Comparative Review of Growth Factors for Induction of Three-Dimensional In Vitro Chondrogenesis in Human Mesenchymal Stem Cells Isolated from Bone Marrow and Adipose Tissue

Abstract

The ability of bone-marrow-derived mesenchymal stem cells (MSCs) and adipose-derived stem cells (ASCs) to undergo chondrogenic differentiation has been studied extensively, and it has been suggested that the chondrogenic potential of these stem cells differ from each other. Here, we provide a comprehensive review and analysis of the various growth factor induction agents for MSC and ASC three-dimensional in vitro chondrogenic differentiation. In general, the most common growth factors for chondrogenic induction come from the transforming growth factor β (TGFβ) superfamily. To date, the most promising growth factors for chondrogenesis appear to be TGFβ-3 and bone morphogenetic protein (BMP)-6. A thorough review of the literature indicates that human MSCs (hMSCs) appear to exhibit the highest chondrogenic potential in three-dimensional culture in the medium containing both dexamethasone and TGFβ-3. Some reports indicate that the addition of BMP-6 to TFGβ-3 and dexamethasone further increases hMSC chondrogenesis, but these results are still not consistently supported. Induction of human ASC (hASC) chondrogenesis appears most successful when dexamethasone, TGFβ-3, and BMP-6 are used in combination. However, to date, current formulations do not always result in stable differentiation to the chondrocytic lineage by hMSCs and hASCs. Continued research must be performed to examine the expression cascades of the TFGβ superfamily to further determine the effects of each growth factor alone and in combination on these stem cell lines.

Introduction

Bone-marrow-derived mesenchymal stem cells (MSCs) and adipose-derived stem cells (ASCs) show promise for use in cartilage repair. However, greater understanding is needed to develop a consistently reproducible approach to potently induce chondrogenesis in these stem cell lines and to inhibit differentiated chondrocytes from further maturation into the hypertrophic lineage. Approaches to induce chondrogenesis in these stem cell lines include appropriate pellet/matrix composition, chondrogenic differentiation media, low oxygen tension, and some forms of mechanical load. This review will focus on the effects of soluble chondrogenic growth factors for three-dimensional (3D) in vitro chondrogenic induction of human MSCs (hMSCs) and human ASCs (hASCs), specifically those in the transforming growth factor β (TGFβ) super family. These include TGFβ-1, -2, and -3, and bone morphogenetic proteins (BMPs)-2, -4, -6, -7, and -9. Other growth factors of note and discussed briefly include fibroblast growth factor 2 (FGF-2) and insulin-like growth factor 1 (IGF-1).

Current methods for articular cartilage repair

Articular cartilage is an avascular connective tissue that functions as an extremely low friction surface layer for diarthrodial joints.¹ Current options for repair of articular cartilage include tissue debridement, joint lavage, microfracture of subchondral bone, subchondral drilling, and transplantation of autologous or allogeneic osteochondral/perichondral/periosteal grafts.^2–20 Tissue engineering approaches to cartilage repair include autologous chondrocyte transplantation, often with cells grown on a biodegradable polymer scaffold. These techniques all retain a high probability of fibrous tissue formation,^{2,5,6,11,13,16,18} cell apoptosis,^5,15 cartilage degeneration,^2,6,7,12,14 incomplete hyaline cartilage generation,^{4,6,9,11,12,16} and/or donor-site morbidity.^4,5,6,17 These challenging issues have encouraged further research for a new method of repair and the use of stem cells is being actively explored. MSCs have been shown to differentiate into multiple musculoskeletal tissues, making them a promising prospect for treatment.²¹ Traditionally, MSCs have been the forerunner in investigations of chondrogenesis; however, ASCs have recently gained increasing interest.

