2010 Nicolas Andry Award: Multipotent Adult Stem Cells from Adipose Tissue for Musculoskeletal Tissue Engineering

Abstract

Background

Cell-based therapies such as tissue engineering provide promising therapeutic possibilities to enhance the repair or regeneration of damaged or diseased tissues but are dependent on the availability and controlled manipulation of appropriate cell sources.

Questions/purposes

The goal of this study was to test the hypothesis that adult subcutaneous fat contains stem cells with multilineage potential and to determine the influence of specific soluble mediators and biomaterial scaffolds on their differentiation into musculoskeletal phenotypes.

Methods

We reviewed recent studies showing the stem-like characteristics and multipotency of adipose-derived stem cells (ASCs), and their potential application in cell-based therapies in orthopaedics.

Results

Under controlled conditions, ASCs show phenotypic characteristics of various cell types, including chondrocytes, osteoblasts, adipocytes, neuronal cells, or muscle cells. In particular, the chondrogenic differentiation of ASCs can be induced by low oxygen tension, growth factors such as bone morphogenetic protein-6 (BMP-6), or biomaterial scaffolds consisting of native tissue matrices derived from cartilage. Finally, focus is given to the development of a functional biomaterial scaffold that can provide ASC-based constructs with mechanical properties similar to native cartilage.

Conclusions

Adipose tissue contains an abundant source of multipotent progenitor cells. These cells show cell surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow mesenchymal stem cells (MSCs).

Clinical Relevance

The availability of an easily accessible and reproducible cell source may greatly facilitate the development of new cell-based therapies for regenerative medicine applications in the musculoskeletal system.

Introduction

Tissue engineering seeks to repair or replace damaged or diseased tissues of the body by implanting combinations of cells, biomaterial scaffolds, biologically active molecules, and genes. An underlying premise of this approach is that exogenously introduced cells will improve the speed and extent of tissue repair. To this end, there is a significant need for potential sources of cells for tissue engineering and other cell-based therapeutic approaches, such as gene therapy.

In recent years, there has been a growing emphasis on the use of undifferentiated progenitor cells for tissue engineering owing to their ability to be expanded in culture and to differentiate into multiple cell types. Although historically there has been controversy regarding the presence of true adult stem cells outside the hematopoietic system, it now is apparent that many adult tissues harbor cells that have the ability to differentiate into multiple cell types, once cultured under specific growth conditions [18]. Depending on their differentiation potential and site of origin, these cells have been described using various terms, such as MSCs, multipotent adult progenitor cells, marrow stromal cells, or mesenchymal progenitors [13, 15, 17, 44, 68, 69, 72–74, 80, 85]. The adult stem cell can be defined as an “undifferentiated (unspecialized) cell that is found in a differentiated (specialized) tissue; it can renew itself and become specialized to yield all of the specialized cell types of the tissue from which it originated” [19]. Because of these characteristics, adult stem cells have been used extensively in various musculoskeletal tissue engineering applications.

We have identified the presence of an abundant source of undifferentiated progenitor cells residing in various adipose tissue sites in the human body. These cells, termed ASCs, or adipose-derived adult stromal (ADAS) cells, show cell surface marker profiles and differentiation characteristics similar to other adult stem cells, such as bone marrow-derived MSCs. Under controlled culture conditions, these cells show phenotypic characteristics of numerous cell types, including chondrocytes, osteoblasts, adipocytes, neuronal cells, or muscle cells. These characteristics are present at the clonal level, suggesting that individual cells have multipotent capabilities. The major advantages of such cells are their ease of availability through standard liposuction procedures and their abundance in normal human fat.

We will review our recent studies that show the stem-like properties and multipotency of ASCs, and their promise for use in cell-based therapies and functional tissue replacement for the musculoskeletal system. In particular, we focus on the application of ASCs for regeneration or repair of cartilaginous tissues, addressing the following questions: (1) Does adult adipose tissue contain multipotent stem cells?; (2) Are these cells capable of chondrogenic differentiation?; (3) What are the influences of various growth factors, biomaterial scaffolds, and environmental cues such as oxygen tension on ASC chondrogenesis?; (4) What effect do these factors have on the functional biomechanical properties of ASC-based constructs?

