Biomaterials for stem cell differentiation

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ews 60 (2008) 215–228www.elsevier.com/locate/addr

Advanced Drug Delivery Revi

Biomaterials for stem cell differentiation☆

Eileen Dawson, Gazell Mapili, Kathryn Erickson, Sabia Taqvi, Krishnendu Roy ⁎

Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712, USA

Received 31 July 2007; accepted 11 August 2007Available online 11 October 2007

Abstract

The promise of cellular therapy lies in the repair of damaged organs and tissues in vivo as well as generating tissue constructs in vitro forsubsequent transplantation. Unfortunately, the lack of available donor cell sources limits its ultimate clinical applicability. Stem cells are a naturalchoice for cell therapy due to their pluripotent nature and self-renewal capacity. Creating reserves of undifferentiated stem cells and subsequentlydriving their differentiation to a lineage of choice in an efficient and scalable manner is critical for the ultimate clinical success of cellulartherapeutics. In recent years, a variety of biomaterials have been incorporated in stem cell cultures, primarily to provide a conducivemicroenvironment for their growth and differentiation and to ultimately mimic the stem cell niche. In this review, we examine applications ofnatural and synthetic materials, their modifications as well as various culture conditions for maintenance and lineage-specific differentiation ofembryonic and adult stem cells.© 2007 Elsevier B.V. All rights reserved.

Keywords: Embryonic stem cells; Adult stem cells; Polymers; Metals; Ceramics; Bioreactors; Differentiation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2152. Biomaterials used for studying stem cells in 3-D culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

2.1. Scaffolds in cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2162.2. Natural materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2162.3. Synthetic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

2.3.1. Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2182.3.2. Ceramics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2182.3.3. Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

3. Modifications to biomaterials for stem cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2193.1. Substrates with altered mechanical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2193.2. Modifications with chemical and biological stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2203.3. Patterned biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on“Emerging Trends in Cell-Based Therapeutics”.⁎ Corresponding author. Department of Biomedical Engineering, The

University Texas at Austin, ENS 610, C0800, 1 University Station, Austin,TX 78712, USA. Tel.: +1 512 232 3477.

E-mail address: [email protected] (K. Roy).

0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2007.08.037

1. Introduction

Current therapies in modern medicine mostly involveprevention, manipulation and control of diseases through

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216 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228

chemical or biological molecules. More recently, the restorationand direct replacement of diseased cells and tissues arebecoming a clinical possibility in large part due to paralleladvances in modifying biomaterials and understanding stemcell (SC) behavior [1]. Complete tissue and organ replacementusing stem cells is still a distant milestone in which currentstudies are laying the necessary groundwork. Recent reviewarticles have discussed how certain types of materials are usedas substrates to mimic the physico-chemical microenvironmentsof cells and tissues [2,3]. Several papers have also carefullyexamined materials that have been used to specifically study thedevelopment of bone, cartilage, and skin [4–8]. Others havereported studies on multiple pre-differentiated stem cellpopulations and how to combine them together usingbiomaterials to form hybrid constructs that closely mimicnative tissue [9,10]. In this review, we focus exclusively onbiomaterials and how they are revolutionizing studies in tissueand organ replacements and providing a greater comprehensionof the underlying mechanism behind stem cell behavior anddifferentiation within their physiological niche.

Stem cells are simply defined as a progeny of cells that havethe potential to differentiate into a variety of different lineages.These cells can be isolated from a variety of sources includingembryos, umbilical cord blood, as well as from adult tissues, asreviewed previously [11]. Since the isolation of mouseembryonic stem cells (mESCs) in 1981 by David and Kaufman,and the subsequent isolation of human embryonic stem cells(hESCs) in 1998, their exploration in regenerative medicinehave received great interest. Embryonic stem cells (ESCs) areisolated from the inner cell mass of the blastocyst duringembryological development [12,13]. Their pluripotent naturegives them the ability to differentiate into any one of the threegerm layers: endoderm, ectoderm, and mesoderm. ESCs alsohave the unique property of indefinite self-renewal i.e. they canbe cultured and maintained in an undifferentiated, pluripotentstate. Thus, ESCs have the potential of providing the biomedicalcommunity with a continuous source of all cell types [13].

Although ESCs are attractive because of their pluripotency,they are also difficult to work with because of this samecharacteristic. Maintaining large number of ESCs in an undiffer-entiated state and subsequently directing them to differentiate, in areliable and reproducible manner, into specific cell types are theforemost complications in ESC-based cell therapy [14]. If ESCsremain undifferentiated after implantation in the body, they willspontaneously differentiate into multiple cell types and form atype of tumor called teratoma [13,15]. To avoid teratomaformation, ESCs must be guided to differentiate into particularlineages prior to implantation [13]. On the other hand, ultimatelarge scale therapeutic application of these cells necessitates theirmaintenance in an undifferentiated state in vitro so that they can bedifferentiated into specific lineages on-demand.

It is now well established that adult tissues carry a variety ofadult stem cells that are less pluripotent and are more committedthan ESCs. Adult stem cells are often referred to as progenitoror multipotent cells since they have limited differentiationpotential. These cells have been found in bone marrow, cordblood, adipose tissues, neural tissues etc. Bone-marrow derived

progenitor cells or mesenchymal stem cells (MSCs), hemato-poietic stem cells (HSCs) from both bone marrow and cordblood as well as adipose-derived stem cells have becomeattractive cell populations for the field of tissue engineeringbecause of their ability to differentiate into variety of cell typesand relative ease in harvest, isolation and expansion in vitro.

In this review we have focused exclusively on the use ofbiomaterials, both natural and synthetic, to maintain anddifferentiate stem cells. Both adult and ESCs are discussed. Amajor focus of this review is on the chemical and biologicalmodification of materials to better mimic the stem cell niche andcreate microenvironments to control stem cell response.

2. Biomaterials used for studying stem cells in 3-D culture

2.1. Scaffolds in cell culture

Although classical 2-D cell cultures on flat surfaces haveprovided us with majority of our knowledge in modern biology,it is now well accepted that cells (including SCs) reside,proliferate and differentiate inside the body within complex 3-Dmicroenvironments. Most of the current research in biomaterial-directed SC manipulation is focused on such 3-D environments.Hence this review will primarily focus on materials andconcepts that involve 3-D culture of SCs.

Biomaterial-based scaffolds have been the most important toolin providing a 3-D environment to cells, both in culture or insidethe body. These 3-D structures provide an ideal platform for cell–cell and cell–material communications and their properties can bevaried to promote differentiation of cells into specific lineages.Scaffolds for tissue engineering serve numerous functions andtheir role during tissue development is dependent upon specificproperties of the chosen biomaterial. 3-D systems have proven toenhance osteogenic [16], hematopoietic [17], neural [18], andchondrogenic [19,20] differentiation. They serve as biointeractivestages promoting cell attachment, proliferation, and organization,in addition to acting as delivery vehicles for bioactive moleculesduring tissue formation.

Properties of biocompatible scaffolds, synthetic or natural, thatmust be taken into careful consideration include optimal fluidtransport, delivery of bioactive molecules, material degradation,cell-recognizable surface chemistries, mechanical integrity andthe ability to induce signal transduction. The overall success oftissue organization and development is highly dependent uponthese properties, since they can ultimately dictate cell adherence,nutrient/waste transport, matrix synthesis, matrix organization andcell differentiation. Most scaffolding materials can be chemicallyand physically modified to adjust all of these critical parameters,and a variety of synthetic and natural materials have been used forstudying SC behavior through specifically manipulating theseproperties. Several articles have reviewed the application ofscaffolds in tissue engineering in general [21,22].

2.2. Natural materials

Natural biomaterials used for developing scaffolds canconsist of components found in the extracellular matrix

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(ECM), such as collagen, fibrinogen, hyaluronic acid, glycosa-minoglycans (GAGs), hydroxyapatite (HA) etc., and thereforehave the advantage of being bioactive, biocompatible, and ofsimilar mechanical properties as native tissue. Other naturalmaterials include those derived from plants, insects or animalcomponents (e.g. cellulose, chitosan, silk fibroin etc.) withproperties to provide favorable microenvironments for SCculture. Disadvantages of using natural materials over syntheticmaterials include limited control over physico-chemical prop-erties, difficult to modify degradation rates, difficulty insterilization and purification as well as pathogen/viral issueswhen isolating from different sources. However, in recent years,several natural materials, e.g. chitosan, hyaluronic acid, silk etc.have become commercially available, are well characterized,and have reproducible, controlled properties.

Matrigel™, a product currently available commercially, iscomprised of a variety of ECM components including laminin,collagen IV, and heparan sulfate proteoglycans [23,24] and hasbeen used extensively in cell culture [23,25]. In addition toMatrigel's application in normal tissue culture, when injectedwith cells isolated from the stromal vascular fraction (SVF) ofadipose tissue (i.e. adipose-derived SCs), improved neovascu-lature formation was observed in an ischemic mouse model[26]. Further studies indicated that cells pre-cultured underproper conditions and then injected with Matrigel could producetubelike vascular structures [26].

Fibrinogen and fibrin are another class of tissue-derived naturalmaterials that can be utilized to create three-dimensional scaffoldmaterials [27]. Fibrin scaffolds have been optimized for neuraldifferentiation of mESCs [18]. These scaffolds, in conjunctionwith various growth factors showed significant increase in neuronand oligodendrocyte production as well as neuronal viability overtime [28]. Suggs and colleagues have also demonstrated the use offibrin as a material for mouse ESC culture [29]. Chondrogenesispotential of MSCs derived from adipose tissue and bone marrowhas also been evaluated in fibrin gels, and while both cell typesdemonstrated cellular functions indicative of chondrogenicdifferentiation, bone marrow derived MSCs appeared to producedifferentiated cells that were more efficient in both collagen IIproduction as well as proteoglycan synthesis [27].

Hyaluronic acid is a highly attractive natural biomaterial due toits participation in cell behavior and cell signaling. It is present intissue as a gel-like substance but can be chemically modified forefficient processing into fibers, membranes, or microspheres. Amodified type of hyaluronic acid is commercially available asHyaff® [30]. Hyaff®-based scaffolds are biodegradable andcombine both the benefit of having a 3-D microenvironmentcomprised of a natural material while allowing for cells to replacethe scaffold with their own ECM. Recently Gerecht et al. reportedthe use of hyaluronic acid hydrogels for maintaining thepluripotency and undifferentiated state of hESCs [31], andshowed that the addition of soluble growth factors to thesehydrogels successfully triggers lineage specific differentiation ofthese hESCs. In addition, MSCs grown on this type of scaffolds,modeled to resemble a tendon, have been shown in vitro toexpress a variety of ligament proteins while suppressingindicators of bone and cartilage differentiation [32].