MSCs versus adipose-derived stem cells

MSCs from trabecular bone or bone marrow have shown promise in chondrogenesis; however, the harvest of bone marrow necessary for isolation of MSCs is itself an invasive and painful procedure. For this reason, interest has increased in the use of ASCs, a relatively more abundant and accessible source of stem cells.^22–24 The harvest procedure may be better tolerated by many patients, more cells may be available, and, like MSCs, ASCs have multipotent differentiation capabilities, including chondrogenesis.^22–24

Markers of chondrogenesis

The extracellular matrix (ECM) of articular cartilage is composed primarily of water, collagen II, collagen VI, collagen IX, collagen XI, cartilage oligomeric matrix protein, and the proteoglycans aggrecan, versican, and fibromodulin.²⁵ Collagen II is the most abundant collagen in articular cartilage and aggrecan the predominant proteoglycan. Thus, commonly accepted indicators of chondrogenesis include expression of these two ECM components. Collagen II is expressed in two variants, A and B, based on alternative splicing of exon 2 in precursor mRNA.²⁵ The A variant contains exon 2 and is characteristic of prechondrocytes, whereas the B variant, lacking exon 2, is characteristic of mature chondrocytes.^22,25 Commonly accepted negative markers of chondrogenesis often include collagen I and collagen X. Collagen X tends to be an indicator of hypertrophic cartilage formation.^22,25 Collagen I is the most abundant collagen in the body found in tissues such as skin, tendon, ligament, fibrocartilage, vascular walls, and bone. Some collagen I is present in the superficial zone of normal articular cartilage; however, it has been shown repeatedly to be down-regulated during chondrogenesis and is thus considered a negative marker of chondrogenesis.²²

3D culture system

A 3D pellet/matrix culture system is believed to be an essential aspect of chondrogenic differentiation.^1,26,27 hMSCs and hASCs cultured in 2D monolayer have been reported to exhibit diminished chondrogenesis relative to those in a 3D matrix or pellet.^1,27 It is thought that a pellet or matrix of cells provides an environment similar to that found in precartilage condensation during embryonic development,²⁶ during which the cells adopt a spherical morphology. The pellet system was originally used for the prevention of phenotypic modulation of chondrocytes and the study of terminal differentiation of growth-plate chondrocytes.^28–31 The most commonly reported pellet sizes are in the range of 2 × 10⁵ to 2.5 × 10⁵ cells,^26,32 but have been reported as high as 5 × 10⁵ cells.³³

Matrix-based systems often include alginate^1,34 and agarose.³⁵ These gels do not support hASCs stably for long periods, and subsequent senescence can occur.²³ It has also been reported that an hASC-seeded alginate bead construct results in hASC upregulation of collagen X in the presence of TGFβ-3.³⁴ In contrast, centrifuging hASCs into a pellet and culturing in combined TGFβ-3 and dexamethasone results in no detectable upregulation of collagen X.³⁶

However, a 3D culture environment alone cannot maintain prolonged chondrogenesis. Growth factors and appropriate physical stimuli are necessary for induction, and may prevent chondrocytes from continued differentiation into a hypertrophic state.³⁵

Medium Supplements

Many studies have been performed on chemical approaches to induce MSC and ASC chondrogenesis.^{1,21–27,32,33,36–57} Table 1 provides a breakdown of the different media and supplements described in a multitude of relevant articles to induce chondrogenic differentiation of MSCs and ASCs. We have listed each medium component as it appeared in its respective publication, accounting for multiple forms of pyruvate and ascorbic acid. The standard base medium formulation in the majority of studies is Dulbecco's modified Eagle's medium (DMEM) with some form of glucose supplement. High-glucose DMEM (4500 mg/L) is implemented more frequently than low glucose (1000 mg/L). ITS+ Premix is also a very common component, along with some form of ascorbate and pyruvate. ITS contains insulin, transferrin, and selenium (+ indicates the addition of bovine serum albumin and linoleic acid). Serum is often still used in differentiation; however, over half of the studies cited in this review do not include serum and no obvious negative effect on the ability of cells to undergo chondrogenesis was reported in those studies. The exclusion of serum can allow for testing of a more defined formulation and is often recommended.^38,58

Table 1.