Background: Articular Cartilage Injury and Repair

Articular cartilage functions as a nearly frictionless load-bearing surface in diarthrodial joints, withstanding loads of several times body weight for decades [67]. The cartilage extracellular matrix is maintained by a sparse population of cells (chondrocytes) but exhibits little capacity for self-repair owing to the lack of a tissue blood supply or a source of undifferentiated cells that can promote repair. Isolated cartilage lesions may be responsible for significant pain or loss of function, and may lead to degenerative arthritis in the joint with time [42, 43]. To promote cartilage repair, several surgical techniques have been developed to induce bleeding and clot formation, including drilling or microfracture of the subchondral bone [1, 8, 29, 31, 43]. Numerous tissue engineering approaches have been proposed for enhancing cartilage regeneration in vitro or in vivo [2, 12, 14, 30, 34, 41, 55, 56, 70, 77, 79, 81].

However, significant challenges continue to exist in the long-term repair or replacement of cartilage, and there is currently one cell-based therapy available clinically for cartilage repair that involves implantation of autologous isolated chondrocytes (Carticel™; Genzyme Biosurgery, Cambridge, MA, USA) [10]. Although clinical studies of autologous chondrocyte transplantation generally have reported significant improvements in pain, swelling, and function after surgery [9, 60], one controlled prospective trial showed no benefit of autologous chondrocyte implantation as compared with the standard treatment of microfracture of the subchondral bone, as based on indices of joint function, pain, or characteristics of the cartilage defect [47]. Furthermore, an animal study suggested the harvest procedure may induce donor-site morbidity and iatrogenic damage that may initiate osteoarthritic degeneration in the joint [50]. Thus, such procedures clearly would benefit from the availability of a safe and abundant source of chondrocyte progenitor cells for cartilage repair.

Adipose Tissue as a Source of Multipotent Adult Stem Cells

The isolation procedure of ASCs from adipose tissue has been detailed [23]. In brief, liposuction tissue is washed thoroughly in phosphate buffered saline to remove contaminating erythrocytes, and then digested using type I collagenase. The stromal-vascular fraction of cells is separated from the mature lipid-laden adipocytes by centrifugation. This fraction, which represents a heterogeneous population of cells, contains the ASCs [37, 86]. A large number of ASCs can be harvested in this manner, with yields of approximately 250,000 cells per gram of tissue [3]. This population of cells can be further expanded and purified on tissue culture plastic, yielding approximately 10⁹ cells after 2 weeks of expansion. During expansion, our studies show that ASCs do not exhibit markers of hematopoietic stem cells (eg, CD45 and CD14) but display a similar marker surface profile to marrow derived MSCs [32, 83], although, it generally is recognized there are no specific markers readily available to identify nonhematopoietic stem cells. Nevertheless, ASCs express the stromal markers CD9, CD10, CD29, CD44, CD73, CD90, and CD166, and with increasing passage, the expression of these markers increases while the presence of hematopoietic markers declines [57, 61].

To examine the multipotency of individual cells, ASCs were cultured and ring cloning was performed to select cells derived from one progenitor cell [36]. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histologic and biochemical analyses. Our findings showed that 81% of the ASC clones differentiated into at least one of the lineages (Fig. 1). In addition, 52% of the ASC clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly owing to the loss of adipogenic ability after repeated subcultures. These findings support the hypothesis that ASCs are a type of multipotent adult stem cell and not simply a mixed population of unipotent progenitor cells.

Fig. 1A–B.

(A) Under specific and controlled culture conditions, ASCs can be induced to express the phenotypic characteristics of chondrocytes, osteoblasts, adipocytes, or neurons. (B) Forty-five individual clones were expanded through four passages in culture and then differentiated to adipogenic, osteogenic, chondrogenic, and neurogenic lineages. Fifty-two percent of the cell clones showed stem cell characteristics by displaying bipotent and tripotent differentiation potential. (Reproduced with permission from Guilak F, Awad H, Fermor B, Leddy HA, Gimble JM. Adipose-derived adult stem cells for cartilage tissue engineering. Biorheology. 2004;41:389–399.) [33]

Chondrogenic Potential of Human ASCs

Our work has focused primarily on the ability of ASCs to produce cartilaginous tissue molecules for potential application in the repair of tissues such as articular cartilage, meniscus, or intervertebral disc (eg, [4, 20, 22, 26, 27, 82, 83]). Through numerous studies, it was shown that under specific conditions, ASCs can express the genes and proteins for several cartilage-specific molecules, including type II collagen and aggrecan, without expression of hypertrophic chondrocyte markers such as type X collagen [26]. Nonetheless, under different defined conditions, ASCs can be induced to synthesize type I and type II collagen, suggesting that a fibrocartilaginous phenotype also is possible [5].

