Hydrogel as Stem Cell Niche for In Vivo Applications in … · 2017-01-26 · 31 2 Hydrogel as Stem Cell Niche for In Vivo Applications in Regenerative Medicine Xiaokang Li, Claudia

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2Hydrogel as Stem Cell Niche for In Vivo Applications in Regenerative Medicine

Xiaokang Li, Claudia Wittkowske, Rui Yao, and Yanan Du

2.1 Introduction

Diseases and injuries can lead to severe and irreversible damage to tissues and organs. Organ transplantation is currently the most effective way to restore tissue function and improve the survival rate of patients. However, over 98,000 people are currently on the waiting list for organ transplanta-tion, and the number is rapidly growing due to the huge shortage of organ donors [1,2]. The field of regenerative medicine holds great promise to meet

Contents

2.1 Introduction .................................................................................................. 312.2 Hydrogels as Scaffolding Materials ..........................................................33

2.2.1 Natural Hydrogels ...........................................................................342.2.2 Synthetic Hydrogels ........................................................................352.2.3 Cross-Linking Strategies .................................................................36

2.3 Hydrogel-Based Stem Cell Engineering for Regenerative Medicine ..... 362.3.1 Cartilage Regeneration ....................................................................362.3.2 Adipose Tissue Regeneration ......................................................... 392.3.3 Bone Regeneration ........................................................................... 412.3.4 Vascular Regeneration.....................................................................422.3.5 Hydrogel-Mediated Stem Cell Engineering for

Regeneration of Other Tissues .......................................................452.3.5.1 Nerve Regeneration ..........................................................462.3.5.2 Retina Regeneration .......................................................... 472.3.5.3 Muscle Tissue Regeneration ............................................48

2.4 Summary and Outlook ...............................................................................48Glossary of Terms ..................................................................................................48References ............................................................................................................... 49

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32 Biomaterials and Stem Cells in Regenerative Medicine

this challenge by replacing damaged tissues with functional engineered counterparts. The success in clinical application using engineered tissues requires reliable cell sources with the ability to regrow into specific cel-lular components of targeted tissues and biomaterials to support cellular functions.

With the unique properties of self-renewal and pluripotency, stem cells have emerged as the most promising cell source for tissue engineering and regenerative medicine. According to their origin, stem cells generally include embryonic stem cells (ESCs) and adult stem cells as well as the recently dis-covered induced pluripotent stem cells (iPSCs) [3]. Currently, adult stem cells are the most commonly used cell source for regenerative medicine which do not pose same ethical issue as ESCs and can be readily harvested from adult tissues, such as bone marrow and adipose tissue.

To enhance the efficacy of stem-cell-based applications, a well-defined microenvironment, often referred to as stem cell niche, is indispensible which includes cellular and acellular components (e.g., matrix and solu-ble factors) [4]. Due to the resemblance to the native extracellular matrix (ECM), hydrogels have been extensively applied as the matrix compo-nent of the stem cell niche. Hydrogels are polymer networks with high water content exhibiting biocompatibility, biodegradability, and tunable mechanical properties which are ideal for cellular support and tissue regeneration [5].

In this chapter, natural and synthetic hydrogels used in stem cell engi-neering were first reviewed followed by exemplary studies utilizing stem-cell-incorporated hydrogels for regenerating various tissues, includ-ing cartilage, adipose, bone, vasculature, etc. In general approaches for hydrogel-mediated stem cell engineering (Figure 2.1), stem cells are first harvested from embryos or adult tissues with human or nonhuman origins and expanded in vitro to obtain sufficient cell numbers. The cells are sub-sequently mixed with hydrogel precursor solution, which is either applied to the patients or animals by direct injection (in the aqueous form or as microspheres) or by implantation (as solidified constructs with cells embed-ded in 3D polymerized hydrogel scaffolds). The differentiation of stem cells into the desired cell lineage can be induced either in vitro or directly in vivo after implantation, which is largely regulated by biophysical and biochemical cues. The hydrogel-mediated delivery of stem cells provides both a physical barrier to stabilize the implanted cells at the defect site and microenvironment for new tissue formation with defined structures and functions. As an emerging field, numerous investigations have been con-ducted in recent years to examine the effects of hydrogel on stem cell fate in vitro as contrasted by limited cases that have been extended to in vivo studies. Here we focus on hydrogel-mediated stem cell engineering for in vivo studies, which are of vital importance to future clinical applications in regenerative medicine.