Breakdown of Different Media and Supplements Used by Relevant Articles to Induce Chondrogenesis in Mesenchymal Stem Cells and Adipose-Derived Stem Cells

Each component was recorded as it appears in each publication, accounting for multiple forms of pyruvate and ascorbic acid. All growth factors as reported in each study are listed; however, multiple combinations of them may have been tested in the study. DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FCS, fetal calf serum; BSA, bovine serum albumin; ITS, insulin, transferrin, and selenium; TGFβ, transforming growth factor β; BMP, bone morphogenetic protein; IGF, insulin-like growth factor; FGF, fibroblast growth factor.

The majority of the studies listed and analyzed in this review were performed with hMSCs and hASCs; however, there are a few animal studies included primarily for historical significance and support. Table 1 provides a detailed listing of cell types investigated in each study.

The addition of various growth factors to the medium has been shown to greatly affect the chondrogenic potential of both hMSCs and hASCs. The remainder of this review will describe these growth factors and their potential impact on hMSC and hASC chondrogenesis.

Medium formulations including TGFβ-1

To date, TGFβ-1 has been the most extensively examined factor for inducing chondrogenesis in hMSCs and hASCs. Early work with TGFβ-1 centered around its role during embryonic, ECM, and cartilage development.^37,59–65 From these early investigations, research progressed to the ability of TGFβ-1 to induce chondrogenic differentiation in chick periosteum-derived cells⁶⁶ and in human and animal MSCs.^1,26,32,38 Johnstone et al.²⁶ are cited repeatedly as the first to successfully induce in vitro chondrogenesis in 3D culture with rabbit MSCs exposed to dexamethasone and TGFβ-1. Studies with hMSCs further confirmed the induction of chondrogenesis in pellet culture when hMSCs were exposed to dexamethasone and TGFβ-1.^1,32,38 Later, TGFβ-1 was successfully used for chondrogenic differentiation of hASCs,^22,24,39 and was also noted to have had a positive effect on cell survival.^38,39

Dose response effects with TGFβ-1

Dose response effects of TGFβ-1 on hMSCs and hASCs have been investigated. About 0.01–0.1 ng/mL of TGFβ-1 has been reported to have no significant effect on hMSC response relative to cultures without the growth factor.²⁷ In contrast, 0.5–1.0 ng/mL of TGFβ-1 has been shown to induce hMSCs to exhibit a spindle morphology and begin detaching from the culture dish, while 10 ng/mL caused hMSC differentiation into chondrocytes.²⁷ In hASCs, a concentration of 10 ng/mL has been shown to induce the fastest cell nodule formation relative to other TGFβ-1 concentrations (1–10 ng/mL) and greatest sulfated proteoglycan and aggrecan expression when in pellet culture.²² At a concentration of 100 ng/mL, TGFβ-1 appeared to reduce chondrogenic potential in hASCs.²²

TGFβ-1-combined medium formulations

Combining TGFβ-1 with dexamethasone has also been found to influence hMSC and hASC chondrogenesis. Yoo et al.²⁷ observed that hMSC aggregates cultured in TGFβ-1 survived past 14 days, whereas aggregates cultured with combined TGFβ-1 and dexamethasone, or dexamethasone alone, did not survive; that is, the aggregates disappeared by day 14. However, they further reported that hMSCs exposed to either TGFβ-1 or dexamethasone alone did not undergo chondrogenesis or produce significant matrix (as determined via toluiodine-blue assay), but cultures containing a combination of the two factors displayed notable ECM formation by day 5.²⁷ Johnstone et al.²⁶ found that TGFβ-1 in the presence of dexamethasone increased the percentage of rabbit MSC pellets undergoing chondrogenesis from 25% (TGFβ-1 alone) to 100% (TGFβ-1 plus dexamethasone). They also reported that pellets in cultures containing fetal bovine serum did not aggregate, but those without fetal bovine serum formed clearly identifiable aggregates. Pellets were stained for collagen II, and those cultured in dexamethasone displayed no collagen II. However, pellets incubated in TGFβ-1 stained positive for collagen II, with or without dexamethasone in the culture medium. They further noted that aggregates exposed to both dexamethasone and TGFβ-1 were much larger than those exposed to TGFβ-1 alone.²⁶