The medium used to induce chondrogenesis was based on that developed by Johnstone et al. for inducing similar differentiation of bone marrow derived MSCs [45]. When maintained in pellet culture or encapsulated in alginate beads and cultured with 10 ng/mL of transforming growth factor beta 1 (TGF-β1), ascorbate, and dexamethasone, ASCs have been shown to express a chondrocyte-like phenotype and synthesize collagen type II, aggrecan, link protein, and chondroitin sulfate in a time-dependent manner based on mRNA analysis, immunohistochemistry, and radiolabel incorporation [4, 22, 83], with significant enhancement of proteoglycan and protein synthesis under chondrogenic conditions. Winter et al. reported that the gene expression profile of ASCs under chondrogenic conditions is similar to that of MSCs [39]. Following an in vitro differentiation process, Erickson et al. showed that human ASCs retain the chondrocyte phenotype and form cartilaginous tissue when implanted subcutaneously in vivo in immunodeficient mice for up to 12 weeks [22].

The Influence of Culture Conditions on ASC Chondrogenesis

The combinations of growth factors and media supplements used to induce chondrogenic differentiation of progenitor cells such as bone marrow MSCs have been defined empirically, and may depend on numerous factors such as concentration, duration of exposure, or cell type. In a series of studies, it was shown that the chondrogenic potential of ASCs depends on numerous culture conditions such as the expansion/growth media used, the number of passages, and the composition of the extracellular matrix [4, 20, 24, 26, 27].

In one study, more than 27 combinations of growth factors, fetal bovine serum (FBS), and other media supplements were studied to examine the influence of these soluble mediators on ASC chondrogenesis [4]. The findings showed that these factors may act in an additive or synergistic manner, depending on concentration and time of exposure. For example, the serum substitute ITS + (insulin, transferring, and selenious acid) and TGF-β1 function to increase ASC proliferation. Similarly, TGF-β1 and dexamethasone promote protein synthesis rates in an additive manner in the presence of ITS + or FBS. Of particular note was the finding that dexamethasone, which often is used as a media supplement for chondrogenesis, suppresses the ability of TGF-β1 to stimulate proteoglycan synthesis and accumulation by 1.5- to twofold.

Using further screens of numerous different growth factors, it was found that BMP-6 applied in solution [26] or through genetic transfection of ASCs [20] greatly enhances the chondrogenic potential of ASC cells encapsulated in alginate beads. In particular, soluble BMP-6 upregulated aggrecan and collagen II expression by an average of 205-fold and 38-fold, respectively, over Day 0 controls, while downregulating collagen X expression (as a marker of hypertrophic differentiation) by twofold. These changes in mRNA expression levels were paralleled at the protein level (Fig. 2). In contrast to bone marrow MSCs, which exhibit increased expression of type X collagen in response to BMP-6, these findings suggest that BMP-6 serves as a potent regulator of ASC chondrogenesis. This finding has been confirmed, and data indicate that BMP-6 may be especially important for ASC chondrogenesis owing to differences in TGF-β receptor expression as compared with MSCs [39].

Fig. 2A–D.

Immunohistochemistry of human ASCs in alginate beads after 7 days in culture showed the presence of chondrogenic markers in the presence of TGF-β1 or BMP-6. (A) The standard growth factor combination of TGF-β1 + dexamethasone (DEX) used for chondrogenesis of MSCs induced the production of type II collagen by ASCs. (B) Collagen II labeling was increased in the presence of BMP-6 as compared with TGF-β1 + DEX. (C) TGF-β1 + DEX increased type X collagen labeling in ASCs, suggestive of a hypertrophic phenotype. (D) Type X collagen expression decreased with BMP-6 treatment. (Reproduced with permission from Estes BT, Wu AW, Guilak F. Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6. Arthritis Rheum. 2006;54:1222–1232.) [26]

The Influence of Biomaterial Scaffolds on ASC Chondrogenesis

In other studies, the influence of different biomaterial scaffolds on chondrogenesis of ASCs was investigated. In addition to the biochemical interactions that scaffolds may have with cell surface receptors, there is growing evidence of the important role that physical interactions between stem cells and their extracellular matrix have in regulating stem cell fate [25, 35]. One of the studies showed that different construct materials or structures can significantly influence the differentiation of ASCs and functional properties of the tissue-engineered construct [5]. For example, scaffolds that maintain the cells with a rounded shape and prevent cell-to-cell contact (eg, alginate or agarose) promote a chondrogenic phenotype and prevent the expression of type I collagen. Conversely, porous gelatin scaffolds (Surgifoam®, Johnson and Johnson, New Brunswick, NJ, USA) or fibrin-based scaffolds (Tisseel®, Baxter Bioscience, Westlake Village, CA, USA) also support the chondrogenic differentiation of ASCs but induce type I and type II collagen expression, suggesting the differentiation of ASCs into a fibrocartilaginous phenotype is associated with a more fibroblastic cell shape [48] (Fig. 3).