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33Hydrogel as Stem Cell Niche for In Vivo Applications

2.2 Hydrogels as scaffolding Materials

Hydrogels have been widely used in tissue engineering, thanks to their structural and compositional resemblances to the ECM, their extensive framework for supporting cellular proliferation, and the convenience for delivery in a minimally invasive manner [2,6]. Hydrogels with vari-ous compositions and forms have been employed as space-filling agents, as 3D constructs for organizing cells, and as scaffolds for delivering bio-molecules [1]. Integrated with microfabrication approaches, dimension-defined hydrogels can be made with precision in micro- or nanoscale to better mimic the exquisite architectures of stem cell niche [7]. Depending on their origins, hydrogels can be categorized into natural hydrogels, syn-thetic hydrogels, and the hybrid combining these two. Hydrogels utilized in tissue engineering and regenerative medicine are required to meet a number of design criteria including biocompatibility, controlled degrada-tion, gelation conditions compatible with physiological applications, and tailored bioactivity (i.e., adhesiveness and specific ligand-receptor interac-tions) and mechanical stiffness [1,2,7,8]. Tables 2.1 and 2.2 summarize the origin and chemical structure of natural and synthetic hydrogels used in stem cell engineering.

AQ1

Donor(animal or human)

Implantation

Injection

Stem cell isolation(embryo, adult tissues)

In vitro expansion or differentiation ofisolated stem cells

Mixing with hydrogel precursor

Cells suspended inprecursor or embedded

in microspheres

Cells embeddedin hydrogels

Growthfactors

Growthfactors

Figure 2.1Schematic of basic strategies for hydrogel-based stem cell engineering.

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2.2.1 Natural Hydrogels

Hydrogels derived from natural polymers have been extensively used in stem cell engineering applications as they either are components of natural ECM or exhibit properties similar to the matrix components in native tissues. As shown in Table 2.1, collagen, for example, is one major ECM protein of mam-malian tissue [9,10]. Similarly, hyaluronic acid (HA) is universally present in mammalian tissue as the simplest glycosaminoglycan (GAG) [9]. Alginate and cellulose can be extracted from plants and are widely used for in vivo applications [11,12]. Other examples for natural hydrogels include Matrigel®,

Table 2.1

Origin and Chemical Structure of Natural Hydrogels Used in Stem Cell Engineering

Hydrogel origin Chemical structure

Collagen Naturally occurring proteins as major components of ECM in mammalian tissues

Three polypeptide chains twined around one another to form triple helix

HA (also hyaluronan)

GAG Repeating β-1,4-d-glucuronic acid-β-1,3-N-acetyl-d-glucosamine disaccharide

OO O

O

nNH

O

O

OH

OHHO

HOHO

Matrigel® (by BD Bioscience)

EHS mouse sarcoma cells Protein mixture secreted by EHS mouse tumor

Fibrin gel Formed by reaction of fibrinogen and thrombin

Fibrinogen and thrombin

Alginate Marine brown algae (1–4)-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) monomers

G G M M

O

OOO

O

O

OOO

O

OH

OH

OH OH

OHCOO–

COO–

–OOC

OH

OH

OH

Cellulose Cell wall of green plants, many algae, and oomycetes

(1–4)-linked β-d-glucose units

n

HO

HO

O

O

OO

O

OHOH

OHOH

Source: Drury, J.L. and Mooney, D.J., Biomaterials, 24(24), 4337, 2003; Slaughter, B.V. et al., Adv. Mater., 21(32–33), 3307, 2009; Mather, M.L. and Tomlins, P.E., Regen. Med., 5(5), 809, 2010; Hoffman, A.S., Bioartificial Organs III: Tissue Sourcing Immunoisolation Clinical Trials, Vol. 944, pp. 62–73, 2001; Lee, K.Y. and Mooney, D.J., Chem. Rev., 101(7), 1869, 2001; Burdick, J.A. and Prestwich, G.D., Adv. Mater., 23(12), H41, 2011.

See Glossary for definition of terms.

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which is a mixture of proteins secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and fibrin gel, which is formed by the clotting reaction of fibrinogen and thrombin [13,14]. Potential problems of applying natural hydrogels for regenerative medicine may arise from undefined compositions, batch-to-batch variation, and risk of immune rejection for animal-derived materials.

2.2.2 Synthetic Hydrogels

Synthetic hydrogels are chemically defined polymers with well-tunable properties such as chemical composition, molecular weights, block struc-tures, cross-linking density, mechanical strength, and degradability [2]. The ability to control and reproduce these properties offers great advantages to achieve optimal and consistent performance in tissue engineering. As shown in Table 2.2, synthetic polymers such as polyethylene glycol (PEG), polyeth-ylene oxide (PEO), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), or polyethylene(glycol)diacrylate (PEGDA) [15–18] have been frequently used in stem-cell-based tissue engineering applications. Synthetic polymers also prevent the potential contamination caused by pathogen originated from animal-derived matrices.

Table 2.2

Origin and Chemical Structure of Synthetic Hydrogels Used in Stem Cell Engineering

Hydrogel Unit Molecular Weight (g/mol)

PEG/PEO (chemically synonymous, but different molecular weight)

HO

On

H<20,000(PEG)>20,000 (PEO)

PLA O

On

Wide range

PLGA O

OO

Ox y

HHO

Wide range

Source: Drury, J.L. and Mooney, D.J., Biomaterials, 24(24), 4337, 2003; Slaughter, B.V. et al., Adv. Mater., 21(32–33), 3307, 2009; Mather, M.L. and Tomlins, P.E., Regen. Med., 5(5), 809, 2010; Hoffman, A.S., Bioartificial Organs III: Tissue Sourcing Immunoisolation Clinical Trials, Vol. 944, pp. 62–73, 2001; Lee, K.Y. and Mooney, D.J., Chem. Rev., 101(7), 1869, 2001; Burdick, J.A. and Prestwich, G.D., Adv. Mater., 23(12), H41, 2011.


