Abstract
The goal of tissue engineering is to create a functional replacement for tissues damaged by injury or disease. In many cases, impaired tissues cannot provide viable cells, leading to the investigation of stem cells as a possible alternative. Cartilage, in particular, may benefit from the use of stem cells since the tissue has low cellularity and cannot effectively repair itself. To address this need, researchers are investigating the chondrogenic capabilities of several multipotent stem cell sources, including adult and extra-embryonic mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Comparative studies indicate that each cell type has advantages and disadvantages, and while direct comparisons are difficult to make, published data suggest some sources may be more promising for cartilage regeneration than others. In this review, we identify current approaches for isolating and chondrogenically differentiating MSCs from bone marrow, fat, synovium, muscle, and peripheral blood, as well as cells from extra-embryonic tissues, ESCs, and iPSCs. Additionally, we assess chondrogenic induction with growth factors, identifying standard cocktails used for each stem cell type. Cell-only (pellet) and scaffold-based studies are also included, as is a discussion of in vivo results.
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Introduction
Stem cells are promising cell sources for many tissue engineering applications, including cartilage regeneration. Embryonic stem cells (ESCs), adult and extra-embryonic mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) have all been investigated as potential cell sources for cartilage applications (Fig. 1). The motivation for exploring the regenerative potential of these cells results from the lack of effective therapies currently available for patients suffering from joint diseases. One example is osteoarthritis, a pathology characterized by the degradation of hyaline cartilage.79 Aside from total joint replacement, possible forms of treatment include microfracture, which allows for subchondral MSCs to populate the defect, and autologous chondrocyte implantation, which is a treatment using ex vivo expanded chondrocytes and, potentially, stem cells. Unfortunately, both procedures can result in the formation of fibrocartilage, a mechanically inferior tissue to healthy hyaline cartilage. Tissue engineering approaches using primary chondrocytes are non-ideal since undamaged cartilage has to be destroyed to obtain the cells, and in vitro expansion is necessary to achieve sufficient cell numbers. This process also takes precious weeks, results in dedifferentiation, and raises the risk of contamination. Stem cells have become an attractive therapeutic alternative due to their relative abundance and multipotent capabilities, specifically their ability to undergo chondrogenesis.148 An ideal stem cell source has yet to be identified, as each has strengths and weaknesses. Studies have characterized these populations extensively, highlighting large variations in the different cell types, such as ease of isolation, differentiation potential, and surface marker expressions. Additional research has led to progress within all stem cell fields to optimize growth factor cocktails and delivery systems, although to varying degrees of success.
Stem cells can be isolated from multiple anatomical locations, encompassing adult, and extra-embryonic tissues. The cell sources shown above have all been investigated for cartilage regeneration, although mesenchymal sources have been studied much more than other tissues
To induce stem cell chondrogenesis, many in vitro strategies have been explored, including mechanical stimulation, the use of scaffolds or growth factors, or a combination of these techniques.110 The most frequently used method of induction is treatment with chondrogenic medium in a pellet culture system.27 Induction medium typically consists of insulin, transferrin, and selenous acid (ITS), dexamethasone, ascorbic acid, and sodium pyruvate, in addition to growth factors.88,148 Many growth factors have been considered for chondrogenic differentiation, as reviewed by Danisovic et al.30 The most well-characterized and implemented growth factors are part of the transforming growth factor-beta (TGF-β) superfamily, including TGF-β1, 2, and 3, as well as bone morphogenic proteins (BMPs). This review includes the reported optimal growth factors for chondrogenesis, identifying specific cocktails for each stem cell type. Following differentiation, chondrogenesis is confirmed by the presence of extracellular matrix, specifically type II collagen, proteoglycans, and glycosaminoglycans (GAGs), as reviewed by Vater et al.155 Many methods are used to assess these components, the most common of which are stains specific to proteoglycans, such as toluidine blue, and stains that bind to GAGs or sulfated GAGs, such as alcian blue and safranin-O. An additional assay commonly used to measure GAG synthesis is 1,9-dimethyl methylene blue (DMMB), which can be used to provide quantitative data via spectrophotometry. We will use these reports of matrix synthesis to evaluate the relative effectiveness of stem cell type and culture environment for inducing the chondrocytic phenotype.
This review also seeks to highlight the differences inherent among human stem cell populations currently being investigated for cartilage applications, though for areas with limited human studies we will report results from animal models. Isolation procedures and surface marker expressions will be summarized for each cell type, as they are stem cell-specific. In addition, due to the extensive use of in vitro differentiation by means of pellet culture and 3D scaffolds, evaluation of studies successfully inducing chondrogenesis by these methods will be included. It has also been shown that in vitro chondrogenic ability is not always indicative of function in vivo, due to loss of the chondrocytic phenotype upon implantation.115 As such, recent in vivo studies will be reviewed to stress the feasibility and variability of stem cell use. While previous articles have assessed stem cells for cartilage tissue engineering, these typically focus on only the most common sources. Consideration of many stem cell types is necessary to evaluate the progress and potential of these populations. A compilation of our findings has been included in Table 1.