Fig. 3A–D.

Cell viability and morphologic features can be seen in different scaffold materials observed using confocal laser scanning microscopy and the Live-Dead fluorescent probes. Cells in (A) agarose and (B) alginate scaffolds had spherical morphologic features that persisted throughout the culture period, regardless of culture conditions. By contrast, the cells in the (C) gelatin scaffolds showed distinct fibroblastic morphologic features at Day 7. (D) By Day 28, the cells in the gelatin scaffolds proliferated and became confluent with significant cell-cell contact as they exerted considerable contraction of the scaffolds. Scale bar = 50 μm (A, B, C) or 200 μm (D). (Reproduced with permission from Awad HA, Wickham MQ, Leddy HA, Gimble JM, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials. 2004;25:3211–3222.) [5]

It also has been shown that a novel genetically engineered biomaterial scaffold can directly influence the chondrogenic potential of ASCs. This material consists of a genetically modified version of native elastin, termed an elastin-like polypeptide (ELP), which is thermally sensitive and undergoes an inverse temperature transition (ie, liquid at room temperature and a solid gel at body temperature) [59]. This property readily allows cellular encapsulation, providing an important advantage for cellular delivery in the context of tissue engineering [7]. Human ASCs were cultured in ELP hydrogels in either chondrogenic or standard media for 2 weeks, at which point, constructs in either medium showed significant increases in sulfated glycosaminoglycans (up to 100%) and collagen content (up to 420%), particularly collagen II with little formation of collagen I. At the mRNA level, ASCs seeded in ELP showed upregulated SOX9 and type II collagen gene expression, whereas type I collagen was downregulated. These findings provide additional evidence of the chondrogenic ability of ASCs and the ability of biomaterial scaffolds to directly control stem cell differentiation [35], even in the absence of exogenous growth factors [6].

In other studies, it was shown that biomaterial scaffolds created solely from native extracellular matrix tissues also can control the differentiation of ASCs [16, 21]. Using a porous scaffold derived exclusively from articular cartilage, chondrogenesis of ASCs in the absence of exogenous growth factors was examined. ASCs showed increased gene expression and biosynthesis of cartilage-specific extracellular matrix components, particularly type II collagen (Fig. 4), and mechanical testing showed significant increases in the mechanical properties of the ASC-seeded constructs with time, with a threefold increase in the aggregate modulus during 6 weeks in culture.

Fig. 4A–D.

A porous scaffold created purely from native articular cartilage supports chondrogenesis of ASCs. Shown are (A) the gross morphologic features of the cartilage-derived matrix scaffold, (B) a scanning electron micrograph of cartilage-derived matrix scaffold, (C) a microCT showing high porosity of cartilage-derived matrix scaffold, and (D) immunohistochemical samples showing chondrogenesis by ASCs in the porous cartilage derived scaffold. In the first row are blank scaffolds before seeding; in the second row are samples from Day 28; and in the third row are samples from Day 42. Scale bar = 1 mm (A, B, C) and 200 μm (D). (Reproduced with permission from Cheng NC, Estes BT, Awad HA, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix. Tissue Eng Part A. 2009;15:231–241.) [16]

The Role of Oxygen Tension in Chondrogenesis

Although it often is acknowledged that cell culture conditions used for tissue engineering fail to mimic the in vivo condition, it is unclear whether reproducing in vivo characteristics can influence chondrogenesis. For example, articular cartilage exists in a reduced oxygen (approximately 1%–5%) environment in vivo; therefore, oxygen tension has been hypothesized to be an important factor that regulates the metabolism of cartilage [62]. There is significant evidence that oxygen tension can influence the differentiation and biosynthetic activity of primary chondrocytes as they undergo passage in culture [67]. To examine the influence of oxygen on ASC chondrogenesis, human ASCs in alginate beads were cultured in control or chondrogenic media at either low oxygen (5%) or atmospheric oxygen tension (20%) for up to 14 days [82]. Low oxygen tension significantly inhibited the proliferation of ASCs, but induced a twofold increase in the rate of protein synthesis and a threefold increase in total collagen synthesis. Low oxygen tension also increased glycosaminoglycan synthesis at certain times. Immunohistochemical analysis showed significant production of cartilage-associated matrix molecules, including collagen type II and chondroitin-4-sulfate. These findings show that oxygen tension can play an important role in regulating the proliferation and differentiation of human ASCs as they undergo chondrogenesis and suggest that manipulation of the physiochemical culture environment may provide additional means of controlling the activity of undifferentiated progenitor cells in the context of bioreactors [28].