It has been similarly reported that hASCs cultured in the presence of dexamethasone, but not TFGβ-1, do not form aggregates; however, a combination of dexamethasone and TGFβ-1 resulted in the formation of larger aggregates than those exposed to TGFβ-1 alone.²² Further, hASCs cultured with combined dexamethasone and TFGβ-1 have been reported to significantly increase [³H]proline protein synthesis late in culture (between days 5 and 9), but suppress [³⁵S]sulfate proteoglycan synthesis in comparison to hASCs cultured with TGFβ-1 alone.³⁸

Chondrogenic potential of hMSCs versus hASCs in the presence of TGFβ-1

Some investigators have reported greater chondrogenic potential of MSCs than ASCs in combined TFGβ-1/dexamethasone medium formulations. Mehlhorn et al.²⁵ compared hMSC and hASC chondrogenesis in the presence of TGFβ-1 and dexamethasone, and reported that cartilage oligomeric matrix protein, collagen II, collagen X, and aggrecan mRNA expression levels were significantly greater on days 3, 6, 9, and 14 in hMSCs than in hASCs. However, it was collagen X expression, which tends to indicate hypertrophy, that displayed the greatest distinction between the cell types with hMSCs expressing 15-fold more collagen X than hASCs. A study by Huang et al.³⁹ comparing hMSC and hASC chondrogenic differentiation in the presence of dexamethasone and TGFβ-1 supported the Mehlhorn²⁵ findings and reported that hMSCs underwent a more extensive chondrogenic induction than hASCs. Although the hASC aggregates were larger than hMSC aggregates, they displayed less toluidine blue metachromasia, indicating decreased ECM and proteoglycan formation. Their immunohistochemistry analyses indicated a greater amount of collagen II and X in hMSC aggregates relative to same day hASC aggregates, while hASC aggregates expressed no collagen X and significantly less collagen II.³⁹ These findings potentially indicated that chondrogenesis in the hASC aggregates was not as extensive as in hMSCs. Although collagen X is generally associated with hypertrophic chondrocytes and for that reason is often considered a negative marker, it has also been found as a component of normal human articular cartilage.^25,39 The observation of cells representing hypertrophic chondrocytes in hMSC aggregates indicates that hMSCs may have been differentiating beyond mature chondrocytes. This phenomenon was not seen in hASCs. Significantly greater glycosaminoglycan (GAG) content was also detected in hMSCs, and collagen II mRNA levels were 5 × 10² to 5 × 10³-fold higher in hMSC than hASC aggregates.³⁹

Transforming growth factor β-3*

The role of TGFβ-3 in cell differentiation and cartilage formation was first discovered in 1986 by similarities found during its comparison to cartilage inducing factor-A.⁶⁷ In 2005, it was observed to also have a role in chondrocyte dedifferentiation.⁴¹ As described previously, a medium formulation for chondrogenic differentiation of rabbit MSCs was established in 1998 using TGFβ-1.²⁶ This formulation was adapted to a hMSC formulation with TGFβ-3, high-glucose DMEM, and proline later in the same year.²¹

It has been shown that hMSCs exposed to dexamethasone and TGFβ-1, -2, or -3 for 21 days exhibited strong toluiodine blue staining along with an increased pellet size.³² That study also reported that hMSC pellets exposed to dexamethasone and TGFβ-1 had less GAG accumulation at 7, 14, and 21 days of chondrogenic induction in comparison to the combination of dexamethasone and TGFβ-2 or -3.³² Collagen II staining and pellet size followed this same trend for TGFβ-1 induction periods of 14 and 21 days.³² TGFβ-3 produced more intense collagen II staining in hMSC³² and hASC³⁴ cultures than TGFβ-1 or TGFβ-2. Even though hMSC aggrecan and collagen II expression was upregulated in the presence of TGFβ-3, collagen X expression was also upregulated, although collagen I remained constant.^32,36