Silk fibroin is another versatile natural material that isisolated from silkworm cocoons. Silk has been developed intoporous scaffolds using gas foaming or salt leaching methods[33–35] and is widely used as suture materials for surgicalapplications. The use of silk as a biomaterial for SC culture hasbeen reviewed recently by Kaplan and colleagues [36].

Other natural materials used as scaffolds for studies in SCdifferentiation include chitosan, HA, alginate and coralline.Chitosan, isolated and processed from crustacean shells, ishydrophilic in nature, biodegradable, biocompatible, and hassimilar properties to GAGs. One particular study combinedchitosan with coralline, exoskeletons of marine species or coral,as a composite scaffold to study MSC-osteogenesis since coralis composed of calcium carbonate, a component found in bone[37]. Another material that has been extensively used as acomposite for bone tissue engineering, both from osteoblasts aswell as MSCs, is HA, a natural, inorganic component of bonemineral [38]. HA has the ability to encourage bone in-growthand the inclusion of HA particles in a nanostructured self-assembling peptide scaffold, encourages osteogenic differenti-ation of mESCs [39]. Alginate (derived from algae cell walls) isa natural polysaccharide that has been evaluated for theencapsulation and differentiation of ESCs [40]. mESCsencapsulated in alginate poly-L-lysine (PLL) was shown tosupport cell proliferation [41]. Moreover, this microencapsu-lated structure prevented embryoid body (EB) formation andpromoted differentiation towards a hepatic lineage without theneed for EB formation [41].

By crosslinking pullulan, dextran, and fucoidan at a ratio of71:24:5 with a total concentration of 25% (w/v), homogenous3-D hydrogels could be created and stored for up to 4 weeks[42]. CD34+ human umbilical cord blood cells (hUCBCs)previously differentiated towards the endothelial lineage,CD133+ human bone marrow cells (hBMCs) also differenti-ated into endothelial cells, and mature endothelial cells that hadbeen isolated from human saphenous veins, were all culturedon this novel hydrogel [42]. While the scaffolds appear tosupport cell adhesion, and lack cytotoxicity, further experi-ments need to be performed before in vivo applications invascular tissue repair can be determined [42].

A classic natural material for tissue engineering is collagenand its derivatives. Type II collagen, isolated from bovinecartilage, has been used to culture human mesenchymal stemcells (hMSCs) in pellet form when combined at the ratio of1.25 mg type II collagen per 2.5⁎105 cells (altering the ratio ofcollagen altered the structural integrity of the pellet) [43]. ThehMSCs cultured under these conditions not only assembled butwere able to reorganize the pellet structure as shown by thedegree of matrix contraction [43]. In vivo results indicate thatthat this type of 3-D culture can provide a system capable ofmaintaining chondrogenesis [43]. Collagen microbeads havealso been studied for the expansion of hUCBCs and shown tonot only improve expansion, as compared to a 2-D system, butalso improve overall cell viability [44]. Though these resultswere not statistically significant, as compared to the 2-D system,the cells isolated from these microbeads demonstrated morefavorable clonogenic ability, suggesting that collagen-beads


maintained cell hematopoietic functions for a longer duration inculture [44]. Additional in vivo results suggested that cellsmaintained within microbeads had the ability to engraft into thebone marrow of NOD/SCID mice even after 12 days of culture,whereas 2-D cultured hUCBCs failed to engraft in three out offour mice [44].

2.3. Synthetic materials

2.3.1. PolymersPolyglycolic acid (PGA), polylactic acid (PLA) and the

copolymer polylactide-co-glycolide (PLGA) have been exten-sively used as synthetic 3-D scaffold materials for evaluating cellbehavior [45,46]. These materials are hydrolytically degradablethrough bulk erosion due to the presence of ester bonds, and theglycolic/lactic acid byproducts are physiologically removed viametabolic pathways. Polymer molecular weight, copolymeriza-tion ratio, and polydispersity can be easily adjusted to control thedegradation rate, making these attractive synthetic materials fortissue engineering. Furthermore, standard methods (e.g., saltleaching, sintering, porogen melting, and nanofiber electrospin-ning) have been well established to prepare a wide variety of 3-Dscaffolds using these materials [45,47,48].

These scaffolds have been shown to support human ESCgrowth as well as encourage 3-D tissue-like organization [49]. Byusing this type of degradable scaffold, it may be possible to allowfor the structural support necessary to control growth of ESCswhile allowing the efficient transport of nutrients and othergrowth factors. As ESCs form a 3-D tissue structure, the scaffoldsdegrade, leaving only the differentiated cells. In such a system,using appropriate growth cues, it may be possible to differentiateESCs into a number of different tissues without altering the initialmatrix material [49]. In our laboratory we have demonstrated thatthe physical properties of the scaffold structure e.g. varying poresizes and polymer composition significantly influence mESCdifferentiation into the hematopoietic lineage. Specifically,scaffold porosity was found to be inversely related to ESChematopoiesis with an optimal pore size of less then 150 μm [50].Hematopoietic progenitor cell (HPC) formation was also shownto be directly proportional to polymer concentration in thescaffold with optimal concentration being 20% w/v.

Non-woven fabrics developed from polyethylene tereptha-late (PET), another widely used synthetic polymer, have beenstudied with MSCs and human cord blood-derived HSCs toevaluate seeding, proliferation, and aggregation for tissueregeneration [51–53]. Nanofibrous scaffolds fabricated usingpoly(ɛ-caprolactone) (PCL) have also been studied foradipogenesis, chondrogenesis, and osteogenesis [54,55].These electrospun polymers produce an interconnected porousmatrix capable of encouraging significant cell proliferation andcell–cell interactions in both MSCs and mESCs [56]. Theintroduction of adipogenic hormones into the system, resultedin specific expression of a transcriptional factor, PPAR-γ,associated with adipocyte differentiation [56].

Acrylated polymers that form hydrogels, such as poly(ethylene glycol) diacrylates (PEGDA) and its derivatives, poly(6-aminohexyl phosphate acryloyl) ((PPE-HA)-acryl) and

acrylated polyanhydrides or polyesters have been widelystudied for MSC and ESC cultures [57–59]. EBs frommESCs encapsulated in a poly(ethylene glycol) (PEG) hydro-gel, and exposed to transforming growth factor beta (TGF-β),have been shown to up regulate the expression of chondrogenicmarkers [19]. Furthermore, chondrogenic differentiation ofmESCs using PEG-diacrylate hydrogels can be augmented withthe addition of glucosamine, an amino monosacchaaride presentin GAGs [20]. Glucosoamine is known to increase theproduction of chondrogenic proteoglycans; therefore its addi-tion to the hydrogel structure may increase the mechanicalproperties of the scaffold by synthesizing new ECM [20].Recently Healy and colleagues demonstrated that photocros-slinked hydrogels incorporating poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAAm-co-AAc)] and an acrylated matrixmetalloproteinase sensitive peptide Gln-Pro-Gln-Gly-Leu-Ala-Lys-NH2 (QPQGLAK-NH2) can support short term selfrenewal and maintenance of hESCs [60].

Though synthetic materials provides the versatility ofcreating 3-D microenvironments with tunable features (i.e.,mechanical properties, degradation rates, porosities), disadvan-tages for choosing such materials include poor inherentbioactivity (e.g. PEG), acidic by-products (e.g. PLA orPLGA), etc. It is thus critical to modify synthetic materialswith biological or chemical entities to achieve appropriatecellular response (discussed in Section 3).

2.3.2. CeramicsCalcium phosphates, bioactive glasses, and other biocera-

mics are desirable materials for studying SC differentiation,especially osteogenesis since these materials have favorablemechanical properties and can integrate with bone to a higherdegree than soft biomaterials, thereby enhancing mineralizationand matrix formation. Biphasic calcium phosphate ceramics canbe commercially purchased (Triosite™) and MSCs grown onthis material demonstrated continued osteoblastic phenotypicproperties even after an entire month of culture [61]. The use ofceramics in tissue engineering has been extensively reviewed inseveral articles [62,63].

2.3.3. MetalsTitanium, an attractive material due to its inertness and

biocompatibility, is used widely in orthopedic and dentalsurgery [64]. The combination of titanium along with theosteogenic differentiation potential of MSCs makes for anattractive bone regeneration material. MSCs are able to attachand proliferate on titanium dishes [64]. Furthermore, afterappropriate differentiation media is applied to the cultures,titanium scaffolds are able to exhibit signs of bone matrixformation [64]. Titanium fiber meshes seeded with rat MSCshave been cultured in a perfusion bioreactor to study effects onosteoblastic differentiation [65]. Levels of mineralizationformed by osteoblasts within the scaffolds were comparedbetween plain titanium meshes and pre-generated bone ECM-deposited titanium meshes. Results indicated that synergisticeffects of both mechanical stimulation through shear stress andthe presence of ECM deposition onto the scaffold substrate


profoundly enhanced osteoblastic differentiation. Layer bylayer (LbL) nano-assembly, a technique which allows for thesurface modification of materials can fabricate layered coatingsto modify any substrate material. Using nanoparticles of TiO2 itis possible to produce a thin surface film which increases theattachment of MSCs by creating a rougher surface [66].Titanium nitride (TiN) can also be deposited on a surface inthe form of a thin film by using a DC magnetron sputteringtechnique [67]. The TiN coating encouraged hMSC adherenceas compared with traditional implant material (TiAlV) [67].

Another metal based material suitable for SC differentiation isCytomatrix™, a tantalum based scaffold material. In studiesperformed in our laboratory, Cytomatrix™ has been shown to beable to effectively induce EB formation in mESCs in both staticconditions and dynamic culture conditions [17]. As compared to a2-D culture, the EBs formed on Cytomatrix™ appeared to havean ECM-like structure that covered not only the EB itself, butextended onto the surface of the scaffold indicating both cell–celland cell–matrix interactions [17]. Additionally, EBs formed onthe Cytomatrix™ scaffolds appeared to be of a smaller size,avoiding the formation of aggregates usually associated with 2-Dcultures [17]. HSC generation in 3-D cultures, especially thosecultured under dynamic conditions, was significantly higher ascompared to 2-D culture and revealed a higher potential forfurther differentiation into the myeloid lineage [17]. ExtensivecDNA microarray analysis of mESCs differentiated in 2-D aswell as 3-D static and dynamic conditions demonstratedsignificant differences in gene expression [68]. Cells differenti-ated in the different culture conditions expressed genes unique totheir scaffold environment as well as to their culture conditions[68].