Adult Stem Cell Sources
Adult stem cells are promising cell sources for cartilage repair due to their multipotency, lack of tumorogenicity, ease of isolation, and applicability to autologous transplantation procedures, which removes the risk of rejection associated with allogeneic sources.62 Unfortunately, these cell types do have disadvantages.111 When compared with alternative sources, such as extra-ESCs or embryonic and embryonic-like stem cells, adult stem cells have limited self-renewal capacities. Additionally, as a person ages, these cells exhibit decreased proliferation rates and lessened differentiation potential and, in some cases, have similar regenerative characteristics as those associated with diseased patients.64,111
Bone Marrow-Derived Stem Cells
Though their existence was discovered much earlier by Friedenstein et al., bone marrow-derived mesenchymal stem cells (BMSCs) were first characterized by Pittenger et al. in the late 1990s and remain the most commonly used stem cell for cartilage tissue engineering.8,16,46,118 The cells were identified by positive expression of STRO-1, CD105, CD44, CD71, CD90, CD106, CD120a, and CD124, and negative expression of hematopoietic markers, though later studies concluded negative expression of endothelial markers as well (Table 2).3,118 Additionally, cells possessed the ability to adhere to tissue culture plastic, as well as expand in vitro, and differentiate along mesenchymal lineages.9 To obtain cells for assessment, Pittenger and colleagues employed a density centrifugation gradient, yielding approximately 0.001–0.01% of cells from the initial population. Low cell yield is still a persistent obstacle within the field, since it necessitates in vitro expansion to obtain sufficient cell numbers for therapies, a process that results in decreased differentiation potential.23 Due to this setback, alternative methods have been pursued in an attempt to enhance cell yield efficiency and purification of cell populations.23,106 Some approaches include frequent medium changes, plastic adherence, cell sorting by surface markers, as well as exposure of cells to cytotoxic reagents during expansion.23,50,56,106 Many physical characteristics have also been used to identify BMSCs, including cell size and mechanical properties.31,96,146,172 To date, however, BMSC isolation from marrow, regularly harvested from the iliac crest, is still typically conducted by density gradient centrifugation.105 Subsequently, to further purify the population, cells are plated on tissue culture plastic where non-adhered cells are later washed away.
The use of chondrogenic induction factors has been heavily investigated in vitro, as reviewed by Puetzer et al.,119 leading to continuous modification of cocktails and culture environments. One of the earliest studies attempting to optimize chondrogenic differentiation compared BMSC pellets exposed to TGF-β1, β2 or β3.12 While chondrogenesis was induced under all conditions, after 21 days, it was discovered that TGF-β2 and β3 promoted the greatest GAG production, about twice that of pellets treated with TGF-β1. To enhance differentiation with TGF-β3, it was found that particular BMPs, most effectively BMP-2, could be incorporated.133 Other uses for growth factors have also been considered, such as delaying impaired potency, a prominent issue after long-term expansion.138 Sochaga et al. expanded BMSCs with fibroblast growth factor-2 (FGF-2) supplemented media and found that not only did this increase proliferation rates, but it also successfully delayed the loss of chondrogenesis. Furthermore, Cooke et al.28 investigated the ability of co-culturing BMSCs with juvenile chondrocytes to enhance differentiation in pellet culture, without additional growth factors. A two-fold increase in GAG levels was observed in comparison to pellets containing only BMSCs. This was a greater degree of induction than BMSC pellets exposed to TGF-β1.
Scaffolds of various materials have successfully induced chondrogenic differentiation of seeded BMSCs in vitro. Many biomaterials have been used to date, including collagen, a material known for its biocompatibility and ability to degrade.121 Ng et al.111 investigated differences between type I collagen and type II collagen scaffolds. After treatment of porcine BMSC-seeded constructs with TGF-β1, type I collagen scaffolds were superior to type II collagen in terms of GAGs per DNA production, 122.6 vs. 100.7 μg GAGs/μg DNA. Other groups have combined scaffold materials to test for enhanced potency. Zhou et al.176 incorporated hydroxyapatite (HA) into their collagen scaffold to better mimic osteochondral tissue. After 4 weeks of treatment with TGF-β3, scaffolds with HA exhibited a two-fold increase of GAG production in comparison with those solely comprising of collagen. Additionally, encapsulation of cells by TGF-β3-incorporated fibrin hydrogels can also be used, as shown by Park et al.115 GAG and relative type II collagen production revealed successful chondrogenic differentiation, 6 and 4.5 μg/μg DNA, respectively. Other materials include, but are not limited to, chitosan, alginate, and polyglycolic acid (PGA).135,159,165