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2.2.3 Cross-linking Strategies

Hydrogels keep their structural integrity while remaining insoluble in aque-ous solutions due to physical or chemical cross-linking between individual polymer chains. Physical treatments such as UV radiation and temperature alterations (warming or cooling) can polymerize gel precursor resulting in the formation of hydrogels with covalent linkages between adjacent poly-meric chains. For example, PEGDA, a diacrylated derivative of PEG, can be photocross-linked by UV light to form biocompatible PEGDA hydrogels with tunable stiffness spanning large ranges. Widely used thermal cross-linkable materials include Matrigel, collagen, and HA, which are present as precursor solution in lower temperatures and form hydrogel when heated [19].

Hydrogel precursor can also be cross-linked by chemical reactions [20]. For example, the divalent metal cations (i.e., Ca2+) can induce the cross-linking of alginate during chelation. Fibrin gel is formed by the enzymatic polym-erization of fibrinogen in the presence of thrombin as seen in the clotting process during wound healing [8]. For certain hydrogels, the polymerization can be triggered by either physical or chemical strategies. For example, cel-lulose hydrogel can be formed by heating a precursor solution of cellulosic or exposing the precursor to high-energy radiation. Besides physical treat-ments, chemical cross-linking with aldehydes or epichlorohydrin can also generate stiff cellulose networks [20].

2.3 Hydrogel-Based stem Cell engineering for Regenerative Medicine

As one of the most well-studied and widely applied biomaterials in tissue engi-neering, hydrogels have been proved to provide an ECM-mimicking microen-vironment as stem cell niche, to regulate stem cell fate both in vitro and in vivo. The integration of hydrogels and stem cells offers both tunable biomaterials and unlimited cell sources which opens new opportunities to regenerate via-ble tissues and organs. Hydrogel-mediated stem cell engineering is particu-larly powerful in regenerating soft tissues, such as cartilage and adipose tissue in vivo which have demonstrated great potential for clinical success in the near future. Attempts have also been made to regenerate other tissues with more complex structures and functions (i.e., bone, nerves, vasculature, muscle, and retina) indicating its wide application for regenerative medicine.

2.3.1 Cartilage regeneration

Articular cartilage is a nonvascularized, noninnervated connective tissue, which is highly hydrated and composed of chondrocytes embedded in

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type II collagen and GAGs [1]. Cell density in cartilage tissue is extremely low which limits its capacity for self-repair. Damage to the articular cartilage caused by osteoarthritis is becoming a significant clinical and social problem due to the increase in the elderly population. Tissue-engineered cartilages represent promising alternatives for cartilage repair. Since the availability of chondrocytes in human is limited and their proliferation capacity decreases with age [21], most studies in cartilage repair focus on using adult stem cells as cell source. For instance, mesenchymal stem cells derived from bone mar-row (BMSCs) exhibited higher chondrogenic potential than stem cells iso-lated from other sources such as adipose tissue [12,22,23].

With similar hydration and softness as cartilage tissue, hydrogels have been the most commonly used scaffold in cartilage tissue engineering. Additionally, hydrogels can be injected in a minimally invasive manner directly in the cartilage defect site. As summarized in Table 2.3, a variety of hydrogels (e.g., fibrin, agarose, gelatin, collagen, and alginate) have already been fabricated to serve as scaffolds to achieve chondrogenesis [22,24].

Growth factors, such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and insulin-like growth factor-I (IGF-I), play an impor-tant role in the lineage-specific differentiation of BMSCs into chondrocytes which has already been well demonstrated in vitro [21,25,26]. Incorporation of growth factors into hydrogel was also shown to enhance the chondrogen-esis of the implanted stem cells in vivo. In one example, TGF-β1 was coupled to HA which has been used to load BMSCs for cartilage repair in a mice model and a rabbit model, respectively. The growth-factor-loaded hydrogel enhanced the formation of cartilaginous matrix in both animal models [27] (Figure 2.2I). In another case, MSCs adhered to the surface of TGF-β3 func-tionalized PLGA microspheres, which were injected subcutaneously and intramuscularly in mice. Cartilage-like tissue was formed after 4 weeks as indicated by upregulation of cartilage-specific markers at the mRNA and protein level [28].

One problem of using growth factors for in vivo studies is the short half-life which hinders their in vivo efficacy. To prolong the activity of growth factors, DNA plasmids encoding corresponding growth factors were incor-porated into the hydrogels and were transfected to the stem cells to maintain sustained expression. Wang et al. [29] established a composite construct for BMSCs which comprised a PLGA sponge/ fibrin hydrogel scaffold loaded with plasmid DNA encoding TGF-β1. Twelve weeks postimplantation, suc-cessful repair of the full-thickness cartilage defect could be achieved in a rabbit model (Figure 2.2II).

