The role of biomaterials in stem cell differentiation: applications in the musculoskeletal system

The capabilities of stem cells continue to be revealed, leading to excitement regarding potential new therapies. Regenerative medicine is an area in which stem cells hold great promise for overcoming the challenge of limited cell sources for tissue repair. Biomaterials play an important role in directing tissue growth and may provide another tool to manipulate and control stem cell behavior. Biomaterials are made from natural or synthetic polymers and can be processed into three-dimensional scaffolds designed to promote cell proliferation and/or differentiation that ultimately produces new tissue. Stem cells will have a significant impact on the fields of regenerative medicine and tissue engineering as a powerful cell source that will work, in conjunction with biomaterials, to treat tissue and organ loss. Herein, we survey our latest research on applying embryonic stem (ES) cells to hydrogel biomaterials for engineering musculoskeletal tissues, emphasizing the unique biomaterial requirements of ES cells for differentiation and tissue development.     

The Advancement of Biomaterials in Regulating Stem Cell Fate

Stem cells are well-known to have prominent roles in tissue engineering applications. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can differentiate into every cell type in the body while adult stem cells such as mesenchymal stem cells (MSCs) can be isolated from various sources. Nevertheless, an utmost limitation in harnessing stem cells for tissue engineering is the supply of cells. The advances in biomaterial technology allows the establishment of ex vivo expansion systems to overcome this bottleneck. The progress of various scaffold fabrication could direct stem cell fate decisions including cell proliferation and differentiation into specific lineages in vitro. Stem cell biology and biomaterial technology promote synergistic effect on stem cell-based regenerative therapies. Therefore, understanding the interaction of stem cell and biomaterials would allow the designation of new biomaterials for future clinical therapeutic applications for tissue regeneration. This review focuses mainly on the advances of natural and synthetic biomaterials in regulating stem cell fate decisions. We have also briefly discussed how biological and biophysical properties of biomaterials including wettability, chemical functionality, biodegradability and stiffness play their roles.     

Human mesenchymal stem cell differentiation on self-assembled monolayers presenting different surface chemistries

Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression and function during hMSC commitment along osteogenic, chondrogenic and adipogenic lineages. Using self-assembled monolayers of ω-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long-term functional differentiation of adult stem cells, and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.     

Three-dimensional collagen fiber remodeling by mesenchymal stem cells requires the integrin-matrix interaction

Tissue engineering aiming to repair or regenerate damaged tissues necessitates fabricating three-dimensional biomaterial scaffolds with controlled porosity for delivering cells. To facilitate cell distribution, a strategy using stem cell-based fabrication of biomaterials was tested in type II collagen fibers. Human mesenchymal stem cells when delivered in type II collagen assembled and reorganized these matrices and differentiated into spherical chondrocytes with the synthesis of cartilage proteins. The cell-mediated assembly and reorganization of collagen fibers was not limitless and only restricted to an appropriate ratio of cell number and collagen amount. The blocking of alpha2 or beta1-integrin function with specific antibodies significantly impeded the collagen-assembly effects. In vitro chondrogenesis or in vivo cartilage formation of human mesenchymal stem cells was also dependent on the interactions between cells and surrounding matrices. This method for three-dimensional fabricating collagen fibers may generally be applied to other biomaterials, when combined with surface modification or ligand addition for cell adhesion.     

Combining adult stem cells and polymeric devices for tissue engineering in infarcted myocardium

An increasing number of studies in cardiac cell therapy have provided encouraging results for cardiac repair. Adult stem cells may overcome ethical and availability concerns, with the additional advantages, in some cases, to allow autologous grafts to be performed. However, the major problems of cell survival, cell fate determination and engraftment after transplantation, still remain. Tissue-engineering strategies combining scaffolds and cells have been developed and have to be adapted for each type of application to enhance stem cell function. Scaffold properties required for cardiac cell therapy are here discussed. New tissue engineering advances that may be implemented in combination with adult stem cells for myocardial infarction therapy are also presented. Biomaterials not only provide a 3D support for the cells but may also mimic the structural architecture of the heart. Using hydrogels or particulate systems, the biophysical and biochemical microenvironments of transplanted cells can also be controlled. Advances in biomaterial engineering have permitted the development of sophisticated drug-releasing materials with a biomimetic 3D support that allow a better control of the microenvironment of transplanted cells.     