TGFβ-3 combined medium formulations

Sekiya et al. have reported that chondrogenic induction of hMSCs via TGFβ-3 and dexamethasone is greatly enhanced by the addition of 500 ng/mL of BMP-6 to the culture medium.⁴² One explanation for this may be that the cell density used for differentiation in their study was significantly less than that reported in previous experiments (several cells/cm² vs. several thousand cells/cm²). This result was further examined in 2004 by Indrawattana et al.⁴³ in a study that also tested IGF-1. The combination of dexamethasone, TGFβ-3, and BMP-6 led to similar mRNA expression of collagen II, aggrecan, and sox9 by hMSCs in pellet culture as a combination that substituted IGF-1 for BMP-6. The collagen II, aggrecan, and sox9 mRNA expression levels from the combination of TGFβ-3, BMP-6, and dexamethasone were slightly higher than those produced by induction with TGFβ-3 and dexamethasone without BMP-6 except for aggrecan, which was higher when BMP-6 and IGF-1 were excluded. That study did not measure the expression of collagen X, but collagen I expression was reported to be approximately 25–35-fold less in the combined medium (BMP-6, TGFβ-3, and dexamethasone) than when TGFβ-3 was added alone. The addition of BMP-6 only slightly increased collagen II and sox9 mRNA expression in hMSCs.⁴³ This result was further supported by Diekman et al.⁵⁷ in a study that observed no change in collagen II, aggrecan, collagen I, or collagen X with the addition of 10 ng/mL of BMP-6 to TGFβ-3 and dexamethasone in pellet culture.

Chondrogenic potential of hMSCs versus hASCs in the presence of TGFβ-3

Repeatedly, hMSCs have been shown to undergo chondrogenesis to a greater extent than hASCs when under the same culture conditions.^{25,33,39,57,68,69} Recently, the gene expression profiles of hASCs and hMSCs were compared, and it was observed that concentrations of 10–50 ng/mL of TGFβ-3, combined with dexamethasone, were not enough to induce a chondrogenic gene expression profile in hASCs similar to that found in hMSCs under the same conditions.³⁶ The expression profile and response to growth factors seen in that and other studies strongly indicate that hMSCs and hASCs are not identical cell populations and thus require distinct growth factor supplementation for successful chondrogenesis.^36,57 A recent study by Kim and Im³³ also reported that greater doses of growth factors in hASCs were necessary to obtain similar chondrogenic results as hMSCs. Specifically, that hASCs required fivefold the TGFβ-2 and IGF-1 dose as hMSCs to produce similar GAG and collagen II expression.³³

Bone morphogenetic protein-6

In 1994, BMP-6 (originally referred to as Vgr-1) was not well understood. Its function remained a mystery until Chinese hamster ovary cells overexpressing the BMP-6 protein were studied as tumors in nude mice.⁷⁰ The tumors formed by the Chinese hamster ovary BMP-6 cells resulted in the presence of BMP-6 in hypertrophic cartilage. BMP-6 was also reported to proportionally increase the chondrogenic differentiation of clonal mouse embryonal carcinoma cells (ATDC5)⁷¹ and to have induced chondrogenic differentiation in immature chicken chondrocytes.⁷² Sekiya et al.⁴² reported in 2001 that BMP-6 combined with TGFβ-3 resulted in a 10-fold increase in hMSC micromass pellet size over the course of 21 days, and caused an increase in the mRNA expression levels of type II procollagen and type X collagen. Interestingly, BMP-6 alone appeared to have no effect on proteoglycan synthesis.⁴²

BMP-6 is usually used at a concentration of 500 ng/mL based on two published studies performed on hMSCs⁴² and hASCs.³⁴ The concentrations tested in those studies ranged from 5 to 500 ng/mL³⁴ and 10 to 500 ng/mL⁴²; 500 ng/mL produced the highest mRNA expression of collagen II and aggrecan in hASCs along with the greatest suppression of collagen X.³⁴ The heaviest proteoglycan staining also occurred at 500 ng/mL.³⁴ These studies did not test at concentrations above 500 ng/mL. As of the date of this review, it is still not known how hMSCs or hASCs will differentiate under higher BMP-6 concentrations, and/or the concentration that will induce the greatest collagen II and aggrecan expression with collagen X suppression.