Hydrogel-mediated stem cell engineering for cartilage repair has already been applied in a clinical trial. In this study, hBMSCs were embedded in a collagen gel and transplanted into the articular cartilage defect in patients suffering from knee osteoarthritis. Cartilage-like tissue could be observed 42 weeks after transplantation. However, the clinical improvement was not significantly different in the stem-cell-transplanted group compared to

AQ2

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Table 2.3

Hydrogel-Mediated Stem Cell Engineering for Cartilage Regeneration In Vivo

Cell source Hydrogel In Vivo Model Results Ref.

MSC Collagen ImplantationFull-thickness cartilage defect

Rabbits

Greater chondrogenic potential of MSCs from synovium and bone marrow than other sources

Synovium-derived MSCs had greatest proliferation potential

[31]

hASC Cross-linked HA with TGF-β1

InjectionSubcutaneouslyNude mice

TGF-β1-conjugated hydrogel produced more type II collagen

[27]

ImplantationFull-thickness defect of knee cartilage

Rabbits

Formation of cartilaginous matrix and maintenance of original volume in the TGF-β1-conjugated hydrogel

hBMSC PLGA microspheres with TGF-β3

InjectionIntramuscularly and subcutaneously

SCID mice

Formation of histologically resembling cartilage, staining positive for chondrocyte markers

[28]

BMSC Fibrin gel, plasmid DNA encoding TGF-β1 and PLGA sponge

ImplantationFull-thickness cartilage defect

Rabbits

Repair of cartilage defectsGood integration with surrounding tissue

[29]

BMSC Collagen and collagen-alginate

ImplantationSubcutaneouslyRabbits

Chondrogenic differentiation of BMSCs

[12]

BMSC PLGA ImplantationJoint cavitySheep

Expression of type II collagen but no differentiation into bone-like cells after implanting to the normal joint cavity

[32]

BMSC Collagen ImplantationArticular cartilage defect in knee joints

Humans

Width, thickness, stiffness, and cell morphology of newly formed tissue were better resembling native cartilage in cell-transplanted group than in cell-free control group

No significant difference in clinical outcome among different experiment groups

[30]


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the cell-free control group [30]. This result indicates that further scientific studies and technical improvements are required before the integration of stem cells and hydrogels can be truly beneficial for cartilage regeneration in clinical studies.

2.3.2 adipose Tissue regeneration

The shortage of adipose tissue in many clinical situations, such as traumatic injury, oncologic surgery, or congenital defects, poses a big challenge for plastic and reconstructive surgeons. Currently, transplantation of autologous adipose tissue is the most widely adopted method for soft tissue reconstruc-tion [33]. However, the clinical outcome of adipose tissue transplantation is often unpredictable due to implant resorption caused by a lack of vascular-ization [34,35]. Tissue-engineered adipose grafts with proper vasculariza-tion and scaffolding support could reduce implant resorption and thus start gaining more attention [34]. Although readily available, mature adipocytes are not suitable for engineering adipose tissue because of their limited pro-liferative capabilities. Preadipocytes, the progenitors which show better pro-liferation and specialized differentiation into adipocytes, are an alternative [34], but still require exogenous endothelial cells to improve the vasculariza-tion of the engineered adipose tissue.

With the ability to differentiate into adipocytes and vascular cells, stem cells are an ideal cell source for engineering vascularized adipose tissue [36]. Since adipose tissue is largely available, harvesting ASCs from adipose tis-sue is more convenient than from bone marrow. However, depending on

Complexes

BMSCs(a)

I II

III

(b)

(c) (d)

Fibrin gel

PLGA sponge

PDM + XLHAPDM

PDM

0.5 mm 0.5mm100 mm 100 mm

PDM PDM

PDM

Hybrid construct

Figure 2.2Hydrogel-mediated stem cell engineering for regenerating cartilage and adipose tissue. AQ3

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the location of the donor tissue as well as the age and gender of the patient, ASCs vary in metabolic activity and in their capacity for proliferation and differentiation [34].

Hydrogel-based scaffolds for adipose tissue engineering are expected to recapitulate the physical and architectural features of the native adipose tis-sue. These features include tunable softness and appropriate pore size facili-tating proliferation and differentiation of stem cells into adipocytes [34]. As summarized in Table 2.4, a variety of hydrogels, including those for clinical use, such as type I collagen sponges, nonwoven PGA, and HA [37], have already been successfully applied for regenerating adipose tissues in vivo.