Engineering articular cartilage with spatially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel

Despite significant advances in stem cell differentiation and tissue engineering, directing progenitor cells into three-dimensionally (3D) organized, native-like complex structures with spatially-varying mechanical properties and extra-cellular matrix (ECM) composition has not yet been achieved. The key innovations needed to achieve this would involve methods for directing a single stem cell population into multiple, spatially distinct phenotypes or lineages within a 3D scaffold structure. We have previously shown that specific combinations of natural and synthetic biomaterials can direct marrow-derived stem cells (MSC) into varying phenotypes of chondrocytes that resemble cells from the superficial, transitional, and deep zones of articular cartilage. In this current study, we demonstrate that layer-by-layer organization of these specific biomaterial compositions creates 3D niches that allow a single MSC population to differentiate into zone-specific chondrocytes and organize into a complex tissue structure. Our results indicate that a three-layer polyethylene glycol (PEG)-based hydrogel with chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) incorporated into the top layer (superficial zone, PEG:CS:MMP-pep), CS incorporated into the middle layer (transitional zone, PEG:CS) and hyaluronic acid incorporated in the bottom layer (deep zone, PEG:HA), creates native-like articular cartilage with spatially-varying mechanical and biochemical properties. Specifically, collagen II levels decreased gradually from the superficial to the deep zone, while collagen X and proteoglycan levels increased, leading to an increasing gradient of compressive modulus from the superficial to the deep zone. We conclude that spatially-varying biomaterial compositions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population.     

Biomaterial selection for tooth regeneration

Biomaterials are native or synthetic polymers that act as carriers for drug delivery or scaffolds for tissue regeneration. When implanted in vivo, biomaterials should be nontoxic and exert intended functions. For tooth regeneration, biomaterials have primarily served as a scaffold for (1) transplanted stem cells and/or (2) recruitment of endogenous stem cells. This article critically synthesizes our knowledge of biomaterial use in tooth regeneration, including the selection of native and/or synthetic polymers, three-dimensional scaffold fabrication, stem cell transplantation, and stem cell homing. A tooth is a complex biological organ. Tooth loss represents the most common organ failure. Tooth regeneration encompasses not only regrowth of an entire tooth as an organ, but also biological restoration of individual components of the tooth including enamel, dentin, cementum, or dental pulp. Regeneration of tooth root represents perhaps more near-term opportunities than the regeneration of the whole tooth. In the adult, a tooth owes its biological vitality, arguably more, to the root than the crown. Biomaterials are indispensible for the regeneration of tooth root, tooth crown, dental pulp, or an entire tooth.     

Stem cell-based tissue engineering with silk biomaterials

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications.     

Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold

Functional engineering of musculoskeletal tissues generally involves the use of differentiated or progenitor cells seeded with specific growth factors in biomaterial scaffolds. Ideally, the scaffold should be a functional and structural biomimetic of the native extracellular matrix and support multiple tissue morphogenesis. We have previously shown that electrospun, three-dimensional nanofibrous scaffolds that morphologically resemble collagen fibrils are capable of promoting favorable biological responses from seeded cells, indicative of their potential application for tissue engineering. In this study, we tested a three-dimensional nanofibrous scaffold fabricated from poly(epsilon-caprolactone) (PCL) for its ability to support and maintain multilineage differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in vitro. hMSCs were seeded onto pre-fabricated nanofibrous scaffolds, and were induced to differentiate along adipogenic, chondrogenic, or osteogenic lineages by culturing in specific differentiation media. Histological and scanning electron microscopy observations, gene expression analysis, and immunohistochemical detection of lineage-specific marker molecules confirmed the formation of three-dimensional constructs containing cells differentiated into the specified cell types. These results suggest that the PCL-based nanofibrous scaffold is a promising candidate scaffold for cell-based, multiphasic tissue engineering.     

Natural ECM as Biomaterial for Scaffold Based Cardiac Regeneration Using Adult Bone Marrow Derived Stem Cells

Cellular therapy using stem cells for cardiac diseases has recently gained much interest in the scientific community due to its potential in regenerating damaged and even dead tissue and thereby restoring the organ function. Stem cells from various sources and origin are being currently used for regeneration studies directly or along with differentiation inducing agents. Long term survival and minimal side effects can be attained by using autologous cells and reduced use of inducing agents. Cardiomyogenic differentiation of adult derived stem cells has been previously reported using various inducing agents but the use of a potentially harmful DNA demethylating agent 5-azacytidine (5-azaC) has been found to be critical in almost all studies. Alternate inducing factors and conditions/stimulant like physical condition including electrical stimulation, chemical inducers and biological agents have been attempted by numerous groups to induce cardiac differentiation. Biomaterials were initially used as artificial scaffold in in vitro studies and later as a delivery vehicle. Natural ECM is the ideal biological scaffold since it contains all the components of the tissue from which it was derived except for the living cells. Constructive remodeling can be performed using such natural ECM scaffolds and stem cells since, the cells can be delivered to the site of infraction and once delivered the cells adhere and are not “lost”. Due to the niche like conditions of ECM, stem cells tend to differentiate into tissue specific cells and attain several characteristics similar to that of functional cells even in absence of any directed differentiation using external inducers. The development of niche mimicking biomaterials and hybrid biomaterial can further advance directed differentiation without specific induction. The mechanical and electrical integration of these materials to the functional tissue is a problem to be addressed. The search for the perfect extracellular matrix for therapeutic applications including engineering cardiac tissue structures for post ischemic cardiac tissue regeneration continues.     