In the field of BMSC cartilage tissue engineering, there have been many encouraging studies using novel approaches that indicate these cells can treat cartilage pathologies in vivo. One study using a leporine (rabbit) model analyzed the use of BMSCs, among other stem cell types, to treat osteochondral defects.91 Scaffolds were comprised of demineralized bone matrix integrated with TGF-β1. After 12 weeks, BMSC-seeded scaffolds were well-integrated into the surrounding tissue and exhibited robust expression of type II collagen. An alternative study by Igarashi et al.,61 conducted without growth factors, treated osteochondral defects in a canine model. Their approach consisted of an in situ forming gel based on ultra-purified alginate containing autologous BMSCs. After 16 weeks of recovery, analysis of the treated area was reported as being smooth, firm, and glossy-white. Mechanical testing of the regenerated cartilage determined structural integrity was still inferior to that of native cartilage (9.2 vs. 12.2 MPa). Advances like these are what have enabled BMSC research in cartilage tissue engineering to reach clinical trials, where many are currently in progress.26
Adipose-Derived Stem Cells
Adipose-derived stem cells (ASCs) were first pursued by Zuk et al.178 in 2001 as an alternative to BMSCs in an attempt to overcome their limitations. In contrast to bone marrow, not only is adipose tissue readily available, but it can be harvested using minimally invasive surgical procedures such as liposuction, and yields significantly more cells overall. Initial studies found that while ASCs exhibit the functional characteristics of stem cells, like multipotency and extended proliferation, they express some surface marker dissimilarities when compared to BMSCs.12,37 Unlike BMSCs, ASCs show positive expression of CD49d and negative expression of CD106, although there is considerable overlap of other markers. To obtain these cells, protocols from the 1960s, originally designed for fat pads, are used and have since been modified for subcutaneous lipoaspirate, the standard source of ASCs.15,71 However, more recently, ASCs from alternative locations have been isolated using similar procedures, concluding that variations in differentiation abilities exist among ASCs derived from epididymis, perinephrium, and subcutaneous adipose tissue.71 Following extraction of fat tissue, the process of ASC isolation involves enzymatic digestion with type I collagenase, followed by subsequent centrifugation and washing steps.15 The isolated cells generate a heterogeneous population including ASCs, fibroblasts, adipocytes, smooth muscle cells, and endothelial cells.169 The negative effects of this cell contamination, including reduced proliferation and differentiation potential, continue to limit the use of ASCs for therapeutic applications.122 Seeking to reduce these effects, protocols that aid in purification have been proposed, such as additional washing steps, density centrifugation, and sorting by surface markers.4,49,122
Many in vitro studies have been conducted to investigate the chondrogenic ability of ASCs. As such, cocktails have been refined to promote optimal differentiation. After reviewing current literature, there are few studies comparing cocktails directly, though many confirming that they have been used successfully, as reviewed by Puetzer et al.119 Commonly used growth factors are TGF-β1, BMP-6, or a combined growth factor cocktail of TGF-β3 and BMP-6.41 Additionally, TGF-β2 in conjunction with BMP-7 has proven to be a potent inducer of chondrogenesis.77 Kim et al. assessed chondrogenic induction after treatment with numerous members of the TGF-β superfamily, including BMP-2, 6, 7, and TGF-β2. Individually, GAG amounts were at least twice those of control pellets for all factors. Combining BMP-2 and TGF-β2 resulted in a three-fold increase in GAG production when compared with controls, similar to BMSC pellets. Additionally, as seen with BMSCs, FGF-2 can be administered at low concentrations during expansion of ASCs to enhance proliferation rates and induce chondrogenesis.21 However, chondrogenic induction can be hindered by FGF-2 when combined with BMP-6 and TGF-β1.54 It has also been shown that chondrocytes can enhance chondrogenesis of ASCs by means of soluble factors, or direct interaction through co-culture.87 Lee et al. assessed differentiation of ASCs in co-culture with chondrocytes, after exposure to chondrocytic conditioned medium, or exposure to a cocktail of TGF-β2 and BMP-7. The co-culture condition resulted in a 37% increase in GAG production as compared with controls, while ASC pellets exposed to conditioned medium resulted in a 25% increase. However, the growth factor condition induced the greatest degree of chondrogenesis, increasing GAG levels by 50% over controls.
Many materials have been employed as scaffolds to obtain chondrogenic differentiation of ASCs. While early groups studied the ability of agarose, and gelatin to function as a scaffold material for ASC chondrogenesis, modification of these materials has since occurred.9 Nieto-Aguilar et al. combined fibrin and agarose to produce a novel hydrogel for such applications. Seeded scaffolds were cultured in chondrogenic induction medium supplemented with TGF-β1 for extended periods. Though early assessment confirmed no differentiation had occurred, after 20 days, 98.7 and 86.7% of cells were positive for expression of GAGs and type II collagen, respectively. Recently, mechanically stimulated chitosan/gelatin scaffolds induced potent chondrogenesis of insulin-like growth factor-1 (IGF-1) induced ASCs.92 Collagen and proteoglycan levels were increased roughly three-fold from controls when assessing scaffolds not exposed to mechanical stimulation. An even greater increase, four-fold, was seen when comparing mechanically stimulated scaffolds with controls. Poly(lactic-co-glycolic acid) (PLGA) has also been used as a scaffold material.113 Park et al. recently observed chondrogenic differentiation of leporine ASCs in PLGA scaffolds incorporating dexamethasone and TGF-β1. Though incorporation of TGF-β1 yielded higher GAGs per DNA production than PLGA scaffolds without growth factors (0.15 vs. 0.25 μg GAGs/μg DNA), seeded scaffolds incorporating only dexamethasone produced the most GAGs per DNA (~0.275 μg GAGs/μg DNA).