Table 2.4

Hydrogel-Mediated Stem Cell Engineering for Adipose Tissue Regeneration In Vivo


hMSC, hASC Collagen, PLA, or silk fibroin

ImplantationMuscle pouchRats

Attachment of hMSCs and hASCs on all scaffolds

Rapid collagen and PLA scaffold degeneration

Silk fibroin scaffolds provided longer-term structural integrity

[40]

hBMSC PEGDA ImplantationDorsumImmunodeficient mice

Expression of adipogenic markers (Oil Red O staining) and adipocyte specific genes

Maintenance of volume

[38]

ASC Type I collagen sponge, nonwoven PGA, or HA gel

ImplantationSubcutaneouslyAthymic mice

Type I collagen sponge provided best scaffold for generation of adipose tissue

[37]

hASC PDM and cross-linked HA

ImplantationSubcutaneouslyAthymic mice

Maintenance of volumeDifferentiation into mature adipocytes

Implant integrated with host tissue and established vascularization

[35]

ASC PLGA microspheres

InjectionSubcutaneouslyNude mice

Enhanced tissue regeneration and adipogenic differentiation as indicated by Oil Red O staining

Complete differentiation of ASCs into adipocytes was confirmed using RT-PCR

[39]

MSC PLGA microspheres

InjectionNeckNude mice

Microsphere diameter of 100–150 μm formed fat pads resembling the native tissue

[39]


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In one study, hydrogel constructs consisting of cross-linked PEGDA were shown to provide favorable microenvironment to promote the differen-tiation of the encapsulated BMSCs into adipocytes in mice. Furthermore, the implanted constructs maintained their volume and architecture for 4 weeks [38]. Hybrid scaffolds which combine functionalities of two or mul-tiple material components have been applied as well. For example, combi-nation of placental decellular matrix (PDM) as the backbone for structural maintenance and cross-linked HA for achieving a high degree of vascular-ization led to a positive effect in terms of angiogenesis and adipogenesis in vivo [35] (Figure 2.2III).

Minimizing postoperation scars is extremely important in plastic and reconstructive surgery. Therefore, injectable hydrogels are preferred com-pared to sheet- or sponge-type scaffolds, which tend to leave scars due to the incision during transplantation. However, most injectable hydrogels are jel-lylike and are insufficient for providing desired rigidity. A possible solution might be the use of injectable microspheres (i.e., PLGA microspheres), which can load stem cells and exhibit enhanced mechanical strength [39]. In addi-tion, controlled degradation of hydrogels is vital for maintaining long-term integrity of adipose tissue in vivo. Rapidly degraded hydrogels (i.e., collagen) have been shown to lack the ability to support the growth of the adipose tissues during the 4 week implantation period, while slower-degraded bio-materials (i.e., silk) improved the long-term maintenance of the engineered adipose tissue [40].

2.3.3 bone regeneration

Birth defects, tumor resections, and serious injury can all lead to bone dam-age, which is normally repaired by transplantation of autologous bone grafts in clinical practice. However, the limited availability of autologous bone and the need for a secondary surgery to harvest the bone from the donor both cause critical problems [42]. Tissue-engineered bone grafts based on stem cells can potentially overcome these problems and offer better therapeutic strategies to repair bone defects. Adult BMSCs can easily differentiate into osteocytes; hence, they are the preferred cell source when engineering bone tissue [43].

To resemble the rigidity of bone, the scaffold utilized in bone tis-sue engineering is expected to present sufficient mechanical strength. Hydrogels, however, do not possess the mechanical properties to be used alone as scaffold in load bearing applications such as bone regeneration [1]. Therefore, most hydrogel-based applications in bone tissue engineering are in combination with stiffer materials such as bioceramics (e.g., coral and hydroxyapatite-tricalcium phosphate) [44]. In vitro studies combining hydroxyapatite with natural hydrogels such as collagen [45] or chitosan [46] have already showed promising results. The successful differentiation of MSCs to bone in vivo has been demonstrated utilizing different hydrogels

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(i.e., fibrin glue, alginate, collagen I) in combination with a stiff, porous scaffold made of tricalcium phosphate ceramic [42]. Extensive bone forma-tion particularly in collagen I hydrogel has been achieved as indicated by mineralization, secretion of bone-specific matrix, and gene expression of bone-specific markers. In this study, the stem-cell-loaded constructs were implanted into the nonload bearing subcutaneous dorsal pockets of nude mice, which is often regarded as a standard technique for evaluating the in vivo ossification. Since mechanical stimulation plays a critical role in bone formation [44], implantation into a load-bearing site to replace the function of bone is necessary to provide an appropriate mechanical stimulation for stem cell development. In another in vivo study, BMSCs were embedded in a bone-like composite consisting of three different materials (nanohydroxyap-atite, collagen, and PLA) and were implanted in a radial defect. The compos-ite showed an enhanced and accelerated bone formation in vivo compared to the use of fresh-frozen allogeneic bone [47].

The encapsulation of growth factors in hydrogels can further improve the formation of bone tissue. Kim et al. performed an in situ polymerization on an HA-based hydrogel to encapsulate human mesenchymal stem cells (hMSCs) and bone morphogenetic protein-2 (BMP-2) [48]. The histological results demonstrated that the hydrogel construct loaded with hMSCs and BMP-2 showed the highest expression of osteocalcin and mature bone forma-tion in vivo compared to hydrogels with only BMP-2 or hMSCs as well as the blank control (Figure 2.3I). Besides growth factors, the encapsulation of oxy-gen carriers in the hydrogel construct can additionally enhance bone forma-tion by supplying newly grown tissues directly with oxygen [49] (Tables 2.5 through 2.7).