BMP-6 may induce a different response in vivo from in vitro. Sheyn et al.⁷³ demonstrated that primary porcine ASCs nonvirally transfected with plasmid rhBMP-6 and injected into lumbar paravertebral muscle in mice can induce functional bone tissue formation. This osteogenic response of ASCs treated with BMP-6 in vivo is in direct contradiction to the observed chondrogenic response in vitro, thus suggesting that the effects of growth factors are highly dependent on local environment.

Temporal effects of growth factor supplementation

The sequential addition of growth factors to induce chondrogenesis may also have an important role on chondrogenic induction. It is still unknown exactly how the transcription cascade of the TGFβ superfamily operates. In an attempt to try and take advantage of the possible cascade effects, BMP-6 has been added sequentially after TGFβ-3 in hMSCs.⁴³ This sequential addition has been reported to cause decreased transcription of the collagen II, aggrecan, and sox9 genes, and increased collagen I transcription. It was also reported with hMSCs that BMP-6 did not cause expression of collagen II mRNA relative to the house keeping gene GAPDH.⁴³ These findings are in contrast to the significant BMP-6-induced upregulation of collagen II mRNA expression by day 7 in comparison to day 0 in hASCs.³⁴

When the undifferentiated profiles of hASCs and hMSCs were examined, it was reported that TGFβ receptor I and BMPs were not present in the hASC profile, but were present in the hMSC profile.³⁶ However, when hASCs were exposed to BMP-6 in the expansion medium, TGFβ receptor I was then expressed.³⁶ This indicates the possibility that BMPs may be required for chondrogenesis. hMSCs normally produce BMPs while hASCs appear to require supplementation. hASCs induced to chondrogenically differentiate with 10 ng/mL BMP-6 and TGFβ-3 exhibited a gene expression profile that matched that of hMSCs differentiated with TGFβ-3 alone.³⁶ In that study, however, induction was performed with 10 ng/mL of BMP-6 instead of 500 ng/mL, which had previously been used in chondrogenic differentiation. This concentration difference may explain the contrast with reports that have stated a need for BMP-6 in hMSC induction.^42,43

To date, it has appeared that TGFβ-3 and BMP-6 are the most effective growth factor combination for chondrogenesis of hASCs in vitro,^34,36 and use of higher passage numbers may also have a positive effect on hASC chondrogenesis.⁷⁴ If BMP-6 is neglected, collagen II and aggrecan expression has been drastically reduced in hASCs.³⁶ However, collagen X expression, a negative indicator of chondrogenesis, has also been increased when BMP-6, TGFβ-3, and dexamethasone are added to hASCs, but not when dexamethasone and TGFβ-3 are added.³⁶ Obviously, there is still much optimization to be performed to maintain the chondrogenic phenotype in differentiated hASCs.

Other members of the TGFβ family

BMP-2, -4, -7, and -9 have also been analyzed for their abilities to induce chondrogenesis.^{36,45,47–52} In hMSCs, addition of BMP-2 alone has appeared to result in inferior collagen II promotion and collagen I suppression compared to TGFβ-3 alone.⁴⁹ However, when BMP-2 was combined with TGFβ-3, collagen II expression has been reported to be greater than that produced by TGFβ-3 alone. Aggrecan expression by hMSCs in response to combined BMP-2 and TGFβ-3 was approximately three times higher than TGFβ-3 alone, and collagen I expression in the combined medium remained approximately the same as BMP-2 alone.⁴⁹ BMP-9 has been reported to induce greater collagen II and aggrecan mRNA expression in hMSCs than BMP-2, but its effects on collagen X and collagen I expression were not reported.⁴⁷

It has also been indicated that short-term BMP exposures have long-term effects on ASCs. ASCs exposed to BMP-2 or BMP-7 for 15 min and then cultured in expansion medium in the absence of BMP-2 or -7 for up to 14 days have been reported to exhibit very different responses. Goat ASCs exposed to BMP-2 for 15 min have been reported to undergo osteogenic differentiation, while those exposed to BMP-7 for 15 min appeared to exhibit enhanced chondrogenesis.⁵⁰

