechanical cues to trigger

desired cellular responses. Hydrogels can also be spatially confined as microscopic

shapes (microgels). Microgels are attractive for TE applications because of their

biological and physical properties (i.e., well defined shapes, mechanical strength, and

biodegradability and resemblance to the natural extracellular matrix (ECM)).

Chapter 1 presents general concepts in hydrogels and microfabrication and the thesis

motivation. Chapter 2 reviews the efforts developed regarding microfabrication of

hydrogels, their applications, strategies for microgels assembly and future perspectives in

the field. This chapter also highlights the advantages in the use of hydrogel

microstructures to engineer hierarchically organized 3D structures. The 3D structures

produced by bottom-up approaches demonstrate better vascularization and diffusion of

nutrients and oxygen when compared with traditional scaffolds. These microstructures

can be used to many applications, such as cell microcarriers for cell expansion, drug

delivery or cell encapsulation.

Detailed materials and methods using during the development of this work are presented

in Chapter 3 of this thesis.

Aiming the design of innovate multifunctional microparticles for cell expansion, in

chapter 4, we propose a method to produce laminarin microgels using a microfluidics

device coupled to a UV lamp. This technique allowed to produce highly monodisperse

microgels with perfect spherical shape. A subsequent reaction with pendent alkene groups

was then utilized in a thiol-ene addition reaction to obtain functionalized microparticles

with thiolated biotin. Here was demonstrated the ability of functionalized particles to

promote cell adhesion by conjugation with biotin-RGD molecules. However, the

versatility of this method allows the combination of the biotin-SaV conjugated microgels

with any biotinylated molecules of interest. Here, we demonstrate that this technique can

also be used for the direct encapsulation of platelet lysates (PL) within the microgels, in

a one-step approach. PL are a cocktail of proteins and growth factors and have been

60


studied as a substitute of animal serum in cell culture. This work offers a novel system

encompassing MeLam microgels functionalized with adhesive peptides to promote cell

adhesion that provide simultaneously a controlled release of PL that should be useful in

cell expansion and cell differentiation protocols. To validate this system, the MeLam

microgels were cultured with L929 cells. The cells cultured with microparticles with

encapsulated PL demonstrated clear enhancement in cell attachment and expansion.

Cultured microgels could assemble to form structures with packing densities, suggesting

potential applications in tissue engineering and regenerative medicine.

Further studies will validate the effective potential of released PL by culturing the

microgels in media without FBS supplementation.

clÁudia isabel rodrigues martins lisados de plaquetas … · 2020. 1. 17. · figure 2.4:...

Documents