2.3.4 Vascular regeneration

Engineering blood vessels for providing sufficient vascularization in engi-neered tissue constructs is one of the main objectives in vascular tissue

AQ4

(a)I II

1000 micron

1000 micron

1000 micron

1000 micron

1000 micron

Day 2 Day 7 Day 11

Day 151Day 22

(b)

(c)

(d)

(e)

Figure 2.3Hydrogel-mediated stem cell engineering for regenerating bone and vasculature.AQ5

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engineering. Endothelial progenitor cells (EPCs), which are committed to differentiate into endothelial cells, are one of the most widely studied cell source for vascular tissue engineering [51,52]. Meanwhile, the rapid advance-ment in using pluripotent ESCs and adult stem cells opens new possibili-ties for vascular regeneration. Both human ESCs and MSCs, for example, have the ability to differentiate into smooth muscle cells or endothelial cells,

Table 2.5

Hydrogel-Mediated Stem Cell Engineering for Bone Regeneration In Vivo


MSC PEGDA ImplantationDorsumImmunodeficient mice

Mandibular condyles formed de novo and showed cartilaginous and osseous phenotypes

[43]

BMSC PEO InjectionSubcutaneouslyExperimental mice

Hard nodes formed in mice with MSC

Hydrogel had been absorbed in mice without MSC

Histological examination showed trabecular bone and neocartilage

[50]

BMSC Nanohydroxyapatite/collagen/PLA (nHAC/PLA)

ImplantationRadial defectRabbits

nHAC/PLA with MSC enhanced and accelerated bone formation

[47]

MSC Fibrin glue, alginate, and collagen I hydrogel applied to β-TCP scaffolds

ImplantationSubcutaneouslyNude mice

Collagen I induced greater mineralization, secretion of bone-specific matrix, and gene expression of bone-specific markers than fibrin glue and alginate

[42]

hMSC HA with BMP-2 ImplantationCalvarial defectRats

BMP-2 and MSCs induced highest expression of osteocalcin and mature bone formation with vascular markers

[48]

MSC Fibrin gel with oxygen carriers

ImplantationSubcutaneously, radial bone defect, lumbar paravertebral muscle

C3H/HeN mice

Increase in bone formation, cell survival, and osteocalcin activity

[49]


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which are present in the media and intima, respectively, in the native blood vessels [36].

To regenerate vasculature from stem cells, it is often required to first differ-entiate the stem cells into endothelial cells in vitro followed by implantation in an animal model. The vasculogenesis is tightly regulated by both biochem-ical and biophysical cues, including growth factors, matrices, oxygen concen-tration (during hypoxia), and hydrodynamic shear [11,53]. Incorporation of growth factors into the hydrogel offers a facile method to induce the differ-entiation of stem cells into vascular lineage (i.e., endothelial cells). However, direct delivery of growth-factor-loaded hydrogels in vivo can potentially cause unwanted side effects, such as vascular leakage, due to uncontrolled release of the growth factors at the target site. Therefore, hydrogel-based stem cell delivery systems which can provide sustained stimulation of vasculo-genesis are preferable. For example, Kasper et al. demonstrated in vitro that mechanically stimulated MSCs can secrete more angiogenic growth factors which lead to enhanced vasculogenesis compared to unstimulated MSCs [54].

Vascular networks have been established in animal models originated from hydrogel constructs incorporated with hESCs-derived endothelial cells. Wang et al. coimplanted hESCs-derived endothelial cells and mouse mesenchymal precursor cells embedded in fibronectin-collagen hydrogel into mice. Functional vasculatures could be observed 11 days after implanta-tion, which were successfully integrated into the host vascular system and

Table 2.6

Hydrogel-Mediated Stem Cell Engineering for Vascular Regeneration In Vivo


hBMSC Alginate with VEGF ImplantationSubcutaneously or femoral artery ligation

SCID mice or NOD mice

Enhanced blood vessel formation in the presence of VEGF and mechanical stimulation

[11]

hESC Fibronectin-collagen ImplantationCranial windowSCID mice

Differentiation of hESCs into endothelial cells

Formation of branching blood vessels

Integration with host vasculature

[55]

hESC Matrigel® InjectionSubcutaneouslyNude mice

Differentiation of hESCs into endothelial and smooth muscle cells

Formation of human microvasculature

Functional integration with host vasculature

[56]


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maintained stable for more than 150 days (Figure 2.3II) [55]. In another study, Matrigel was used as scaffold for transplanting hESCs-derived endothelial-like cells into nude mice. The formation of nascent microvessels could be observed which contain mouse blood cells and support blood flow indicat-ing successful integration with the host circulation [56].