3. Modifications to biomaterials for stem cell culture

Chemical and biological modifications to biomaterials candirectly influence SC behavior by altering substrate properties,surface interactions, scaffold degradation rate, microenviron-ment architecture and ultimately manipulating the signaltransduction pathways in SCs. Biomaterials can be designedto fine-tune their degradation kinetics, present specific ligand-based signals and/or release of biological molecules in responseto their microenvironment. These influence cell–matrix inter-actions and should lead to altered gene expression and lineagespecificity. Numerous studies have demonstrated how modifiedbiomaterials and scaffold surfaces introduce specific biologicalresponses in SCs. Ultimately, the goal of biomaterial-directedSC culture is to mimic the properties, both physical andbiochemical, of the physiological SC niche. This sectionprovides a comprehensive review of the various concepts inmodifying biomaterials for maintenance and differentiation ofSCs.

3.1. Substrates with altered mechanical properties

The intrinsic mechanical properties of a biomaterial are ofsignificant interest since they influence the forces exerted bycells on their substrates. For instance, chondrocytes exert

contractile forces when integrated onto substrates and canchange the overall microstructure of the scaffold during tissuedevelopment depending on the mechanical properties of thematerial [69,70]. Recently Discher and colleagues elegantlydemonstrated the importance of substrate mechanical propertieson SC differentiation using MSCs as a model [71]. Thereforethe overall mechanical integrity of scaffolding materials is a keyelement that needs to be addressed when evaluating materialproperties that effect differentiation pathways of SCs.

Bone marrow-derived MSCs are highly sensitive andresponsive to mechanical stimulation in vitro [71,72]. It isspeculated that mechanical stimuli activates cell surfacereceptors and focal adhesion sites, which in turn triggersintracellular signaling cascades. This leads to specific geneactivation that modulates ECM secretion. MSC differentiationis influenced both by their physical microenvironment e.g. bymechanical cyclic stresses applied directly to the cells [73] aswell as by the inherent biomaterial properties (i.e. materialintegrity, crystallinity, crosslinking density, overall micro- andmacro-porosity etc.).

Substrates such as natural or synthetic hydrogels, closelyresemble the consistency of soft, native tissues, making themattractive scaffold materials for soft tissue engineering. On theother hand, MSC differentiation into connective tissue lineages(i.e. bone, cartilage, ligaments, and tendons) require materialswith higher mechanical strength to closely mimic the tissuemechanical properties. However, hydrogel-like materials, canbe modified to have increased modulus of elasticity, makingthem more suitable for applications in connective tissueengineering. For example, collagen gels can be adjusted tohave a higher modulus by adding HA, thereby mimicking thecomposition of bone which is mostly composed of collagenfibers and phosphate minerals. Adding HA to collagen at a 1:1ratio increases the modulus from 0.392 MPa to 0.422 MPa,which is comparable to trabecular bone (E=0.443 MPa) [74].Other studies have fabricated collagen composites to containPLA and chitin fibers to provide increased mechanical integrityand have demonstrated higher human MSC attachment [75].

Silk-based materials have also been commonly used forMSC culture [76–78]. Silk exhibits higher modulus of elasticityover other natural materials, such as collagen. However de-contamination and purification methods of silk, prior to theiruse in vivo, are extremely critical in order to avoid inflammatoryand immunogenic reactions. Silk-based scaffolds seeded withhMSCs were shown to induce bone formation in critical-sized,cranial defects (larger than 4 mm) of nude mice, indicated by thepresence of bone sialoprotein, osteopontin, and osteocalcin[76]. Furthermore, efficient cartilage formation was also seenwhen differentiating MSCs into the chondrogenic pathwaywithin silk scaffolds [35].

In addition to natural materials, synthetic materials can also bechemically modified to enhance mechanical properties. Forexample, simply increasing the macromer concentration in photo-crosslinked hydrogel scaffolds has been shown to increase themodulus of elasticity, such as within (PPE-HA)-acryl hydrogels[57]. A four times increase in the amount of acrylated-PEGreacted with PPE-HA showed an almost 10-fold increase in shear


modulus (3 to 26 kPa) [57]. Additionally this biodegradable,phosphate-based synthetic material is highly conducive to bonetissue engineering due to the phosphate degradation product,which could aid in overall scaffold mineralization duringosteogenesis [57].

Scaffolds have been increasingly applied to study thedifferentiation of ESCs. While most studies focus on fluidtransport and other material properties of the scaffold, recentstudies suggest that the mechanical stiffness may also play a keyrole in controlling differentiation [49,50,82]. Studies reported byBattista et al. using scaffolds of collagen, fibronectin (FN) andlaminin, have indicated that alteration of mechanical propertiesof scaffolds can drastically alter EB formation. When the elasticmodulus was increased from 16 to 34 Pa the formation of EBswas severely inhibited suggesting that the increase in elasticmodulus resulted in an inhibition of apoptosis [82]. A secondpossible explanation may be that the denser network of thesestiffer gels may have altered ESC growth. In our lab, however,we were able to show similar trends with HSC differentiation[50]. Our results indicated that scaffolds with higher moduluswere more conductive towards HSC generation.

Although not a focus of this review, MSC and ESCdifferentiation induced by mechanical stimulation has alsobeen extensively investigated using bioreactors [65,79–81], orin vitro systems that provide dynamic culturing conditions.Cells in vivo consistently undergo fluid shear stress andmechanical strain, thereby influencing cellular interactions andresponses. In our lab, the significance of mimicking this in vivoenvironment was demonstrated with the differentiation ofmESCs into HSCs. Cytomatrix, a porous, tantalum-basedbiocompatible scaffold was used to culture mESCs [68]. It wasshown that in comparison to 2-D culture, the 3-D tantalumscaffold was more efficient at promoting HSC formation [68].Furthermore, when combining 3-D environment with a dynamicculture in a spinner flask, the efficiency of HSC formation waseven greater (1.4–2.2 times) than that of 3-D culture alone [68].

3.2. Modifications with chemical and biological stimuli

SC differentiation can be directly mediated by presentingappropriate biological or chemical signals in their microenvi-ronment. It is well established that specific growth factors,hormones and cytokines can enhance proliferation and lineage-specific differentiation of SCs. For example, fibroblast growthfactor-2 (FGF-2) has been shown to increase self-renewal ofMSCs and maintenance of their multi-lineage differentiationpotential [83–86]. Additionally, bone morphogenetic proteins(BMPs), have shown to have significant role in the regenerationof skeletal tissues, especially bone [87,88].

Propagation of ESCs without a feeder layer can bemaintained in the presence of a variety of cytokines includingleukemia inhibitory factor (LIF), stem cell factor (SCF), andFMS-like tyrosine kinase 3 ligand (Flt3-ligand). Recently it wasalso demonstrated that FGF-2 could allow long term selfrenewal of hESCs and maintain their pluripotent status [89,90].Addition of other growth factors can induce ES differentiationand ultimately formation of a variety of different tissue types.

These growth factors include retinoic acid (RA), activin-A,TGF-β, and insulin-like growth factor 1 (IGF-I). Activin-A hasbeen shown to promote the differentiation of mESC derivedEBs towards the endoderm germ layer [91]. Introducing activin-A into EB cultures has been used in creating lung epithelialprogenitor cells [91]. Both RA and TGF-β are involved inpancreatic formation [92]. The addition of RA and activin-Ahave demonstrated success in promoting in vitro differentiationof mESCs into α, β, γ, and δ cells, all of which are pancreaticendocrine cells [92].

Growth factors, hormones, and chemicals have classicallybeen directly added into the culture medium. More recentlythese bio-molecules have been directly incorporated within thescaffold structure or into the scaffold biomaterial in a variety ofways. Fig. 1 provides a schematic representation of some of themethods used in incorporating these molecules into a 3-Dscaffold. Soluble growth factors can be directly encapsulated orincorporated during the scaffold fabrication process [93,94] andhas been used widely. However, in order to mimic the patterneddistribution of growth factors and ECM molecules in the SCniche, it is necessary to develop methods to sequester these bio-factors in a localized microenvironment to prevent theirdiffusion into other regions of the scaffolds. Heparan sulfateshave been known to bind and protect growth factors, especiallyFGF-2, in the ECM. In our studies, we have successfullydemonstrated the effective binding of FGF-2 within photo-polymerizable PEGDA scaffolds by covalently conjugatingacrylated-PEG moieties to heparan sulfate [95]. Using immuno-histochemistry we effectively demonstrated that FGF-2 wasonly localized or sequestered in a region of a multi-layeredscaffold that had heparin-PEG-acrylate.

A widely used method of growth factor delivery in tissueengineering has been simple physical adsorption of biomole-cules on the biomaterial or scaffold surface. Carstens et al.,using absorbable collagen sponges as delivery vehicles forrecombinant human BMP-2 (rhBMP-2), showed in situosteogenesis in a craniofacial mandibular defect [96]. Thechemotactic effects of rhBMP-2, attracted MSCs to the vicinityof the implants. These cells were characterized to be spindle-shaped and having a pre-osteoblastic phenotype. In anotherstudy titanium fiber mesh scaffolds were coated with arginine-glycine-aspartic acid (RGD) [97], a cell adhesive, integrin-binding peptide found in FN and laminin. MSCs were shown toattach more strongly to these RGD-coated scaffolds, howeverno change was observed in ECM secretion.

Although protein adsorption to scaffold surfaces can be aneffective route for presentation of bioactive molecules [98,99],desorption of the protein during culture period and the inherentpoor reproducibility of adsorption processes limits its applica-bility. Covalent conjugation of bioactive molecules to thebiomaterial surface should provide a more reproducible andcontrolled method of presentation since both ligand amount andligand density can be controlled. By incorporating methacrylicacid within a PEGDA macromer solution prior to photo-polymerizing, we have shown that the surface of 3-D hydrogelscaffolds can be functionalized with carboxylic acid groups.These free carboxyl groups can be subsequently activated to

Fig. 1. Schematic of biochemical modification and spatial patterning of scaffolds for stem cell culture.


attach FN or other ligands [100]. Murine MSCs seeded ontosuch FN-functionalized scaffolds created by an LbL micro-fabrication system, and cells effectively adhered and trans-formed into osteoblasts.