In addition to cartilage assessment in vitro, past studies have demonstrated that ASCs are capable of producing cartilage in vivo using numerous approaches. Jung et al.70 examined the influence of fibrin glue on the in vivo chondrogenesis of ASCs when implanted subcutaneously in a nude murine model. Prior to implantation, the stem cells were differentiated in vitro by media supplemented with TGF-β1. Post-operation, constructs were qualitatively analyzed at 12 weeks, with the resulting tissue appearing to be structurally sound and glossy. Mehlhorn et al.101 assessed chondrogenesis of ASCs seeded on PLGA scaffolds. Differentiation was induced in vitro by means of TGF-β1 supplemented medium after cells had migrated into the polymer matrix. Analysis of implants after 8 weeks revealed the presence of type II collagen and proteoglycans in scaffolds, suggesting successful differentiation and maintained chondrogenic phenotype. TGF-β1 was also investigated during an in vivo study to assess treatment of leporine defects with various MSC sources.91 The growth factor was incorporated into demineralized bone matrix scaffolds, and subsequently seeded with MSCs. While ASC-treated defects were superior to controls, defects treated with BMSCs were observed to have greater surface architecture and integration.
Synovial Membrane-Derived Stem Cells
De Bari et al.32 first characterized synovial membrane-derived mesenchymal stem cells (SDSCs) in 2001 by examining their multipotency, proliferation rates, and surface marker expressions. Many commonalities with BMSCs were found, though later studies determined SDSCs to express CD49d, whereas BMSCs do not.32,102 After collagenase digestion and subsequent expansion, which is the isolation procedure predominantly used today, analysis by De Bari et al.32 revealed many advantageous characteristics of these cells for tissue engineering applications. SDSCs possessed considerable proliferative abilities, as seen by maintained growth rates over 30 population doublings, and multi-lineage differentiation capabilities. Since this early study, many groups have determined and confirmed superior chondrogenic and proliferative abilities compared to other MSC types, further supporting the importance of SDSC research.42,128 Like with many adult stem cell types, isolating SDSCs yields a heterogeneous population, of which fibroblasts and macrophages are persistent contaminants.43,69 To overcome this concern, many studies have investigated enrichment through surface marker expression. For this approach, cells are either sorted for negative expression of CD14 shortly after cell isolation or are expanded prior to sorting to exclude macrophages.43
In terms of in vitro differentiation, beginning with De Bari et al.,32 TGF-β1 was shown to enhance chondrogenesis, as evidenced by GAG production. Later, in vitro differentiation was optimized after treatment with several cytokines, including TGF-β3 and BMP-2.137 Induction with TGF-β3, alone, resulted in pellets averaging 0.8 mm in diameter, larger than controls averaging roughly 0.5 mm in diameter. With the addition of BMP-2, average pellet diameter strikingly increased to roughly 1.7 mm. Significant GAG and type II collagen production was also noted. Alternatively, BMP-2 has been combined with TGF-β1 to induce differentiation of SDSCs. To discern the influence of passage on chondrogenesis, Han et al.51 differentiated cells from P0 to P8, determining the maximum potential at P1, yielding roughly 37 μg GAGs/μg DNA. Additionally, chondrogenic differentiation of porcine-derived cells by treatment with TGF-β1 has been reported.13
Few recent studies have investigated the chondrogenic differentiation of SDSC-seeded scaffolds. Qi et al.120 recently determined chondrogenesis of CD105+ murine SDSCs integrated into chitosan-alginate scaffolds. Following a two-week culture period in medium containing TGF-β3 and BMP-2, expression of type II collagen was observed, as well as GAG production, averaging 5.5 μg GAGs/μg DNA. SDSCs have also been seeded in polyethylene glycol diacrylate-based hydrogels and phosphoester-polyethylene glycol-based hydrogels.42 While both successfully induced chondrogenic differentiation, the latter proved to be superior, as shown by type II collagen and proteoglycan production.
In vivo studies have further confirmed the chondrogenic abilities of these cells. Koga et al.81 treated cartilage defects in a leporine model after suspending SDSCs in a collagen gel. Defects exhibited profuse matrix production after 4 weeks, increasing after 12 weeks. This repair was similar to BMSC-treated defects. Li et al.91 investigated SDSC treatment of defects in a leporine model, as well, with demineralized bone matrix-seeded scaffolds, incorporating TGF-β1. The results from this group were not as successful as Koga et al., as BMSC-treated defects were superior in integration as well as cartilage production. Studies have also been conducted specifically to enhance integration of SDSCs in leporine knee osteochondral defects.136 It was found that not only did cells adhere faster with the addition of magnesium, but cartilage production of SDSCs was expedited, resulting in greater matrix production.