2.3.5 Hydrogel-Mediated Stem Cell engineering for regeneration of Other Tissues

Stem-cell-based tissue engineering can ideally regenerate all the tissue types. In this section, we provide examples on hydrogel-based stem cell

Table 2.7

Hydrogel-Mediated Stem Cell Engineering for Neural, Retina, and Muscle Regeneration


Nerve regeneration

NSC HA and collagen composite with neurotrophin-3

ImplantationFacial nerve damage

Rabbits

Repair of PNSReinnervations of damaged facial nerve on the defect site

[62]

Neural progenitor cell (NPC)

HA-heparin-collagen

ImplantationCortical photothrombotic stroke

C57BL/6 mice

Repair of CNSCells survived twofold in the hydrogel matrix compared to cells without hydrogel

Hydrogel reduced infiltration of inflammatory cells by forming zone around transplanted stem cells

[70]

Retina regeneration

Retinal stem-progenitor cell (RSPC)

HAMC InjectionMice

Cells survived better and distributed evenly in subretinal space in mice

[71]

Mouse ESC Matrigel® No in vivo 3D neural retinal tissues self-formed in vitro

[75]

Muscle regeneration

MuSC PEG ImplantationImmunodeficient mice

Substrate elasticity is a critical factor of MuSCs fate in culture

MuSCs cultured on hydrogel substrates mimicking the rigidity of native muscle self-renewed in vitro and regenerated novel muscle tissues in vivo

[76]


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engineering for nerve, retina, and muscle regeneration. Although, only pre-liminary results have been obtained, the great diversity of applications in this field demonstrates its great potential for regenerative medicine in the future.

2.3.5.1 Nerve Regeneration

The nervous system, consisting of the central nervous system (CNS) and peripheral nervous system (PNS), possesses different abilities for self-repair. Neurons of the PNS exhibit a greater capacity to regrow than those of the CNS after damage.

For PNS repair, formation of new axons across the injury site is the key requirement [57]. A graft is needed to bridge the transected nerve when the injury is extensive. Autologous nerve grafts (autografts) have been widely used and considered as the gold standard in clinical surgery for PNS regen-eration. However, several limitations still exist for autografts, such as extra incision, sacrifice of the donor nerve, and the risk of neuroma formation [58]. Therefore, synthetic grafts, namely, nerve conduits, provide a promising alternative to autografts [59]. Both natural and synthetic hydrogels (e.g., HA, chitosan, laminin, or PEG) were fabricated into nerve conduits and were proved effective for guiding and supporting the ingrowth of novel neurites from transected nerves [59–61]. Furthermore, hydrogels incorporating mul-tipotent neural stem cells (NSCs), which have the potential to differenti-ate into neurons, astrocytes, and oligodendrocyte [62], showed enhanced PNS regeneration compared to hydrogels alone. For example, Zhang et al. encapsulated NSCs in an HA-collagen hydrogel conduit and achieved bet-ter reinnervation of damaged facial nerve compared to blank hydrogel in a rabbit model.

In contrast, for CNS repair, it is nearly impossible to restore the lost func-tions which usually lead to CNS degenerative disorders such as Parkinson’s and Alzheimer’s diseases. Regeneration in the CNS based on hydrogels alone usually shows limited efficacy [63,64], and it requires multiple cell types to function synergistically. Therefore, the use of multipotent NSCs [62], which can be harvested from multiple regions in mammalian brain at different ages [65,66], shows great promise in CNS regeneration. Transplantation of NSCs alone was proved to promote functional restoration in animal models [67–69]. However, the transplanted cells integrated poorly into the adult CNS due to the lack of various bioactive cues which induce cell migration and formation of new axons or synapses [61]. Hydrogels incorporated with bio-active cues can serve as stem cell niche for CNS repair. For example, Zhong et al. [70] fabricated HA hydrogel containing bioactive heparin sulfate and encapsulated NSCs to form a stem cell-hydrogel complex. High survival of embedded cells in vivo was demonstrated, resulting in regrowth of nerve tissues in a mouse stroke model (Figure 2.4I).

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2.3.5.2 Retina Regeneration

Vision loss, often caused by the damage of the retina (i.e., retinitis pig-mentosa, diabetic retinopathy, age-related macular degeneration), has a big impact on the quality of life and can affect the entire age spectrum [71]. The present therapy toward retinal degeneration focuses on pharmacolog-ical treatments, but this approach is unable to restore the impaired retina. An alternative strategy based on cell therapy relies on transplantation of retinal cells (fetal retinal pigmented epithelium cells and neural retinal cells) directly to the damaged regions of the retina [72–74], which usually suffers from poor cellular survival, distribution, and tissue integration. Ballios et al. overcame these limitations by developing a hyaluronan and methylcellulose hybrid hydrogel (HAMC) as stem cell delivery system, which was biodegradable and easily injectable in a minimally invasive manner [71]. This novel hydrogel-based delivery system supported retinal stem cell survival and proliferation in vitro and enabled contiguous dis-tribution of stem cells in vivo with reduced cell aggregation (Figure 2.4I). Another study conducted by Eiraku et al. heralded a new therapeutic method for regenerating damaged retina [75]. An optic cup structure was reconstructed in 3D Matrigel system using mouse ESC aggregates, and fully stratified 3D neural retinal tissue sheets were formed spontaneously in vitro. The formation of retinal sheets in vitro recapitulated the com-plex morphogenesis of retinal anlage in vivo (Figure 2.4II). Therefore, this approach opens novel avenue to create retinal tissue using stem-cell-based tissue engineering.