RGD can be covalently conjugated to PEG-macromers usingthe widely used N-hydroxysuccinimidyl-ester (NHS) chemistry[101–104]. This has been reported for functionalization ofPEGDA scaffolds for hMSC differentiation into osteoblasts[105]. Cell–matrix interactions were enhanced in RGD-functionalized hydrogels leading to increased MSCs viability.Nuttelman and colleagues showed that the viability of theencapsulated hMSCs increases from 15% to 75% when RGD isincorporated into PEGDA hydrogels [105]. It is also possible todifferentiate hESCs into chondrocytic like cells in an RGDmodified PEGDA hydrogel [106]. The hESC-derived cells notonly morphologically resembled chondrocytes but also RT-PCRanalysis indicated that the cells expressed a number ofchondrocytic markers and demonstrated the ability to produce7% w/v of GAG after three weeks in culture [106]. MSCscultured under similar conditions without RGD produced 3.5%w/v of GAG accumulation [59].

Semi-interpenetrating polymer networks (sIPN) which form ahydrogel material can be chemically altered to mimic ECM in avariety of ways, notably, the mechanical properties can be alteredas well as adding functional cell-adhesion ligands (at varieddensities). p(NIPAAm-co-AAc) was crosslinked with Gln-Pro-Gln-GLY-Leu-Ala-Lys-NH2, which can be cleaved by matrixmetalloproteinase-13 [60]. The sIPN was further functionalizedwith a polyacrylic acid-graft-Ac-CGGNGEPRGD-TYRAY-NH2

[p(AAc)-g-RGD] [60]. The p(AAc) chains were modified with(Ac-CGGNGEPRGDTYRAY-NH2) which provides an activeRGD site to promote cellular adhesion [60]. hESCs cultured onthis fully synthetic ECM replacement, functionalized with 0 to150 μM of RGD complexes, were shown to be morphologicallysimilar to hESCs cultured on an embryonic fibroblast feeder layerand continued to produce markers characteristic of undifferenti-ated hESCs [60].

In another study, incorporation of RGD in oligo(PEG-fumarate) hydrogels enhanced alkaline phosphatase (ALP)activity of osteoblasts when compared to non-functionalizedhydrogels [107]. Shin et al. compared RGD with anosteopontin-derived peptide, and determined that hydrogels


modified with the osteopontin-derived peptide triggeredsignificantly higher osteoblast migration than RGD-modifiedhydrogels. However, RGD remained the dominating peptide forcell-attachment [108].

A study completed by Feng et al. compared the effect ofadsorbed, conjugated, and soluble FN on hHSC expansion inPET non-woven scaffolds. Their study indicated that conjuga-tion of FN directly to the scaffolds resulted in a higherexpansion percentage of CD34+ cells [51]. Because the FN wasdirectly conjugated to the scaffold material, detachment of FNfrom the scaffold could be prevented, and perhaps aid instabilizing FN in the culture system [51].

In addition to presenting ligands to control differentiation viaa covalently linked hydrogel, it may be possible to utilize othermaterials to mimic the cellular environment. HSCs located inthe thymus receive specific microenvironmental signals direct-ing them toward the T lineage [111]. Traditionally, studiesfocusing on T cell differentiation employ the usage ofretrovirally transfected stromal cells to present the necessarycues to cause T lineage commitment. In our lab we haveemployed a biomaterial based magnetic microbead system toeffectively present ligands necessary to promote T cellformation (e.g. the notch ligand DLL4) [111]. Mousehematopoietic stem cells (mHSCs) isolated from the bonemarrow of mice and cultured with this microbead systemeffectively produced Thy1.2+ early T cells [111]. This presents aversatile biomaterial-based method to quantitatively presentligands to SCs in order to direct lineage-specific differentiationand study cellular processes during cell differentiation.

Scaffold mineralization during osteogenic differentiation ofMSCs can also be enhanced through chemical modification ofthe biomaterial structure. For example, adding a phosphoestergroup to photo-polymerizable PEG-based hydrogels not onlyprovides biodegradability but has also been shown to promotemineralization of encapsulated MSCs. The use of suchphosphoester-containing hydrogels significantly increasesALP and osteocalcin levels in differentiated cells [112,113]. Itwas shown that cell–matrix interactions and the viability ofencapsulated MSCs increase in the presence of phosphatemolecules within these hydrogel scaffolds [105]. Phosphatemoieties contribute to the adsorption of osteopontin, asialoprotein that binds to bone mineralization and mediatescell adhesion.

Natural materials can also be chemically altered to improvespecific properties for tissue engineering. For example, Zhangand colleagues have created PEGylated fibrin patches to studyin vitro differentiation of MSCs into endothelial cell lineagefor potential use in myocardial repair [114]. Chitosan, apolysaccharide, is another example of a natural biomaterialthat has been modified to be thermo-responsive [115,116] byincorporating hydroxybutyl groups onto the polymer backbone.The modified water soluble polymer undergoes sol–geltransition when exposed to 37 °C [117]. This modified chitosanhas been used in encapsulation and in vitro culture of hMSCswith the ultimate goal of developing an injectable cell-biomaterial composite for degenerative disk diseases. MSCswere effectively encapsulated within these chitosan gels, with

minimal cell toxicity, and showed gene expression of bone-specific markers [117].

In addition to the supplementation of bio-chemical stimuli bygrowth factors and other cytokines, it may be possible tostimulate alterations in the micro-environment by modifying thematerial surface chemistry. By altering hydrophobicity ofpeptides used to create scaffolds it may be possible to encouragea variety of cellular interactions [118]. Hydrophilicty of poly(DL-lactide) (PDLLA), PLA, PGA and PLGA can be altered bysubmerging the scaffolds in various concentrations of potassiumhydroxide [118]. As compared to non-surface treated scaffolds,all poly(α-hydroxyl ester) scaffolds treated with potassiumhydroxide (0.1 M) were able to support a larger number ofmESCs [118] indicating that for each polymer used, there mayexist an optimized hydrophobicity that will best promotecellular growth.

Konno et al. studied the effects of electrostatic charge onmESCs by culturing mESCs on photoimmobilized polymerswith LIF [109]. It was found that only one polymer surface,gelatin coupled with azidophenyl groups, improved the growthand maintenance of mESCs [109]. The other polymers (coupledwith azidophenyl groups), poly(acrylic acid) and poly(2-methacryloyl-oxyethyl phosphorylcholine-co-methacrylicacid) (PMAc50) did not advance the growth of mESCs. Infact, these polymers prevented cell adhesion resulting in EBformation, and induced cell differentiation [109]. Carbonatedapatite surfaces may also have the potential to inducedifferentiation as mESCs cultured on this material proliferatedin a differentiated state [110].

In addition to proteins, scaffolds can also operate as effectivedelivery vehicles for small bioactive molecules that direct SCgrowth and organization. Covalent conjugation and controlled-release of dexamethasone, a synthetic corticosteroid crucial forMSC osteogenesis in vitro, has been achieved through ahydrolytically-labile lactide component incorporated withinPEG hydrogels [119]. Human MSCs were encapsulated withinthese gels, which lead to their efficient osteogenic differenti-ation. The gene expression levels for two common osteogenicmarkers, ALP and core binding factor alpha 1 (Cbfa1),significantly increased in MSCs cultured in dexamethasone-functionalized scaffolds.

Degradable thin films may also act as an efficient deliveryvehicle of SCs [120]. Films coated with FN enabled adiposederived SCs to adhere to poly(L-lactide-co-ɛ-caprolactone)films as efficiently as cells adhering to tissue culture plasticsurfaces, with no significant difference in number of cellsattached even up to 24 hours after seeding [120]. The polymerfilm itself degraded over a prolonged period of time, with asharp increase in degradation rate at week 6 [120].

Delivery of growth factors and chemicals can also bemediated using degradable particles, entrapped within thebiomaterial scaffold, which could provide temporal releasekinetics for signaling biomolecules over a prolonged period oftime [121–124]. Osteogenic studies of rat MSCs usingrecombinant human TGF-β1 encapsulated in polymer blendsof PEG-PLGA particles (sized at an average of 158 μm) hasbeen reported by Peter et al. [125]. Here they show that a

Table 1Application of natural biomaterials in stem cell culture

Biomaterial Chemical modifications Application in stem cell culture Cell source References

Matrigel™ Cell culture applications [22,23,25]Vascularization mADSC [26]

Fibrin Addition of growth factors Neural differentiation mESC [18,29]Cell culture mESC [27]Chondrogenesis hMSC

Hyaff® (hyaluron) Ligament formation Sheep MSC [32]Hyaluron Maintenance of pluripotency hESC [31]

Addition of soluble growth factors Lineage specific differentiationModified with photoreactive groups Cell proliferation hESC [31]Application of hyaluronidase Cell removal

Silk fibroin Medical suture material N/A [33–35]Chitosan with coralline Osteogenesis Murine MSC [37]Hydroxyapatite (HA) Osteogenesis mESC [39]Alginate Cell encapsulation and differentiation ESC [40]Pullulan, Dextran, and

FucoidanAddition of poly-L-lysine cross-linked with sodiumtrimetaphosphate

Hepatic possible applications to vascular repair mESC [41]hESC [42]

Collagen Type II Chondrogenesis hMSC [43]Collagen microbeads Cell expansion and viability,

HematopoiesishUCBC [44]

Collagen Addition of HA MSC seeding and proliferation Rat MSC [74]Addition of crosslinked chitin and PLA Cell attachment hMSC [75]Addition of recombinant human BMP-2 (rhBMP-2) Osteogenesis Porcine MSC [96]

Silk Osteogenesis hMSC [76]Chondrogenesis hMSC [35]

Chitosan Conjugation of hydroxybutyl groups Potential degenerative disk therapies hMSC [117]


loading of 6.0 ng TGF-β1/ mg of microparticle provided anoptimal dose for growth factor delivery for enhancing MSCproliferation and transformation into osteoblasts [125]. Tables 1and 2 summarizes the various natural and synthetic biomaterialsused in SC culture, their chemical modifications (as discussed inSection 3) as well as their specific applications.

3.3. Patterned biomaterials

Recent developments in micro and nanofabrication techni-ques have opened up a myriad of possibilities in studying andcontrolling the differentiation of SCs. These techniques allowfor the controlled design of highly reproducible features on acellular level as well as the possibility of creating spatially andtemporally patterned scaffold structures that might ultimatelylead to generation of complex, hybrid tissue structures.