Muscle-Derived Stem Cells
The discovery of muscle-derived stem cells (MDSCs) in mammals, isolated using a freeze/thaw procedure, occurred in the mid-1990s.117 Though this study confirmed potency of these cells, it was not until years later that their surface markers were extensively characterized.67 Seeking to expedite the isolation procedure, Young et al.171 determined that MDSCs express CD10, CD13, CD56, and Sca-1, while lacking CD45, similar to BMSCs. As reviewed by Wu et al.,163 additional surface marker expressions include CD34, desmin, MyoD, CD144, and c-Kit, though papers have also contradicted this positive expression. Additional markers, distinct from BMSCs, have yet to be determined. Because of their unique surface marker expression within muscle-derived populations, fluorescence-activated cell sorting (FACS) is occasionally used to select for MDSCs, though can be unreliable due to overlapping expression with non-MDSCs.19 However, the most common isolation method is the modified preplate technique. Preplating attempts to isolate MDSC populations based on their adherence rates to tissue culture plastic or collagen-coated flasks, whereby stem cells are the slowly adhering fraction, obtained 4–5 days post-harvest.47 Cells are initially released from biopsies by means of enzymatic digestion.103 A recent study aiming to purify MDSC populations further discovered that Percoll density gradient centrifugation should also be considered as a viable option.19
In vitro chondrogenesis of MDSCs has been accomplished in a few studies, generally incorporating BMP-4. Kuroda et al.85 isolated cells from murine muscle biopsies and transduced them with BMP-4. Colonies were then formed and subsequently differentiated with and without the addition of TGF-β1. In control media, cultures of BMP-4-transduced cells had more type II collagen-producing colonies than those of non-transduced cells. Additionally, exposure to TGF-β1 did not enhance this effect. Similar to the Kuroda et al., another group transduced murine MDSCs with BMP-4, and then co-pelleted these cells with either vascular endothelial growth factor (VEGF)-blocked or VEGF-producing MDSCs and subsequently treated with TGF-β3.84 It was found that pelleting with VEGF-blocked MDSCs enhanced differentiation two-fold when compared to pelleting with VEGF-producing cells or pellets consisting solely of unmodified MDSCs. Recently, Ye et al.167 isolated murine MDSCs for chondrogenic assessment. Following induction of chondrogenesis with TGF-β1 for 2 weeks, a 25-fold increase in type II collagen gene expression was observed over controls. Alternatively, Lu et al.93 confirmed that TGF-β1 induced chondrogenesis in human MDSCs.
Few groups have recently explored scaffolds for chondrogenic differentiation of MDSCs. Alginate beads have been used successfully, while treating MDSCs with TGF-β1.6 Following 21 days, staining with alcian blue and assaying for aggrecan revealed presence of chondrogenic markers. A study by Nawata et al.109 incorporated BMP-2 into type I collagen scaffolds. Toluidine blue staining and type II collagen expression confirmed chondrogenesis.
Many of the previously described MDSC studies investigated the chondrogenic potential of MDSCs in vivo, in addition to in vitro. Kuroda et al.85 determined the ability of differentiated MDSCs to treat osteochondral defects in a murine model when mixed with fibrin glue. After 24 weeks, the cells were well-integrated into the surrounding tissue, and regenerated cartilage looked white, glossy, and smooth. Similar repair was seen by Kubo et al.84 after treating osteochondral defects with VEGF-blocked cells, in addition to fibrin-embedded BMP-4-transduced cells. In a gender study by Matsumoto et al., cells derived from both males and females were transduced with BMP-4 and mixed with fibrin glue before treating osteochondral defects in nude rats. Unlike defects treated with male MDSCs, those treated with female-derived MDSCs did not heal effectively.98 A more recent study by Li et al.91 compared in vivo chondrogenesis of cells derived from various source tissues. Leporine knee defects were treated with demineralized bone matrix scaffolds seeded with undifferentiated MSCs. BMSCs proved to be superior to MDSCs on many levels including integration and surface architecture.
Peripheral Blood Stem Cells
The field of peripheral blood mesenchymal stem cells (PBMSCs), an idea initially proposed in 2000 by Zvaifler et al., is relatively new, as their existence is still being investigated due to contradicting reports in early studies.58,127,179 These non-hematopoietic cells isolated from peripheral blood have been determined to lack STRO-1, unique from BMSCs, and can be isolated from blood samples using Ficoll or Percoll gradients and subsequent plating to further isolate by means of plastic adherence.52,53,58,151 While this method successfully isolates PBMSCs, it results in extremely low yields (0.0002% of initial population), thus, groups have made many modifications to address this issue.94 FACS has been explored to isolate cells based on expression of various surface markers, specifically positive expression of CD133 or negative expression of CD14.58,151 Fibrin microbeads have also been used successfully, due to their ability to bind to matrix-dependent cells.73 In this study, treating patients with granulocyte colony-stimulating factor increased cell yield dramatically, although yield was still only 0.5% of the initial population.