NPCs/No hydrogel

I II

InfarctInfarct

DAPI

GFP-labeled NPCs

(a) (c)

(d)(b)

20 µm

20 µm 20 µm

RPE

RPE

BM

BM/RPE BM

20 µm

NPCs/ Hydrogel

Figure 2.4Hydrogel-mediated stem cell engineering for regenerating nerve and retina. (From Zhong, J. et al., Neurorehabil. Neural Repair, 24(7), 636, 2010; Ballios, B.G. et al., Biomaterials, 31(9), 2555, 2010.)

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2.3.5.3 Muscle Tissue Regeneration

Treatment of muscle degenerations, such as muscular dystrophy, requires new muscle tissue formation. Muscle stem cells (MuSCs) are an ideal source to regrow muscle tissue. However, this ability is lost when MuSCs are expanded in vitro for clinical use. Gilbert et al. [76] found that substrate elas-ticity was a critical factor to regulate MuSC self-renewal in vitro culture by varying the rigidity of the PEG hydrogel substrate. MuSCs expanded with high efficiency in vitro when cultured on hydrogel substrates mimicking the elasticity of native muscle and contributed extensively to muscle regenera-tion when subsequently transplanted into mice.

2.4 summary and outlook

Hydrogels provide a versatile and robust platform to maintain stem cell self-renewal and induce lineage-specific differentiation. As an ideal matrix component for stem cell niche, hydrogels can be easily customized by incor-porating biochemically functional moieties or adjusting mechanical proper-ties and physical states. Hydrogel-mediated stem cell engineering has been proved to be effective in regenerating various tissue types (e.g., cartilage, adi-pose, bone, vascular, and nerves) in vivo mainly in animal models. Clinical trials involving both hydrogel and stem cell components have been also con-ducted for soft tissue regeneration. Despite the great potential in this field, a number of challenges are faced related to common problems involved with stem cell therapy, such as how to achieve efficient and reliable differentiation of stem cells with high specificity, how to prevent the risk of teratocarcinoma formation and unwanted immune responses [77], and how to integrate the regenerated tissue with the host tissues. The encapsulation of stem cells in hydrogels might be beneficial to overcome these challenges by providing microenvironment to locally improve the differentiation efficiency and as a protecting zone to isolate from the immunocytes [70] as well as facilitating the remodeling of the regenerated tissue. With the future advancement in this field, more in vivo studies are expected to conduct using hydrogel-mediated stem cell engineering for regenerating various tissues. And their therapeutic efficacy and long-term safety will be ultimately assessed in clinical trials.

Glossary of terms

ASC adipose-derived stem cellBMP bone morphogenetic protein

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BMSC bone-marrow-derived mesenchymal stem cellCNS central nervous systemECM extracellular matrixEPC endothelial progenitor cellFGF fibroblast growth factorGAG glycosaminoglycanHA hyaluronic acidhESC human embryonic stem cellhMSC human mesenchymal stem cellIGF insulin-like growth factoriPSC induced pluripotent stem cellMSC mesenchymal stem cellMuSC muscle stem cellnHAC nanohydroxyapatite/collagenNPC neural progenitor cellNSC neural stem cellPCL polycaprolactonePDM placental decellular matrixPEG polyethylene glycolPEGDA polyethylene glycol diacrylatePEO polyethylene oxidePGA polyglycolic acidPLA poly lactic acidPLGA poly(lactic-co-glycolic acid)PNS peripheral nervous systemTCP tricalcium phosphate ceramicTGF transforming growth factorVEGF vascular endothelial growth factor

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AQ10

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AUTHOR QUERIES[AQ1] Please check the inserted shortened running head for correctness.[AQ2] The term “cartilage-specific makers” has been changed to “cartilage-

specific markers.” Please check if appropriate.[AQ3] Please define part labels “a–d and I to II” in the caption of Figure 2.2.[AQ4] Please check the citation provided for Tables 2.5 through 2.7 and also

check the placement of the tables.[AQ5] Please define part labels “a–e and I to II” in the caption of Figure 2.3.[AQ6] Please define part labels “a–d and I to II” in the caption of Figure 2.4.[AQ7] Please provide publisher and location details for ref. 7 and update

the source lines of Tables 2.1 and 2.2 accordingly.[AQ8] Please provide complete details for Ref. 22.[AQ9] Please check in-text citation for Ref. 41.[AQ10] Please confirm the page range for refs. 72 and 75.

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