ESC lineage commitment of individual cells depends on anumber of variables. One important factor is the EB shapeand size [126]. The ability to effectively control the size ofEBs in a reproducible manner may have a large impact on theability to control and scale up ESC differentiation. One of theapproaches to culturing hESCs is to create a co-culture ofhESCs on top of inactivated murine embryonic fibroblasts(MEFs). Using polydimethylsiloxane (PDMS) molds, Kha-demhosseini et al. were able to create microwells on a glasssurface [127]. MEFs were then seeded onto the PDMSsurface and subsequently inactivated to allow for hESC seed-ing. Results indicated that hESCs grew in highly homoge-neous aggregates and demonstrated both high cell viability aswell as markers indicative of an undifferentiated cell state[127].

By creating a microwell array system using photolithographyand plasma etching techniques, Mohr and colleagues were ableto create spatially uniform aggregates of undifferentiated hESCswithout the use of MEFs [128]. After culturing in microwells,the hESCs could be removed from the microwells and furthercultured under standard conditions (plates coated with Matrigel)showing little sign of prior differentiation [128]. EBs formedfrom cells isolated from this microwell system ranged in sizefrom 200 to 359 μm, with a majority of EBs between 280 and359 μm (78% of the total cell population) [128]. In comparison,EBs that were derived on tissue culture polystyrene were morevariable in size, with only 31% of the total cell populationfalling in a range of size between 280 and 359 μm in diameter[128]. This system can be further altered by creating microwellsof different sizes and depths in order to form a variety of EBstructures allowing for the optimization of EB size [128].

Khademhosseini et al. reported micropatterned hydrogels ofhyaluronic acid, with photoreactive methacrylates [129]. Acomposite of hyaluronic acid microwells was formed andmESCs were either “docked” in these wells or encapsulated inthe hydrogel [129]. Both conditions promoted cell viability andalso illustrated how cells can be manipulated to colonize inspecific patterns and shapes based on the microarchitecture ofthe wells [129].

The microenvironment comprising complex tissues includesa variety of cellular signaling moieties presented in 3-D spatialpatterns. While many scaffolds incorporate various biomole-cules into the scaffold construct, they do so in bulk, i.e. arandom distribution of factors throughout the scaffold. UsingLbL microfabrication and a digital micro-mirror (DMD)-basedsystem we have incorporated precise, pre-designed spatial

Table 2Application of synthetic biomaterials in stem cell culture

Biomaterial Chemical modifications Application in stem cellculture

Cellsource

References

poly(ɛ-caprolactone) (PCL) Cell propagation mESC [56]Addition of adipogenic promoting factors Adipogenesis mESC [56]

Adipogenesis hMSC [54]Chondrogenesis andOsteogenesis

RatMSC

[55]

Poly(L-lactic acid) (PLLA) Hematopoiesis mESC [50]Polyglycolic acid (PGA), polylactic acid,(PLA), polylactide-co-glycolide (PLGA)

Cell proliferation and 3-Dorganization

hESC [49]

Poly(ethylene glycol) diacrylates(PEGDA) hydrogels

Cell and bio-chemical GoatMSC

[59]

molecule encapsulation hMSC [58]Addition of glucosamine Chondrogenesis mESC [20]Incorporation of RGD-PEG-acrylates Cell viability, Osteogenesis hMSC [105]Modified with RGD Chondrogenesis hESC [106]Release of dexamethasone Osteogenesis hMSC [119]

poly(N-isopropylacrylamide-co-acrylic acid)[p(NIPAAm-co-AAc)]

Incorporated into a photocrosslinkedhydrogel with a metalloproteinase sensitive peptide

Cell self-renewal and maintenance hESC [60]

PEGDA scaffolds Incorporation of methacrylic acid Osteogenesis MurineMSC

[100]

poly(6-aminohexyl phosphate acryloyl)(PPE-HA-acryl)

Cell and bio-chemicalmolecule encapsulation

GoatMSC

[57]

Polyethylene terephthalate (PET) Cell seeding, proliferation, and aggregation RatMSC

[53]

hMSC [52]hHSC [51]

Conjugated with FN CD34+ proliferation hHSC [51]Poly(ethylene glycol) (PEG) hydrogel Exposed to TGF-β Chondrogenesis mESC [19]

Addition of a phosphoester Osteogenesis GoatMSC

[112]

Biphasic calcium phosphate(Triosite™) Osteogenesis hMSC [61]Titanium Cell attachment and

proliferationRatMSC

[64]

Differentiation media Osteogenesis RatMSC

[64,65]

Coated with RGD MSC attachment RatMSC

[97]

Surface modification with TiO2 Cell adherence MurineMSC

[66]

Surface modification with TiO2 Cell adherence hMSC [64,67]Tantalum (Cytomatrix) Hematopoiesis mESC [17,66,68]PPE-HA-acrl Increase acrylated-PEG Osteogenesis Goat

MSC[57]

sIPN p(NIPAAm-coI-AAc) crosslinked withGln-Pro-Gln-GLY-Leu-Ala-Lys-NH2 andfunctionalized with p(AAc) and RGD complexes

Cell propagation hESC [60]

Oligo(PEG-fumarate) hydrogels Modified with osteopontin-derived peptide Osteoblast migration RatMSC

[108]

Modified with RGD Cell attachment RatMSC

[108]

Gelatin Coupled with azidophenyl groups Cell growth mESC [109]Magnetic microbeads T cell formation mHSC [111]Poly(α-hydroxyl ester) scaffolds Treated with potassium hydroxide Cell growth mESC [118]Poly(L-lactide-co-ɛ-caprolactone) films Coated with FN ESC adherence hADSC [120]PEG-PLGA polymer blends Encapsulation of recombinant human TGF-β1 MSC proliferation and

osteogenesisRatMSC

[125]

PDMS molds Seeded with MEFs Cell viability andproliferation

hESC [127]

Microwell array system Create spatially uniformaggregates of undifferentiated cells

hESC [128]

HA microwell system Cell viability, controlled cell patterningand shaping

mESC [129]

Biomaterial microarray Time efficient cell viability anddifferentiation studies

hESC [130]



patterning of molecules into 3-D scaffolds to create complexmicroenvironments more indicative of native tissues [95,100].Using these techniques it may be possible to create a scaffoldcontaining regions conducive to directing a single stempopulation to multiple, spatially organized tissues. By utilizingECM components capable of sequestering growth factors(heparan), we demonstrated that the spatial pattern can bemaintained during the culture period. [95]. Fig. 1 provides aschematic representation of such spatial patterning concept.

4. Concluding remarks

The use of SCs for cellular therapy offers enormous prospectin replacing damaged or lost tissue function. By utilizing SCs,the creation of an unlimited supply of fully differentiated cellscan be obtained, thus making cellular therapy a reality.Ultimately there are a number of obstacles researchers mustovercome before this potential is reached. In addition toovercoming the difficulty of obtaining a therapeuticallymeaningful number of cells, a large variety of in vivo and invitrowork is left to be done prior to the application of these cellson a clinical level. Specifically, the safety of using SCs,especially when considering the usage of allogenic or evenxenogenic donor cells must be carefully established beforeusing SC therapy in a clinical setting [11].

The cell microenvironment is known to play a significantrole in determining progenitor cell fate and function. Theprecise coordination of interpreting spatial and temporal cuesfrom their microenvironment is highly essential for SCs tocreate complex, functional tissues. Advanced and high-throughput assays, such as extracellular microarrays and othertechnologies extensively reviewed by Khademhosseini et al.,[131–133], have uncovered specific cell interactions with ECMcomponents and polymers that can directly stimulate SCsignaling and response. As biomaterial research advances,new materials as well as innovations in their manipulation andusage continue. By utilizing high throughput arrays todistinguish biomaterial functionality the assessment of thesematerials for ultimate usage can occur in short periods of time,wasting few materials and ultimately allowing for a more rapidend product [130]. For SC based cellular therapy to be a viabletherapeutic option, major advances in both the understanding ofthe local cues necessary for lineage commitment and thebiomaterials necessary to promote differentiation is necessary.

References

[1] J.P. Vacanti, R. Langer, Tissue engineering: the design and fabrication ofliving replacement devices for surgical reconstruction and transplantation,Lancet 354 (1) (1999) SI32–SI34.

[2] P.K. Yarlagadda, M. Chandrasekharan, J.Y. Shyan, Recent advances andcurrent developments in tissue scaffolding, Biomed. Mater. Eng. 15 (2005)159–177.

[3] T. Ahsan, R.M. Nerem, Bioengineered tissues: the science, thetechnology, and the industry, Orthod. Craniofac. Res. 8 (2005) 134–140.

[4] A. El-Ghannam, Bone reconstruction: from bioceramics to tissueengineering, Expert. Rev. Med. Devices 2 (2005) 87–101.

[5] R.E. Horch, J. Kopp, U. Kneser, J. Beier, A.D. Bach, Tissue engineeringof cultured skin substitutes, J. Cell. Mol. Med. 9 (2005) 592–608.

[6] J.Y. Lim, H.J. Donahue, Biomaterial characteristics important to skeletaltissue engineering, J. Musculoskelet. Neuronal. Interact. 4 (2004) 396–398.

[7] J. Raghunath, H.J. Salacinski, K.M. Sales, P.E. Butler, A.M. Seifalian,Advancing cartilage tissue engineering: the application of stem celltechnology, Curr. Opin. Biotechnol. 16 (2005) 503–509.

[8] H. Yoshikawa, A. Myoui, Bone tissue engineering with poroushydroxyapatite ceramics, J. Artif. Organs 8 (2005) 131–136.

[9] J. Elisseeff, C. Puleo, F. Yang, B. Sharma, Advances in skeletal tissueengineering with hydrogels, Orthod. Craniofac. Res. 8 (2005) 150–161.

[10] M.N. Rahaman, J.J. Mao, Stem cell-based composite tissue constructs forregenerative medicine, Biotechnol. Bioeng. 91 (2005) 261–284.

[11] M. Mimeault, S.K. Batra, Concise review: recent advances on thesignificance of stem cells in tissue regeneration and cancer therapies,Stem Cells 24 (2006) 2319–2345.

[12] N.D. Evans, E. Gentleman, J.M. Polak, Scaffolds for stem cells, MaterialsToday 9 (2006) 26–33.

[13] M.T. Mitjavila-Garcia, C. Simonin, M. Peschanski, Embryonic stemcells: meeting the needs for cell therapy, Adv. Drug. Deliv. Rev. 57 (2005)1935–1943.