Chondrogenesis of these cells has been achieved in vitro using a variety of methods, many similar to those used for BMSCs, though without the incorporation of scaffolds. It was determined that PBMSCs in 2-D culture induced along the chondrogenic lineage by TGF-β3 express type II collagen precursors, though contradicting results of this finding exist.22,123 Chong et al.25 also analyzed the effect of TGF-β3 on the differentiation of pelleted human PBMSCs. Average GAG concentrations of PBMSCs pellets were found to be similar to those of BMSCs, roughly 3 and 3.25 μg GAGs/μg DNA, respectively. Unfortunately, both of these levels were still lower than those of chondrocyte pellets, 4 μg GAGs/μg DNA. Tondreau et al. induced chondrogenesis of PBMSCs by treatment with TGF-β1. Following 2–3 weeks of culture, the presence of GAGs indicated successful differentiation. Though few groups have assessed the function of these cells in vivo, an encouraging study was recently conducted by Saw et al.130 Patients with chondral defects first underwent subchondral drilling. PBMSCs were harvested one week after the procedure, and then re-injected intraarticularly with hyaluronic acid once a week for 5 weeks. Long-term, post-operative assessment concluded regeneration of hyaline cartilage at the treatment site. Patients reported minimal adverse side effects apart from discomfort.
Extra-ESC Sources
Cells from extra-embryonic sources have recently been proposed for the purposes of regenerative medicine. These cells are isolated from tissue discarded after birth and are characterized as being in a more developmentally primitive state.62 This translates into higher proliferation rates and, potentially, increased multi-lineage differentiation capabilities. Unfortunately, extra-ESCs must be cryogenically stored prior to autologous use, which can be associated with high costs and potentially result in deleterious effects.
Wharton’s Jelly Stem Cells
Though isolation of cells was first achieved in 1991, characterization of MSCs derived from Wharton’s jelly, the mucoid connective tissue surrounding the umbilical vein, was first conducted by Wang et al. in 2004.99,157 These cells isolated from Wharton’s jelly, also termed umbilical cord matrix, were determined to express surface markers similar to BMSCs, though a more recent study reported lack of CD18 expression (Table 3).44,86 In their initial study, Wang et al.157 investigated not only surface marker expression, but also the differentiation potential of Wharton’s jelly stem cells (WJSCs) along the chondrogenic lineage. Pelleted cells were exposed to medium supplemented with TGF-β1 for 3 weeks and then assessed by immunohistochemical analysis. Staining illustrated rich deposits of type II collagen in both control and experimental pellets. Alternatively, Baksh et al.11 analyzed the differentiation of TGF-β3 treated pellets. Compared with controls, GAG levels of WJSC pellets were 4 times as high. Additional studies have been conducted incorporating the use of scaffolds. Fong et al.45 fabricated polycaprolactone (PCL)/type I collagen nanofibrous scaffolds prior to seeding WJSCs and observed significant production of cartilage matrix molecules.
Though these studies suggest the therapeutic potential of WJSCs for hyaline cartilage applications, more promising results were observed for successful fibrocartilage fabrication, indicated by type I collagen production.10,158 An early study by Bailey et al.10 explored the potential of PGA scaffolds seeded with WJSCs to generate temporomandibular joint condylar cartilage. Four weeks after seeding scaffolds with TGF-β1-differentiated cells, quantification of GAG content revealed higher levels in WJSC scaffolds than those seeded with chondrocytes, approximately 3 and 1 μg, respectively. Additionally, WJSC scaffolds exhibited a significant amount of type I collagen, more than was seen in chondrocyte pellets, whereas type II collagen production was nominal in both. These results inspired further investigation by the same group to determine optimal seeding density.158 Concentrations higher than 25 million cells/mL successfully sustained GAG production while simultaneously enhancing the mechanical integrity of the construct.
Umbilical Cord Blood Stem Cells
Confirmed homing capacity of MSCs and circulatory properties of hematopoietic stem cells inspired Erices et al. to investigate whether umbilical cord blood could also serve as a stem cell source, though the presence of mesenchymal-like cells had previously been reported.40,166 Expression of common mesenchymal stem cell surface markers was seen, and again confirmed in a study directly comparing those of BMSCs and umbilical cord blood mesenchymal stem cells, but expression of CD49d has been observed in umbilical cord blood stem cells.89,100,156 Further assessment of surface markers by Chang et al.18 determined the existence of two MSC subpopulations within umbilical cord blood, one being positive for expression of CD90 and the other being negative. Similar chondrogenic and osteogenic abilities were observed between populations. Conversely, adipogenic differentiation potential was greater in the population expressing CD90. While these studies have successfully obtained MSCs from umbilical cord blood for analysis, low or even undetectable levels of MSCs in cord blood continue to hinder their clinical use.40,64,174 Research within the field has continued, however, due to the availability of the tissue and their superior proliferative capacity.74 In vitro chondrogenesis has been investigated and achieved using various growth cocktails. In the presence of TGF-β1, pelleted umbilical cord blood stem cells are capable of producing twice as much type II collagen as control pellets, similar to ASCs, though BMSCs under the same conditions produce three times as much as their controls.124 Differentiation with TGF-β3 and BMP-2 was also analyzed by Zhang et al.174 The pellet mass obtained from three different donors was quantified following differentiation with TGF-β3 and BMP-2, TGF-β3 alone, and BMP-2 alone. It was noted consistently for all donors that BMP-2 promoted the largest pellets. However, this also led to production of hypertrophic chondrocytes. Collagen scaffolds were also seeded with differentiated umbilical cord blood stem cells and then implanted subcutaneously in a murine model. Post-operative assessment 3 weeks later confirmed matrix section, as well as type II collagen production. The constructs were visually white, stiff, and glistening, features resembling cartilage. Most recently, De Mara et al.34 compared the chondrogenic induction efficiencies of BMP-2 and BMP-6 on umbilical cord blood stem cells. Type II collagen expression was superior in cell monolayers exposed to BMP-2, as shown by western blotting. No comments on manifestation of hypertrophic chondrocytes were made.