Functional Properties of Tissue-engineered Cartilage Constructs using ASCs

Changes in the biochemical composition of these tissue-engineered cartilage constructs with time also can influence their functional biomechanical and transport properties. For example, increases in the shear and compressive moduli were correlated with increased accumulation of matrix components such as proteoglycan and collagen content. It was shown that the composition and structure of the biomaterial scaffold also affect the diffusion properties of cartilage engineered from ASCs [48]. As cartilage (native or tissue engineered) is avascular, diffusion serves as the primary mechanism for macromolecular transport. The diffusion coefficients of fluorescent, uncharged dextran molecules (ranging from 3 to 500 KDa) were measured using a novel method for fluorescence recovery after photobleaching (FRAP) [49]. The findings showed that independent of the size of the molecule, the diffusion properties of ASC-engineered constructs depended on the biomaterial composition of the construct, presence of cells, growth factor culture conditions, and culture duration [48]. For example, after 4 weeks in culture, construct diffusivity decreased greater than 40% under chondrogenic conditions, and was associated with a net increase in the biosynthesis and retention of matrix macromolecules. Of note was the finding that the diffusivities of all the different constructs tested in culture were significantly greater than those of native articular cartilage, suggesting that nutrient and metabolite transport to cells in the constructs is not hindered during the early stages of tissue formation, as compared with native tissue [49]. From a functional standpoint, however, constructs created from gel-seeded ASCs or MSCs generally are unable to achieve the mechanical properties of native articular cartilage in a short period of culture time, particularly with respect to tension ([4, 40, 71, 78]). Thus there is an important need to develop scaffold materials that can provide biomechanical function for engineered constructs until the cells can synthesize and assemble a functional matrix [64]. To address this issue, a novel three-dimensionally woven fiber scaffold was developed that replicates the mechanical properties of native cartilage in tension, compression, and shear, while readily allowing consolidation and cell seeding [63]. These porous composite scaffolds can be designed using various combinations of bioresorbable fibers such as poly(glycolic acid) with initial properties that match the anisotropy, viscoelasticity, and tension-compression nonlinearity of native cartilage. Such scaffolds thus provide the potential for load-bearing immediately after cell seeding and implantation, allowing further maturation of the construct in vivo while minimizing in vitro culture times. In one study, a scaffold composed of three- dimensionally woven poly(ε-caprolactone) (PCL) was used that was consolidated with human ASCs encapsulated in fibrin gel and cultured for up to 4 weeks [65]. For all times tested, these scaffolds maintained shear, compressive, and frictional biomechanical properties similar to those of native cartilage (Fig. 5), while exhibiting the synthesis of a collagen-rich extracellular matrix [65]. These findings suggest that ASC-based constructs can be designed and created using biomaterial scaffolds that provide native biomechanical function, allowing for the potential use of these technologies in clinically based applications for cartilage repair and regeneration.

Fig. 5A–D.

Three-dimensional structures were woven by interlocking multiple layers of two perpendicularly oriented sets of in-plane fibers (x- or warp direction, and y- or weft direction) with a third set of fibers in the z-direction. (A) A surface view of the X-Y plane (SEM), (B) cross-sectional view of the X-Z plane, and (C) cross-sectional view of the Y-Z plane are shown. (D) The structural stiffness of three-dimensional PCL scaffolds was increased when consolidated with fibrin. The aggregate modulus (H_A) and Young’s modulus (E) are shown at Day 0 as determined by confined and unconfined compression, respectively. Three-dimensional PCL/fibrin composite scaffolds had significantly higher H_A and E values than did unconsolidated three-dimensional PCL scaffolds (ANOVA, *p < 0.05. **p < 0.0001); data presented as mean ± SEM; scale bar = 1 mm. (Reproduced with permission from Moutos FT, Guilak F. Functional properties of cell-seeded three-dimensionally woven poly(epsilon-caprolactone) scaffolds for cartilage tissue engineering. Tissue Eng Part A. 2010;16:1291–1301.) [65]