[14] Y. Li, D.A. Kniss, L.C. Lasky, S.T. Yang, Culturing and differentiation ofmurine embryonic stem cells in a three-dimensional fibrous matrix,Cytotechnology 41 (2003) 23–35.

[15] C. Chai, K.W. Leong, Biomaterials approach to expand and directdifferentiation of stem cells, Molec. Ther. 15 (2007) 467–480.

[16] E. Garreta, E. Genove, S. Borros, C.E. Semino, Osteogenic differentiationof mouse embryonic stem cells and mouse embryonic fibroblasts in athree-dimensional self-assembling peptide scaffold, Tissue Eng. 12(2006) 2215–2227.

[17] H. Liu, K. Roy, Biomimetic three-dimensional cultures significantlyincrease hematopoietic differentiation efficacy of embryonic stem cells,Tissue Eng. 11 (2005) 319–330.

[18] S.M. Willerth, K.J. Arendas, D.I. Gottlieb, S.E. Sakiyama-Elbert,Optimization of fibrin scaffolds for differentiation of murine embryonicstem cells into neural lineage cells, Biomaterials 27 (2006) 5990–6003.

[19] N.S. Hwang, M.S. Kim, S. Sampattavanich, J.H. Baek, Z. Zhang, J.Elisseeff, Effects of three-dimensional culture and growth factors on thechondrogenic differentiation of murine embryonic stem cells, Stem Cells24 (2006) 284–291.

[20] N.S. Hwang, S. Varghese, P. Theprungsirikul, A. Canver, J. Elisseeff,Enhanced chondrogenic differentiation of murine embryonic stem cells inhydrogels with glucosamine, Biomaterials 27 (2006) 6015–6023.

[21] H. Shin, S. Jo, A.G. Mikos, Biomimetic materials for tissue engineering,Biomaterials 24 (2003) 4353–4364.

[22] J.L. Drury, D.J. Mooney, Hydrogels for tissue engineering: scaffolddesign variables and applications, Biomaterials 24 (2003) 4337–4351.

[23] D.M. Bissell, D.M. Arenson, J.J. Maher, F.J. Roll, Support of culturedhepatocytes by a laminin-rich gel. Evidence for a functionally significantsubendothelialmatrix in normal rat liver, J. Clin. Invest. 79 (1987) 801–812.

[24] H.K. Kleinman, M.L. McGarvey, L.A. Liotta, P.G. Robey, K.Tryggvason, G.R. Martin, Isolation and characterization of type IVprocollagen, laminin, and heparan sulfate proteoglycan from the EHSsarcoma, Biochemistry 21 (1982) 6188–6193.

[25] C. Xu, M.S. Inokuma, J. Denham, K. Golds, P. Kundu, J.D. Gold, M.K.Carpenter, Feeder-free growth of undifferentiated human embryonic stemcells, Nat. Biotechnol. 19 (2001) 971–974.

[26] V. Planat-Benard, J.S. Silvestre, B. Cousin, M. Andre, M. Nibbelink, R.Tamarat, M. Clergue, C. Manneville, C. Saillan-Barreau, M. Duriez, A.Tedgui, B. Levy, L. Penicaud, L. Casteilla, Plasticity of human adiposelineage cells toward endothelial cells: physiological and therapeuticperspectives, Circulation 109 (2004) 656–663.

[27] G.I. Im, Chondrogenesis from mesenchymal stem cells derived fromadipose tissue on the fibrin scaffold, Curr. Appl. Phys. 5 (2005) 438–443.

[28] S.M. Willerth, T.E. Faxel, D.I. Gottlieb, S.E. Sakiyama-Elbert, Theeffects of soluble growth factors on embryonic stem cell differentiationinside of fibrin scaffolds, Stem Cells 25 (2007) 2235–2244.

[29] H. Liu, S.F. Collins, L.J. Suggs, Three-dimensional culture for expansionand differentiation of mouse embryonic stem cells, Biomaterials 27 (2006)6004–6014.


[30] P. Brun, G. Abatangelo, M. Radice, V. Zacchi, D. Guidolin, D. DagaGordini, R. Cortivo, Chondrocyte aggregation and reorganization intothree-dimensional scaffolds, J. Biomed. Mater. Res. 46 (1999) 337–346.

[31] S. Gerecht, J.A. Burdick, L.S. Ferreira, S.A. Townsend, R. Langer, G.Vunjak-Novakovic, Hyaluronic acid hydrogel for controlled self-renewaland differentiation of human embryonic stem cells, Proc. Natl. Acad. Sci.U.S.A. 104 (2007) 11298–11303.

[32] S. Cristino, F. Grassi, S. Toneguzzi, A. Piacentini, B. Grigolo, S. Santi, M.Riccio, E. Tognana, A. Facchini, G. Lisignoli, Analysis of mesenchymalstem cells grown on a three-dimensional HYAFF 11-based prototypeligament scaffold, J. Biomed. Mater. Res. A 73 (2005) 275–283.

[33] S. Hofmann, H. Hagenmuller, A.M. Koch, R. Muller, G. Vunjak-Novakovic, D.L. Kaplan, H.P. Merkle, L. Meinel, Control of in vitrotissue-engineered bone-like structures using human mesenchymal stemcells and porous silk scaffolds, Biomaterials 28 (2007) 1152–1162.

[34] R. Nazarov, H.J. Jin, D.L. Kaplan, Porous 3-D scaffolds from regeneratedsilk fibroin, Biomacromolecules 5 (2004) 718–726.

[35] Y. Wang, U.J. Kim, D.J. Blasioli, H.J. Kim, D.L. Kaplan, In vitro cartilagetissue engineering with 3D porous aqueous-derived silk scaffolds andmesenchymal stem cells, Biomaterials 26 (2005) 7082–7094.

[36] Y. Wang, H.J. Kim, G. Vunjak-Novakovic, D.L. Kaplan, Stem cell-basedtissue engineering with silk biomaterials, Biomaterials 27 (2006)6064–6082.

[37] M. Gravel, T. Gross, R. Vago, M. Tabrizian, Responses of mesenchymalstem cell to chitosan-coralline composites microstructured using corallineas gas forming agent, Biomaterials 27 (2006) 1899–1906.

[38] F. Zhao,W.L.Grayson, T.Ma,B.Bunnell,W.W.Lu, Effects of hydroxyapatitein 3-D chitosan-gelatin polymer network on human mesenchymal stem cellconstruct development, Biomaterials 27 (2006) 1859–1867.

[39] E. Garreta, D. Gasset, C. Semino, S. Borros, Fabrication of a three-dimensional nanostructured biomaterial for tissue engineering of bone,Biomol. Eng. 24 (2007) 75–80.

[40] S.K. Dean, Y. Yulyana, G. Williams, K.S. Sidhu, B.E. Tuch,Differentiation of encapsulated embryonic stem cells after transplanta-tion, Transplantation 82 (2006) 1175–1184.

[41] T. Maguire, E. Novik, R. Schloss, M. Yarmush, Alginate-PLLmicroencapsulation: effect on the differentiation of embryonic stemcells into hepatocytes, Biotechnol. Bioeng. 93 (2006) 581–591.

[42] N.B. Thebaud, D. Pierron, R. Bareille, C. Le Visage, D. Letourneur, L.Bordenave, Human endothelial progenitor cell attachment to polysac-charide-based hydrogels: a pre-requisite for vascular tissue engineering,J. Mater. Sci., Mater. Med. 18 (2007) 339–345.

[43] C.F. Chang, M.W. Lee, P.Y. Kuo, Y.J. Wang, Y.H. Tu, S.C. Hung, Three-dimensional collagen fiber remodeling by mesenchymal stem cells requiresthe integrin–matrix interaction, J. Biomed. Mater. Res. A. 80 (2007)466–474.

[44] H.S. Kim, J.B. Lim, Y.H. Min, S.T. Lee, C.J. Lyu, E.S. Kim, H.O. Kim,Ex vivo expansion of human umbilical cord blood CD34+ cells in acollagen bead-containing 3-dimensional culture system, Int. J. Hematol.78 (2003) 126–132.

[45] M.J. Mondrinos, S. Koutzaki, E. Jiwanmall, M. Li, J.P. Dechadarevian, P.I.Lelkes, C.M. Finck, Engineering three-dimensional pulmonary tissueconstructs, Tissue Eng. 12 (2006) 717–728.

[46] C.S. Young, H. Abukawa, R. Asrican, M. Ravens, M.J. Troulis, L.B.Kaban, J.P. Vacanti, P.C. Yelick, Tissue-engineered hybrid tooth andbone, Tissue Eng. 11 (2005) 1599–1610.

[47] M. Borden, M. Attawia, C.T. Laurencin, The sintered microsphere matrixfor bone tissue engineering: in vitro osteoconductivity studies, J. Biomed.Mater. Res. 61 (2002) 421–429.

[48] A.S. Lin, T.H. Barrows, S.H. Cartmell, R.E. Guldberg, Microarchitecturaland mechanical characterization of oriented porous polymer scaffolds,Biomaterials 24 (2003) 481–489.

[49] S. Levenberg, N.F. Huang, E. Lavik, A.B. Rogers, J. Itskovitz-Eldor, R.Langer, Differentiation of human embryonic stem cells on three-dimensionalpolymer scaffolds, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 12741–12746.

[50] S. Taqvi, K. Roy, Influence of scaffold physical properties and stromalcell coculture on hematopoietic differentiation of mouse embryonic stemcells, Biomaterials 27 (2006) 6024–6031.

[51] Q. Feng, C. Chai, X.S. Jiang, K.W. Leong, H.Q. Mao, Expansion ofengrafting human hematopoietic stem/progenitor cells in three-dimen-sional scaffolds with surface-immobilized fibronectin, J. Biomed. Mater.Res. A 78 (2006) 781–791.

[52] W.L. Grayson, T. Ma, B. Bunnell, Human mesenchymal stem cells tissuedevelopment in 3D PET matrices, Biotechnol. Prog. 20 (2004) 905–912.

[53] Y. Takahashi, Y. Tabata, Homogeneous seeding of mesenchymal stem cellsinto nonwoven fabric for tissue engineering, Tissue Eng. 9 (2003) 931–938.

[54] W.J. Li, R. Tuli, X. Huang, P. Laquerriere, R.S. Tuan, Multilineagedifferentiation of human mesenchymal stem cells in a three-dimensionalnanofibrous scaffold, Biomaterials 26 (2005) 5158–5166.