Amniotic Fluid Stem Cells
The earliest report of amniotic fluid-derived stem cells (AFSCs) is by In’t Anker in 2003, though amniotic fluid cells expressing OCT-4 were publicized just prior and were further characterized by De Coppi et al. a few years later.33,65 Cells were isolated from second trimester fluid after centrifugation and subsequent culturing. Flow cytometry revealed positive expression of MSC markers, however, positive expression of CD117 and negative expression of CD10 were also determined, unique from BMSCs.7,33,112,153 The ability of AFSCs to undergo chondrogenesis was studied in depth by Kolambkar et al.82 in 2007 by comparing the effects of TGF-β1, TGF-β3, and BMP-2. The combination of TGF-β3 and BMP-2, and TGF-β3 alone, produced the lowest increase in GAG production, 1.9- and 2.2-fold greater than controls, whereas TGF-β1 alone produced a 2.7-fold increase. Staining for type II collagen was substantial in pellets exposed to TGF-β1 or β3. However, minimal staining was found after treatment with the combination of TGF-β3 and BMP-2. Tsai et al.152 also differentiated AFSCs using TGF-β3 and confirmed proteoglycan production after 3 weeks. Because AFSC research is still preliminary, aside from the few studies investigating in vitro chondrogenesis, cartilage repair applications have not been investigated thoroughly.162 An in vivo study has been conducted by Park et al.,114 encapsulating cells with TGF-β3-containing fibrin gels. While in vitro analysis revealed substantial GAG levels, on par with BMSCs, post-implantation assessment determined GAG production of BMSCs to be superior to AFSCs in vivo, roughly 7 and 4 μg GAGs/μg DNA.
Placenta-Derived Mesenchymal Stem Cells
In 2003, Zhang et al.175 first identified MSCs in term placenta. Soon after, further characterization was performed by In ‘t Anker et al.,64 when they investigated the specific membranes of the placenta, as the amnion and chorion are of fetal origin, and the decidua portions are of maternal origin. Cells were isolated simply by mincing the membranes and then filtering the resulting suspension. Their surface markers have been characterized to include many BMSC markers, with the exception of negative expression of STRO-1 and SSEA-4.134 Since that time, though many encouraging studies have been conducted in the field of placenta-derived mesenchymal stem cells (PMSCs), very few targeted cartilage applications.57 Groups have studied the amnion and the chorion, determining characteristic differences between the two sources, as well as their ability to differentiate. Portman-Lanz et al. determined that chondrogenesis of chorion-derived cells with TGF-β1 was superior to amnion-derived cells. Conversely, Cavallo et al. discovered that with the addition of 10% serum, chorion-derived cells do not undergo chondrogenesis. However, type II collagen gene expression of amnion-derived cells was determined to increase by roughly 15-fold.17 Alternatively, amnion-derived cells have been differentiated successfully after exposure to TGF-β3, as seen by type II collagen production.5
Embryonic and Embryonic-Like Stem Cell Sources
Embryonic and embryonic-like stem cells, such as iPSCs, have been considered for regenerative medicine applications due to their pluripotency and capacity to continuously self-renew.111 Use of ESCs requires immunosuppressants, but iPSCs can be transformed from patient-specific cells into embryonic-like cells such that autologous transplantation is possible.39 However, tumorogenicity is an unwavering concern for both cell types, in addition to transmission of zoonotic factors introduced during the expansion process, since murine feeder layers are essential for maintaining proliferation rates and pluripotency.39,125,148 The therapeutic versatility of ESCs and iPSCs may outweigh these concerns in the long run for tissues that cannot be repaired using other cell types.