A functional biphasic biomaterial homing mesenchymal stem cells for in vivo cartilage regeneration

Cartilage regeneration after trauma is still a great challenge for clinicians and researchers due to many reasons, such as joint load-bearing, synovial movement and the paucity of endogenous repair cells. To overcome these limitations, we constructed a functional biomaterial using a biphasic scaffold platform and a bone-derived mesenchymal stem cells (BMSCs)-specific affinity peptide. The biphasic scaffold platform retains more cells homogeneously within the sol–gel transition of chitosan and provides sufficient solid matrix strength. This biphasic scaffold platform is functionalized with an affinity peptide targeting a cell source of interest, BMSCs. The presence of conjugated peptide gives this system a biological functionality towards BMSC-specific homing both in vitro and in vivo. The functional biomaterial can stimulate stem cell proliferation and chondrogenic differentiation during in vitro culture. Six months after in vivo implantation, compared with routine surgery or control scaffolds, the functional biomaterials induced superior cartilage repair without complications, as indicated by histological observations, magnetic resonance imaging and biomechanical properties. Beyond cartilage repair, this functional biphasic scaffold may provide a biomaterial framework for one-step tissue engineering strategy by homing endogenous cells to stimulate tissue regeneration.     

Therapeutic application of mesenchymal stem cells in orthopaedics

Stem cell therapy of skeletal tissues involves the transplantation of stem cells to the tissues that have been damaged by injury or disease. Although these cells can be derived from embryos, the preferred source of skeletal stem cells is the bone marrow as it contains adult stem cells that can be easily driven towards a bone phenotype. More recently, cells with similar potentialities have also been derived from adipose tissue, muscle, or blood. A biomaterial (ceramics or polymers) is often required as a scaffold to promote cell adhesion, proliferation and differentiation as well as encourage vascular invasion and ultimately new bone formation. The first clinical studies are encouraging and suggests that stem cell therapy could be a prime method for bone reconstruction.     

Analysis of progenitor cell-scaffold combinations by in vivo non-invasive photonic imaging

Recent developments in stem cell research have promoted a flourishing of new biomaterials and scaffolds for tissue repair. However, there is a scarcity of procedures to monitor the performance of scaffold-stem cell combinations implanted in live animals, avoiding the inherent artefacts associated with in vitro assay conditions. We report the implementation of a procedure based on the use of the luciferase gene as a cell proliferation tracer to monitor, by in vivo non-invasive imaging, the performance of stem cell-biomaterial combinations used for tissue regeneration. In a model system using immunodepressed mice we show preference of a mouse embryonic mesenchymal cell line (C3H/10T1/2) for specific implantation sites and biomaterials during a prolonged in vivo growth period (3 months). Moreover, we analyzed the safety of implanted cells using a sensitive luminometric procedure and showed that the implanted cells did not spread to other organs. Our results demonstrate the utility of this simple and resource-saving procedure in the development and screening of biomaterials for tissue engineering.     

The effect of cerium valence states at cerium oxide nanoparticle surfaces on cell proliferation

Understanding and controlling cell proliferation on biomaterial surfaces is critical for scaffold/artificial-niche design in tissue engineering. The mechanism by which underlying integrin ligates with functionalized biomaterials to induce cell proliferation is still not completely understood. In this study, poly-l-lactide (PL) scaffold surfaces were functionalized using layers of cerium oxide nanoparticles (CNPs), which have recently attracted attention for use in therapeutic application due to their catalytic ability of Ce⁴⁺ and Ce³⁺ sites. To isolate the influence of Ce valance states of CNPs on cell proliferation, human mesenchymal stem cells (hMSCs) and osteoblast-like cells (MG63) were cultured on the PL/CNP surfaces with dominant Ce⁴⁺ and Ce³⁺ regions. Despite cell type (hMSCs and MG63 cells), different surface features of Ce⁴⁺ and Ce³⁺ regions clearly promoted and inhibited cell spreading, migration and adhesion behavior, resulting in rapid and slow cell proliferation, respectively. Cell proliferation results of various modified CNPs with different surface charge and hydrophobicity/hydrophilicity, indicate that Ce valence states closely correlated with the specific cell morphologies and cell–material interactions that trigger cell proliferation. This finding suggests that the cell–material interactions, which influence cell proliferation, may be controlled by introduction of metal elements with different valence states onto the biomaterial surface.     