As with the other growth factors described previously, combinations of growth factors have appeared to work in a synergistic fashion. When hASCs have been exposed to a combination of BMP-2 and TGFβ-1, the effects of TGFβ-1 appeared to be amplified in the presence of BMP-2.^25,52 However, at concentrations of 10 ng/mL each, BMP-2, -4, and -7 alone and in combination with TGFβ-3 have been reported to result in decreased immunohistochemical staining for collagen II in hASCs when compared to BMP-6 + TGFβ-3.³⁶ In contrast, a more recent study indicated that concentrations of 100 ng/mL BMP-7 + 10 ng/mL TGFβ-2 worked more effectively to induce proliferation and chondrogenic differentiation in hASCs than BMP-2 and -6 alone and in combination with TGFβ-2.⁵³ Since these two studies used different members of the TFGβ superfamily and different concentrations of BMPs, it is still not clear which combination is preferred for inducing chondrogenesis in hASCs.

Fibroblast growth factor 2

In humans, there are over 20 members of the FGF family. In this review we concentrate on FGF-2 alone since it has been the most extensively investigated member of the FGF family for its role in induction of chondrogenesis in MSCs and ASCs. FGF-2, used in conjunction with TGFβ-1 either in vivo,³⁷ or in the expansion medium in vitro,^46,54 appeared to induce proliferation and chondrogenic differentiation of MSCs. In the expansion medium, it has been reported that hMSCs exposed to FGF-2 are 30% smaller than control cells, and proliferated almost twice as fast after the first passage.⁴⁶ FGF-2 concentrations tested in that study ranged from 0 to 10 ng/mL in culture. At 10 ng/mL FGF-2, microarray data appeared to confirm the positive proliferation effects of FGF-2 on hMSCs. Over 50% of positive proliferation genes were upregulated, while over 50% of negative proliferation genes were downregulated.⁴⁶ Interestingly, 8 out of 10 ECM-related genes were also downregulated, and the microarray data indicated a negative feedback on FGF-2's mRNA and receptor protein.⁴⁶ Stewart et al.⁵⁴ performed a similar experiment but used a concentration of 100 ng/mL FGF-2 on equine MSCs. They reported that 100 ng/mL FGF-2 significantly increased pellet DNA and GAG content.⁵⁴ Equine MSC pellets treated with 10 or 100 ng/mL FGF-2 induced a fourfold increase in collagen II mRNA expression relative to MSC pellets not treated with FGF-2.⁵⁴

Pelleted hMSCs exposed to 0–10 g/mL FGF-2 in the expansion medium have also been reported to form larger pellets after 1, 2, and 3 weeks than hMSCs expanded without FGF-2 (control cultures).⁴⁶ The FGF-2-treated hMSC pellets also displayed a cartilaginous matrix more evenly distributed throughout the pellet. Further, no fibrous outer layer was observed, unlike in control pellets. Increased GAG formation was also observed in FGF-2-exposed cells relative to non-FGF controls, and no collagen I was found in staining. However, both collagen II and X stained positive in both FGF and control cultures.⁴⁶ GAG expression is not necessarily a marker of chondrogenesis since GAGs are also associated with fibrous connective tissues. Expression of collagen II, the most abundant collagen in articular cartilage, and expression of aggrecan, the most predominant proteoglycan, are generally considered more compelling indicators of chondrogenesis.

FGF-2-treated MSC pellets expanded in a TGFβ-3-supplemented medium have also been reported to increase the growth rate and chondrogenic differentiation of hMSCs.^55,56 Human MSC pellets treated with 1 ng/mL FGF-2 were reported to have significant increases in GAG content and mRNA expression of collagens II and X.^55,56

Discussion and Conclusions

To develop a reproducible method of inducing chondrogenesis in MSCs and ASCs, expansion medium, pellet/matrix composition, chondrogenic differentiation medium, oxygen tension, and appropriate mechanical environments must be analyzed. This review focuses on one of those components, the soluble inductive growth factors in chondrogenic differentiation medium, to provide an analysis of our understanding to date of the different growth factors known to induce chondrogenesis in hMSCs and hASCs. We attempt to provide further understanding of how such factors work to promote and nurture the chondrogenic lineage in these stem cell lines. Due to space limitations, the exceedingly important role of mechanical load on chondrogenic differentiation is not included, but others and we have shown its importance.^35,75–79 Some reviews have touched on the importance of mechanical load in the chondrogenesis of MSCs.^80,81 The effects of mechanical load on the chondrogenic differentiation of ASCs should be a topic of another review.