[55] M. Shin, H. Yoshimoto, J.P. Vacanti, In vivo bone tissue engineeringusing mesenchymal stem cells on a novel electrospun nanofibrousscaffold, Tissue Eng. 10 (2004) 33–41.

[56] X.Kang,Y.Xie,H.M. Powell, L. JamesLee,M.A.Belury, J.J. Lannutti, D.A.Kniss, Adipogenesis of murine embryonic stem cells in a three-dimensionalculture system using electrospun polymer scaffolds, Biomaterials 28 (2007)450–458.

[57] Q. Li, J. Wang, S. Shahani, D.D. Sun, B. Sharma, J.H. Elisseeff, K.W.Leong, Biodegradable and photocrosslinkable polyphosphoester hydro-gel, Biomaterials 27 (2006) 1027–1034.

[58] C.R. Nuttelman, M.C. Tripodi, K.S. Anseth, In vitro osteogenicdifferentiation of human mesenchymal stem cells photoencapsulated inPEG hydrogels, J. Biomed. Mater. Res. A 68 (2004) 773–782.

[59] C.G. Williams, T.K. Kim, A. Taboas, A. Malik, P. Manson, J. Elisseeff,In vitro chondrogenesis of bone marrow-derived mesenchymal stemcells in a photopolymerizing hydrogel, Tissue Eng. 9 (2003) 679–688.

[60] Y.J. Li, E.H. Chung, R.T. Rodriguez, M.T. Firpo, K.E. Healy, Hydrogelsas artificial matrices for human embryonic stem cell self-renewal,J. Biomed. Mater. Res. A 79 (2006) 1–5.

[61] J. Toquet, R. Rohanizadeh, J. Guicheux, S. Couillaud, N. Passuti, G.Daculsi, D. Heymann, Osteogenic potential in vitro of human bonemarrow cells cultured on macroporous biphasic calcium phosphateceramic, J. Biomed. Mater. Res. 44 (1999) 98–108.

[62] H. Ohgushi, A.I. Caplan, Stem cell technology and bioceramics: from cellto gene engineering, J. Biomed. Mater. Res. 48 (1999) 913–927.

[63] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biodegradable andbioactive porous polymer/inorganic composite scaffolds for bone tissueengineering, Biomaterials 27 (2006) 3413–3431.

[64] M. Maeda, M. Hirose, H. Ohgushi, T. Kirita, In vitro mineralization bymesenchymal stem cells cultured on titanium scaffolds, J. Biochem.(Tokyo) 141 (2007) 729–736.

[65] N. Datta, Q.P. Pham, U. Sharma, V.I. Sikavitsas, J.A. Jansen, A.G. Mikos,In vitro generated extracellular matrix and fluid shear stress synergisticallyenhance 3D osteoblastic differentiation, Proc. Natl. Acad. Sci. U. S. A.103 (2006) 2488–2493.

[66] D.S. Kommireddy, S.M. Sriram, Y.M. Lvov, D.K. Mills, Stem cellattachment to layer-by-layer assembled TiO2 nanoparticle thin films,Biomaterials 27 (2006) 4296–4303.

[67] M.Manso-Silvan, J.M.Martinez-Duart, S. Ogueta, P. Garcia-Ruiz, J. Perez-Rigueiro, Development of humanmesenchymal stem cells on DC sputteredtitanium nitride thin films, J. Mater. Sci., Mater. Med. 13 (2002) 289–293.

[68] H. Liu, J. Lin, K. Roy, Effect of 3D scaffold and dynamic culturecondition on the global gene expression profile of mouse embryonic stemcells, Biomaterials 27 (2006) 5978–5989.

[69] A. Subramanian, H.Y. Lin, Crosslinked chitosan: its physical propertiesand the effects of matrix stiffness on chondrocyte cell morphology andproliferation, J. Biomed. Mater. Res. A 75 (2005) 742–753.

[70] J.M. Zaleskas, B. Kinner, T.M. Freyman, I.V. Yannas, L.J. Gibson, M.Spector, Contractile forces generated by articular chondrocytes in collagen-glycosaminoglycan matrices, Biomaterials 25 (2004) 1299–1308.

[71] A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directsstem cell lineage specification, Cell 126 (2006) 677–689.

[72] G.H. Altman, R.L. Horan, I. Martin, J. Farhadi, P.R. Stark, V. Volloch, J.C.Richmond, G. Vunjak-Novakovic, D.L. Kaplan, Cell differentiation bymechanical stress, FASEB J. 16 (2002) 270–272.

[73] C.A. Simmons, S. Matlis, A.J. Thornton, S. Chen, C.Y. Wang, D.J.Mooney, Cyclic strain enhances matrix mineralization by adult human


mesenchymal stem cells via the extracellular signal-regulated kinase(ERK1/2) signaling pathway, J. Biomech. 36 (2003) 1087–1096.

[74] L. Liu, L. Zhang, B. Ren, F. Wang, Q. Zhang, Preparation and charac-terization of collagen-hydroxyapatite composite used for bone tissueengineering scaffold, Artif. Cells Blood Substit. Immobil. Biotechnol. 31(2003) 435–448.

[75] X. Li, Q. Feng, W. Wang, F. Cui, Chemical characteristics andcytocompatibility of collagen-based scaffold reinforced by chitin fibers forbone tissue engineering, J. Biomed.Mater. Res. BAppl. Biomater. 77 (2006)219–226.

[76] L. Meinel, R. Fajardo, S. Hofmann, R. Langer, J. Chen, B. Snyder, G.Vunjak-Novakovic, D. Kaplan, Silk implants for the healing of criticalsize bone defects, Bone 37 (2005) 688–698.

[77] L. Meinel, S. Hofmann, V. Karageorgiou, L. Zichner, R. Langer, D.Kaplan, G. Vunjak-Novakovic, Engineering cartilage-like tissue usinghuman mesenchymal stem cells and silk protein scaffolds, Biotechnol.Bioeng. 88 (2004) 379–391.

[78] L. Meinel, V. Karageorgiou, S. Hofmann, R. Fajardo, B. Snyder, C. Li, L.Zichner, R. Langer, G. Vunjak-Novakovic, D.L. Kaplan, Engineeringbone-like tissue in vitro using human bone marrow stem cells and silkscaffolds, J. Biomed. Mater. Res. A 71 (2004) 25–34.

[79] M.E. Gomes, C.M. Bossano, C.M. Johnston, R.L. Reis, A.G. Mikos,In vitro localization of bone growth factors in constructs of bio-degradable scaffolds seeded with marrow stromal cells and cultured ina flow perfusion bioreactor, Tissue Eng. 12 (2006) 177–188.

[80] H.L. Holtorf, N. Datta, J.A. Jansen, A.G. Mikos, Scaffold mesh size affectsthe osteoblastic differentiation of seeded marrow stromal cells cultured in aflow perfusion bioreactor, J. Biomed. Mater. Res. A 74 (2005) 171–180.

[81] H.L. Holtorf, J.A. Jansen, A.G. Mikos, Flow perfusion culture inducesthe osteoblastic differentiation of marrow stroma cell-scaffold constructsin the absence of dexamethasone, J. Biomed. Mater. Res. A 72 (2005)326–334.

[82] S. Battista, D. Guarnieri, C. Borselli, S. Zeppetelli, A. Borzacchiello, L.Mayol, D. Gerbasio, D.R. Keene, L. Ambrosio, P.A. Netti, The effect ofmatrix composition of 3D constructs on embryonic stem cell differen-tiation, Biomaterials 26 (2005) 6194–6207.

[83] G. Bianchi, A. Banfi, M. Mastrogiacomo, R. Notaro, L. Luzzatto, R.Cancedda, R. Quarto, Ex vivo enrichment of mesenchymal cell progenitorsby fibroblast growth factor 2, Exp. Cell Res. 287 (2003) 98–105.

[84] I. Martin, A. Muraglia, G. Campanile, R. Cancedda, R. Quarto, Fibroblastgrowth factor-2 supports ex vivo expansion and maintenance of osteogenicprecursors from human bone marrow, Endocrinology 138 (1997)4456–4462.

[85] L.A. Solchaga, K. Penick, J.D. Porter, V.M. Goldberg, A.I. Caplan, J.F.Welter, FGF-2 enhances the mitotic and chondrogenic potentials ofhuman adult bone marrow-derived mesenchymal stem cells, J. Cell.Physiol. 203 (2005) 398–409.

[86] S. Tsutsumi, A. Shimazu, K. Miyazaki, H. Pan, C. Koike, E. Yoshida, K.Takagishi, Y. Kato, Retention of multilineage differentiation potential ofmesenchymal cells during proliferation in response to FGF, Biochem.Biophys. Res. Commun. 288 (2001) 413–419.

[87] A.H. Reddi, N.S. Cunningham, Initiation and promotion of bonedifferentiation by bone morphogenetic proteins, J. Bone Miner. Res.8 (2) (1993) S499–S502.

[88] J.M. Wozney, The bone morphogenetic protein family and osteogenesis,Mol. Reprod. Dev. 32 (1992) 160–167.

[89] M.E. Levenstein, T.E. Ludwig, R.H. Xu, R.A. Llanas, K. VanDenHeuvel-Kramer, D. Manning, J.A. Thomson, Basic fibroblast growth factor supportof human embryonic stem cell self-renewal, StemCells 24 (2006) 568–574.

[90] R.H. Xu, R.M. Peck, D.S. Li, X. Feng, T. Ludwig, J.A. Thomson, BasicFGF and suppression of BMP signaling sustain undifferentiatedproliferation of human ES cells, Nat. Methods 2 (2005) 185–190.

[91] H.J. Rippon, J.M. Polak, M. Qin, A.E. Bishop, Derivation of distal lungepithelial progenitors from murine embryonic stem cells using a novelthree-step differentiation protocol, Stem Cells 24 (2006) 1389–1398.

[92] M. Nakanishi, T.S. Hamazaki, S. Komazaki, H. Okochi, M. Asashima,Pancreatic tissue formation from murine embryonic stem cells in vitro,Differentiation 75 (2007) 1–11.

[93] J.A. Jansen, J.W. Vehof, P.Q. Ruhe, H. Kroeze-Deutman, Y. Kuboki, H.Takita, E.L. Hedberg, A.G. Mikos, Growth factor-loaded scaffolds forbone engineering, J. Control. Release 101 (2005) 127–136.