Intervertebral disc cell-mediated mesenchymal stem cell differentiation

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.     

Compatibility of embryonic stem cells with biomaterials

Periodontal bone defects and atrophy of the jaws in an aging population are of special concern. Tissue engineering using embryonic stem cells (ESCs) and biomaterials may offer new therapeutic options. The purpose of this study is to evaluate the compatibility of ESCs with biomaterials and the influence of biomaterials on the osteogenic gene expression profile.Therefore, ESCs are cultured with various biomaterials. The cytocompatibility of murine ESCs is measured regarding the proliferation of the cells on the materials by CyQUANT assay, the morphology by scanning electron microscopy, and the influence on the gene expression by real time PCR.The results show that insoluble collagenous bone matrix, followed by beta-tricalciumphosphate, is most suitable for bone tissue engineering regarding cell proliferation, and phenotype. The gene expression analysis indicates that biomaterials do influence the gene expression of ESCs.Our results provide new insight into the cytocompatibility of ESCs on different scaffolds.     

Multifunctional cell-instructive materials for tissue regeneration

Current approaches in tissue engineering and regenerative medicine have focused on controlling the presentation of various factors that influence cellular behavior and tissue formation. Numerous biomaterials have been utilized as sites for new tissue growth by migrating or transplanted cells, nanoscale control of cellular behavior through the presentation of specific peptide sequences, and depots for growth factor release. More recently, the development of bioresponsive materials has emerged as a promising approach to cede control of temporal macromolecule presentation and material degradation to invading cell populations. Biomaterials now have the potential of possessing multiple functions in the process of tissue regeneration. This review summarizes some of the recent advances in the use of multifunctional biomaterials in the arena of tissue engineering. Specifically, the potential of various materials is described as it pertains to the control of cellular behavior, integration of engineered materials with host or transplanted tissue, and inductive factor presentation.     

Mimicking cell-cell interactions at the biomaterial-cell interface for control of stem cell differentiation

The ability to regulate stem cell proliferation and differentiation has relevance in numerous medical applications, including medical devices, tissue engineering, and regenerative medicine. To control cellular behavior at the biomaterial or scaffold interface, many studies have employed surface modifications that mimic the extracellular matrix. Strikingly absent is the immobilization of cell-surface ligands to the biomaterial surface. One cell-to-cell signaling pathway that has been shown to regulate tissue development and stem cell fate is the Notch pathway. Recently, the Notch signaling pathway was identified as a key regulator of epithelial differentiation. Utilizing this knowledge, we applied an affinity immobilization scheme designed to attach and orient the Notch ligand, Jagged-1, in an active conformation on a biomaterial surface. When epithelial stem cells were plated on the bound ligand, the Notch/CBF-1 signaling pathway was stimulated and the cells upregulated both intermediate- and late-stage differentiation markers. In addition, the ligand promoted tight clustering and extensive stratification. Soluble Jagged-1 showed no Notch/CBF-1 signaling and very little, if any, cell differentiating activity. The high potency of bound Jagged-1 suggests that modification of a surface with a Notch ligand presents a powerful method to control stem cell differentiation at the cell-biomaterial interface.     

Biomaterials based strategies for skeletal muscle tissue engineering: Existing technologies and future trends

Skeletal muscles have a robust capacity to regenerate, but under compromised conditions, such as severe trauma, the loss of muscle functionality is inevitable. Research carried out in the field of skeletal muscle tissue engineering has elucidated multiple intrinsic mechanisms of skeletal muscle repair, and has thus sought to identify various types of cells and bioactive factors which play an important role during regeneration. In order to maximize the potential therapeutic effects of cells and growth factors, several biomaterial based strategies have been developed and successfully implemented in animal muscle injury models. A suitable biomaterial can be utilized as a template to guide tissue reorganization, as a matrix that provides optimum micro-environmental conditions to cells, as a delivery vehicle to carry bioactive factors which can be released in a controlled manner, and as local niches to orchestrate in situ tissue regeneration. A myriad of biomaterials, varying in geometrical structure, physical form, chemical properties, and biofunctionality have been investigated for skeletal muscle tissue engineering applications. In the current review, we present a detailed summary of studies where the use of biomaterials favorably influenced muscle repair. Biomaterials in the form of porous three-dimensional scaffolds, hydrogels, fibrous meshes, and patterned substrates with defined topographies, have each displayed unique benefits, and are discussed herein. Additionally, several biomaterial based approaches aimed specifically at stimulating vascularization, innervation, and inducing contractility in regenerating muscle tissues are also discussed. Finally, we outline promising future trends in the field of muscle regeneration involving a deeper understanding of the endogenous healing cascades and utilization of this knowledge for the development of multifunctional, hybrid, biomaterials which support and enable muscle regeneration under compromised conditions.