It has been shown that a 3D pellet/matrix system plays a large role in allowing MSCs and ASCs to undergo chondrogenesis. An interesting phenomenon consistently observed in pellet culture induction is the formation of layers within the pellets. The cells within the pellet tend to take a spherical shape, whereas those on the periphery are more elongated. One hypothesis explaining this observation is that the cells are induced to take on a specific characteristic based on placement within the pellet. This may be achieved through a number of cell–cell interactions, growth factor gradients formed by the cells as seen in embryonic development, or a combination of the two. A second hypothesis is that the cells sort themselves based on the characteristics already possessed; that is, cells possessing characteristics of stromal cells are shunted to the outside, while those more prone to chondrogenesis are moved inward. Or, it could be a component of the mechanical environment ensuing from cell growth and proliferation while in the pellet form. Cells in the middle of the pellet might be exposed predominantly to a hydrostatic pressure environment, whereas those on the periphery experience higher levels of tensile strain. Although the exact reason is still an area of investigation, it is generally agreed that 3D culture is an essential component of chondrogenesis.

The analysis of the expression cascades of the TGF-β superfamily will dramatically aid in determining how each growth factor affects gene expression in hMSCs and hASCs; however, the complexity of these sequences requires the blunt methods of chondrogenesis research performed today. As a result, the systematic comparison of growth factor combinations is key to determining how to consistently and optimally induce chondrogenesis in these stem cells. Growth factors may induce specific characteristics while alone; however, combined they may promote and/or inhibit others.^34,42

This review focused on known growth hormones that have been used in the differentiation medium to induce chondrogenesis in hMSCs and hASCs. From our review of the vast literature on this topic, it appears that the most potent methods of chondrogenic induction in hMSCs and hASCs include dexamethasone, TGFβ-3, and BMP-6. In light of this, the fact that other members of the TGF-β superfamily induce chondrogenesis to some extent indicates that they may also play a chondrogenic role. Our analyses of previous studies lead us to conclude that hMSC induction should incorporate dexamethasone and 10 ng/mL TGFβ-3 at a minimum, as the need of BMP-6 supplementation in hMSC chondrogenesis is still not consistently supported.^36,42,74 On the basis of the work performed by Hennig³⁶ and Estes,³⁴ it is recommended, and our review of the data support, that chondrogenic induction of hASCs should likely be performed using a combination of dexamethasone, at least 10 ng/mL TGFβ-3, and 500 ng/mL BMP-6. That being said, the current optimal formulations do not yet produce stable chondrocytes, either as a result of incorrect growth factors or the omission of a key external component such as mechanical load. The effects of the expansion medium on chondrogenesis must also be determined. The expansion medium can genetically prime cells for the ability to utilize growth factors that induce chondrogenesis, and it is frequently modified.^{25,34,52,74,82}

Repeatedly, hMSCs have been shown to undergo chondrogenesis more thoroughly than hASCs when under the same culture conditions.^{33,39,57,68,69} The expression profile and response to growth factors reported in these and other studies strongly indicate that hMSCs and hASCs are not identical cell populations and thus will require distinct growth factor supplementation for successful chondrogenesis.^36,57

Footnotes

Acknowledgments

The authors would like to acknowledge funding contribution from the NCSU Undergraduate Research Awards to both Jennifer L. Puetzer and John N. Petitte.

Disclosure Statement

No competing financial interests exist.

*

In the TGFβ-3 section, dexamethasone is included in each induction medium described unless specifically stated otherwise.

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