Discussion

Tissue engineering, gene therapy, and other cell-based approaches in regenerative medicine have highlighted the need for an abundant source of undifferentiated progenitor cells. The purpose of this study was to determine the utility of adipose tissue as a source of stem cells for musculoskeletal regeneration and the optimal scaffolds and culture conditions to promote their chondrogenic differentiation. Studies show subcutaneous fat and other adipose tissue depots in the adult human body contain large numbers of stem cells that can be readily isolated from standard liposuction waste, providing an easily accessible source of autologous or allogeneic cells. Under appropriate environmental cues, as detailed herein, these cells can exhibit phenotypic characteristics of multiple cell types, and thus provide a unique and promising cell source for applications in regenerative medicine.

Relative to other adult stem cell sources, ASCs can be harvested with high yields of cells, although some limitations to their use still exist. First, it can be difficult to determine the optimal in vitro culture period required for a specific application. The stromal vascular fraction that initially is isolated represents a heterogeneous mix of cells types that can include endothelial cells, pericytes, and immune cells. Some studies have shown that after controlled induction or serial passaging, ASCs become more uniform in phenotype and differentiation potential [24], suggesting that in vitro expansion preferentially selects for a relatively homogeneous cell population enriched for cells expressing a stromal immunophenotype [61]. However, as extensive expansion in culture may introduce the rare possibility of tumorigenesis, the in vitro culture conditions and selection of early progenitors may strongly influence the phenotype of the cells before differentiation [24, 76], and therefore should be selected carefully before use in a clinical application [75]. Regardless, most applications of ASCs are focusing on minimizing or even eliminating cell expansion in vitro before implantation of the cells [46]. Thus, although limitations exist, ASCs continue to meet many, if not all, of the requisite characteristics for a clinically applicable cell therapeutic.

Our findings and those of others have shown that ASCs exhibit multipotent differentiation capabilities in the mesenchymal lineage, similar to other adult stem cells such as MSCs, with evidence of adipogenic, chondrogenic, myogenic, neurogenic, osteogenic, and tenogenic differentiation [36, 53, 86]. Similarly, the culture conditions used to induce lineage-specific differentiation of ASCs generally are similar to those used for MSCs; although increasing evidence indicates that different adult stem cells possess unique characteristics and properties depending on their site of origin. By measures of cell surface markers, ASCs show expression profiles that are similar but distinct in comparison to bone marrow MSCs. However, the response of ASCs to growth factors and biomaterial scaffolds may differ significantly from MSCs [21, 26]. For example, it has been shown that the addition of BMP-6 significantly enhances ASC chondrogenesis, suggesting that conditions for optimal ASC differentiation may be distinct from those required for MSCs [26, 39]. Thus, based on these data, investigators should be cognizant of such differences between ASC and MSC behavior as they optimize cell-based therapeutics for musculoskeletal disease.

The growth and differentiation of these cells, particularly as they undergo chondrogenesis, is sensitive to multiple environmental factors such as soluble mediators (ie, growth factors and cytokines), physicochemical factors such as mechanical loading, pH, or oxygen tension, and their interactions with biomaterial matrices. In particular, findings suggest that a rounded cell shape (via three-dimensional scaffolds or micromass culture) is a critical factor in the induction of the chondrogenic phenotype, and thus physical interactions with the extracellular matrix may have a significant effect on maintaining ASC phenotype over the long term [35]. This evidence suggests that it will be possible to exploit such mechanoinductive stimuli to modulate the chondrogenic differentiation and function of ASCs in conjunction with more traditional biochemical growth factors [38, 51, 54].

Ultimately, the ability of ASCs to provide a basis for tissue regeneration in the musculoskeletal system likely will depend on the ability for engineered constructs to provide the appropriate functional properties over the life of the implant [11]. In this respect, the mechanical and biophysical properties of biomaterial scaffolds used in stem cell-based tissue engineering are critical design parameters that will influence the clinical translation of such approaches. To achieve these goals, continued studies involving biomaterial and biomechanical scientists, manufacturing process engineers, orthopaedic clinicians, and stem cell biologists are needed. By fostering collaborative relationships between teams from academia, biotechnology startups, and established biomaterial industry companies, the development of ASC-based regenerative medical therapies for cartilage and bone defects can be accomplished in compliance with existing federal and international regulatory requirements. Evidence already exists suggesting that these goals are feasible since the successful use of autologous ASCs to repair critical-sized craniofacial defects has been published as case reports in the clinical literature [52, 58].