[94] T.P. Richardson, M.C. Peters, A.B. Ennett, D.J. Mooney, Polymeric systemfor dual growth factor delivery, Nat. Biotechnol. 19 (2001) 1029–1034.

[95] G. Mapili, Y. Lu, S. Chen, K. Roy, Laser-layered microfabrication ofspatially patterned functionalized tissue-engineering scaffolds, J. Biomed.Mater. Res. B Appl. Biomater. 75 (2005) 414–424.

[96] M.H. Carstens,M.Chin, X.J. Li, In situ osteogenesis: regeneration of 10-cmmandibular defect in porcine model using recombinant human bonemorphogenetic protein-2 (rhBMP-2) and Helistat absorbable collagensponge, J. Craniofac. Surg. 16 (2005) 1033–1042.

[97] H.L. Holtorf, J.A. Jansen, A.G. Mikos, Ectopic bone formation in ratmarrow stromal cell/titanium fiber mesh scaffold constructs: effect ofinitial cell phenotype, Biomaterials 26 (2005) 6208–6216.

[98] Y. Takahashi, M. Yamamoto, Y. Tabata, Enhanced osteoinduction bycontrolled release of bone morphogenetic protein-2 from biodegradablesponge composed of gelatin and beta-tricalcium phosphate, Biomaterials26 (2005) 4856–4865.

[99] X.B. Yang, H.I. Roach, N.M. Clarke, S.M. Howdle, R. Quirk, K.M.Shakesheff, R.O. Oreffo, Human osteoprogenitor growth and differen-tiation on synthetic biodegradable structures after surface modification,Bone 29 (2001) 523–531.

[100] Y. Lu, G.Mapili, G. Suhali, S. Chen, K. Roy, A digital micro-mirror device-based system for the microfabrication of complex, spatially patterned tissueengineering scaffolds, J. Biomed. Mater. Res. A 77 (2006) 396–405.

[101] B.K. Brandley, R.L. Schnaar, Covalent attachment of an Arg-Gly-Aspsequence peptide to derivatizable polyacrylamide surfaces: support of fibro-blast adhesion and long-term growth, Anal. Biochem. 172 (1988) 270–278.

[102] D.L. Hern, J.A. Hubbell, Incorporation of adhesion peptides intononadhesive hydrogels useful for tissue resurfacing, J. Biomed. Mater.Res. 39 (1998) 266–276.

[103] U.Hersel, C.Dahmen,H.Kessler, RGDmodified polymers: biomaterials forstimulated cell adhesion and beyond, Biomaterials 24 (2003) 4385–4415.

[104] R.L. Schnaar, B.G. Langer, B.K. Brandley, Reversible covalentimmobilization of ligands and proteins on polyacrylamide gels, Anal.Biochem. 151 (1985) 268–281.

[105] C.R. Nuttelman, M.C. Tripodi, K.S. Anseth, Synthetic hydrogel nichesthat promote hMSC viability, Matrix Biol. 24 (2005) 208–218.

[106] N.S. Hwang, S. Varghese, Z. Zhang, J. Elisseeff, Chondrogenicdifferentiation of human embryonic stem cell-derived cells in arginine-glycine-aspartate-modified hydrogels, Tissue Eng. 12 (2006) 2695–2706.

[107] H. Shin, J.S. Temenoff, G.C. Bowden, K. Zygourakis, M.C. Farach-Carson, M.J. Yaszemski, A.G. Mikos, Osteogenic differentiation of ratbone marrow stromal cells cultured on Arg-Gly-Asp modified hy-drogels without dexamethasone and beta-glycerol phosphate, Bioma-terials 26 (2005) 3645–3654.

[108] H. Shin, K. Zygourakis, M.C. Farach-Carson,M.J. Yaszemski, A.G.Mikos,Attachment, proliferation, and migration of marrow stromal osteoblastscultured on biomimetic hydrogels modified with an osteopontin-derivedpeptide, Biomaterials 25 (2004) 895–906.

[109] T. Konno, N. Kawazoe, G. Chen, Y. Ito, Culture of mouse embryonic stemcells on photoimmobilized polymers, J. Biosci. Bioeng. 102 (2006) 304–310.

[110] A.J. Melville, J. Harrison, K.A. Gross, J.S. Forsythe, A.O. Trounson, R.Mollard, Mouse embryonic stem cell colonisation of carbonated apatitesurfaces, Biomaterials 27 (2006) 615–622.

[111] S. Taqvi, L. Dixit, K. Roy, Biomaterial-based notch signaling for thedifferentiation of hematopoietic stem cells into T cells, J. Biomed. Mater.Res. A 79 (2006) 689–697.

[112] D.A. Wang, C.G. Williams, F. Yang, N. Cher, H. Lee, J.H. Elisseeff,Bioresponsive phosphoester hydrogels for bone tissue engineering,Tissue Eng. 11 (2005) 201–213.

[113] J. Wang, H.Q. Mao, K.W. Leong, A novel biodegradable gene carrierbased on polyphosphoester, J. Am. Chem. Soc. 123 (2001) 9480–9481.

[114] G. Zhang, X. Wang, Z. Wang, J. Zhang, L. Suggs, A PEGylated fibrinpatch for mesenchymal stem cell delivery, Tissue Eng. 12 (2006) 9–19.

[115] J.H. Cho, S.H. Kim, K.D. Park, M.C. Jung, W.I. Yang, S.W. Han, J.Y.Noh, J.W. Lee, Chondrogenic differentiation of human mesenchymal


stem cells using a thermosensitive poly(N-isopropylacrylamide) andwater-soluble chitosan copolymer, Biomaterials 25 (2004) 5743–5751.

[116] E. Ruel-Gariepy, M. Shive, A. Bichara, M. Berrada, D. Le Garrec, A.Chenite, J.C. Leroux, A thermosensitive chitosan-based hydrogel for thelocal delivery of paclitaxel, Eur. J. Pharm. Biopharm. 57 (2004) 53–63.

[117] J.M. Dang, D.D. Sun, Y. Shin-Ya, A.N. Sieber, J.P. Kostuik, K.W. Leong,Temperature-responsive hydroxybutyl chitosan for the culture of mesen-chymal stem cells and intervertebral disk cells, Biomaterials 27 (2006)406–418.

[118] J. Harrison, S. Pattanawong, J.S. Forsythe, K.A. Gross, D.R. Nisbet, H.Beh, T.F. Scott, A.O. Trounson, R. Mollard, Colonization andmaintenance of murine embryonic stem cells on poly(alpha-hydroxyesters), Biomaterials 25 (2004) 4963–4970.

[119] C.R. Nuttelman, M.C. Tripodi, K.S. Anseth, Dexamethasone-functiona-lized gels induce osteogenic differentiation of encapsulated hMSCs, J.Biomed. Mater. Res. A 76 (2006) 183–195.

[120] C.A. Burks, K. Bundy, P. Fotuhi, E. Alt, Characterization of 75:25 poly(L-lactide-co-epsilon-caprolactone) thin films for the endoluminaldelivery of adipose-derived stem cells to abdominal aortic aneurysms,Tissue Eng. 12 (2006) 2591–2600.

[121] T.A. Holland, Y. Tabata, A.G. Mikos, Dual growth factor delivery fromdegradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds forcartilage tissue engineering, J. Control. Release 101 (2005) 111–125.

[122] Z. Lalani, M. Wong, E.M. Brey, A.G. Mikos, P.J. Duke, Spatial andtemporal localization of transforming growth factor-beta1, bonemorphogenetic protein-2, and platelet-derived growth factor-A inhealing tooth extraction sockets in a rabbit model, J. Oral Maxillofac.Surg. 61 (2003) 1061–1072.

[123] H. Park, J.S. Temenoff, T.A. Holland, Y. Tabata, A.G. Mikos, Delivery ofTGF-beta1 and chondrocytes via injectable, biodegradable hydrogels forcartilage tissue engineering applications, Biomaterials 26 (2005)7095–7103.

[124] M.C. Wake, P.D. Gerecht, L. Lu, A.G. Mikos, Effects of biodegradablepolymer particles on rat marrow-derived stromal osteoblasts in vitro,Biomaterials 19 (1998) 1255–1268.

[125] S.J. Peter, L. Lu, D.J. Kim, G.N. Stamatas, M.J. Miller, M.J. Yaszemski,A.G. Mikos, Effects of transforming growth factor beta1 released frombiodegradable polymer microparticles on marrow stromal osteoblastscultured on poly(propylene fumarate) substrates, J. Biomed. Mater. Res.50 (2000) 452–462.

[126] E.S. Ng, R.P. Davis, L. Azzola, E.G. Stanley, A.G. Elefanty, Forcedaggregation of defined numbers of human embryonic stem cells intoembryoid bodies fosters robust, reproducible hematopoietic differentia-tion, Blood 106 (2005) 1601–1603.

[127] A. Khademhosseini, L. Ferreira, J. Blumling III, J. Yeh, J.M. Karp, J.Fukuda, R. Langer, Co-culture of human embryonic stem cells withmurine embryonic fibroblasts on microwell-patterned substrates, Bioma-terials 27 (2006) 5968–5977.

[128] J.C. Mohr, J.J. de Pablo, S.P. Palecek, 3-D microwell culture of humanembryonic stem cells, Biomaterials 27 (2006) 6032–6042.

[129] A. Khademhosseini, G. Eng, J. Yeh, J. Fukuda, J. Blumling III, R. Langer, J.A. Burdick, Micromolding of photocrosslinkable hyaluronic acid for cellencapsulation and entrapment, J. Biomed.Mater. Res. A 79 (2006) 522–532.

[130] D.G. Anderson, S. Levenberg, R. Langer, Nanoliter-scale synthesis ofarrayed biomaterials and application to human embryonic stem cells, Nat.Biotechnol. 22 (2004) 863–866.

[131] D.G. Anderson, D. Putnam, E.B. Lavik, T.A. Mahmood, R. Langer,Biomaterial microarrays: rapid, microscale screening of polymer–cellinteraction, Biomaterials 26 (2005) 4892–4897.

[132] C.J. Flaim, S. Chien, S.N. Bhatia, An extracellular matrix microarray forprobing cellular differentiation, Nat. Methods 2 (2005) 119–125.

[133] A. Khademhosseini, R. Langer, J. Borenstein, J.P. Vacanti, Microscaletechnologies for tissue engineering and biology, Proc.Natl. Acad. Sci. U. S. A.103 (2006) 2480–2487.

Biomaterials for stem cell differentiation

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