ESCs
The field of human ESC research stems from their discovery by Thomson et al.,144 who successfully isolated lines of human ESCs and determined their pluripotent characteristics. Characteristically, ESCs express SSEA3, SSEA4, TRA-1-60, TRA-1-8, OCT4, SOX2, and NANOG as reviewed by Draper et al.38 and De Miguel et al.35 (Table 4). Steps to isolate these cells from an in vitro fertilization-produced embryo include culturing until it reaches the blastocyst stage, whereby cell masses can then be isolated.144 Currently, mechanical isolation, by which the blastocyst is plated and trophectoderm cells are subsequently removed, is used to obtain these cells without the use of zoonotic components, a feature of other techniques such as immunosurgery that involve use of animal enzymes.140 Expansion of ESCs on tissue culture plastic, unlike adult stem cells, adversely leads to rapid differentiation. Early preventative measures, including the addition of a mouse embryonic fibroblast feeder layer or conditioned media, were proven to be effective, though risk of zoonotic pathogens were a concern for clinical applications.125 Alternatives, such as use of human-derived cells, were proven to be a safer method.139
Due to their tumorigenic tendencies upon implantation, well-controlled ESC differentiation along the chondrogenic lineage in vitro is essential.148 While recent findings using ESCs for cartilage engineering are inferior to BMSC, ASC, and SDSC results, elucidation of optimal chondrogenic conditions may make ESCs a viable option in the future (Fig. 2). Many approaches incorporating growth factors have been assessed, most commonly employing the use of embryoid body (EB) systems, a method highly dependent on the stage of development and frequently leading to heterogeneous differentiation.48,148 A few groups have attempted to promote chondrogenesis without EB formation. An initial study conducted by Yang et al.164 compared stem cells directly differentiated, without EB formation, by TGF-β1. Under both conditions, chondrogenesis did not occur. In contrast, Gong et al.48 successfully induced chondrogenesis with TGF-β1 using a high-density adherent micromass culture system. However, this induction was accomplished by supplementing media with BMP-2, as well. Moreover, in the same study, it was shown that BMP-2 alone was capable of stimulating chondrogenic differentiation. Type II collagen production of BMP-2-treated cells was seven times that of control pellets. Chondrogenesis has also been induced by BMP-2, using EB formation, after first treating with TGF-β3.80 However, it was found that treatment with TGF-β1 and IGF-1 after TGF-β3 was more effective, as seen by a two-fold increase in type II collagen production. Alternatively, using a gelatin-coated flask, Nakagawa et al.107 induced chondrogenesis of ESCs cultured with BMP-7, and even more so when combined with TGF-β1. Analysis determined generation of high levels of GAGs per DNA, reaching roughly 21 μg GAGs/μg DNA with BMP-7 alone, and 41 μg GAGs/μg DNA when treated with BMP-7 combined with TGF-β1. As normalized to GAPDH levels, RT-PCR also revealed a seven-fold increase in mRNA expression of type II collagen.
Comparison of reported GAG synthesis amounts per DNA after chondrogenic induction of human-derived cells using box-and-whisker plots. Results were calculated from recent literature by averaging the largest GAG value reported from each study. Cell types and their respective citations include bone marrow stem cells (BMSC, n = 16),1,2,37,55,72,75,76,78,114,116,135,154,159,165,170,176 adipose-derived stem cells (ASC, n = 11),1,20,24,37,54,63,68,76,77,114,168 synovial membrane-derived stem cells (SDSC, n = 5),51,82,88,97,104 and embryonic stem cells (ESC, n = 6).90,107,145,149,150,164
Incorporating scaffolds into the differentiation process has been conducted with much success. Seda Tigli et al.131 assessed ESC differentiation after seeding onto either chitosan or silk-fibroin scaffolds incorporated with BMP-6. Following 4 weeks of differentiation, histological, and RT-PCR analysis identified chitosan to be inferior in terms of chondrogenesis. Upon investigating the effect of modulus on chondrogenic differentiation, Nam et al.108 cited the ability of low modulus PCL scaffolds to induce chondrogenesis, as seen by upregulated type II collagen and aggrecan expression. Conversely, neither type I collagen nor type II collagen scaffolds successfully induced differentiation of ESCs.111 While constructs were also treated with TGF-β1, type II collagen and GAG production were not observed.
Successful demonstration of ESC chondrogenesis in vivo has been conducted numerous times. Hwang et al.60 obtained chondrogenic-committed cells using an ESC-chondrocyte co-culture system and subsequently seeded PEG-RGD scaffolds prior to subcutaneous implantation in a murine model. Analysis of implanted hydrogels after 24 weeks confirmed an increase in pellet weight and long-term chondrogenic commitment. A more recent study reported encouraging results when treating osteochondral defects in a murine model using ESCs embedded in a hyaluronic acid-based hydrogel.149 Prior to implantation, scaffolds were treated in vitro with various cocktails, whereby BMP-7 and TGF-β1 were deemed superior. Assessment after 12 weeks revealed exceptional integration of the scaffold, and successful treatment of the defect, as seen by regenerated, hyaline-like cartilage.
iPSCs
In the late 1990s, Wilmut et al.161 transferred cell nuclei from an embryonic cell line to adult lamb somatic cells, thereby successfully producing a viable offspring. This study confirmed the potential of an adult cell to be reprogrammed into an embryonic cell. These results inspired Takahashi, a decade later, to investigate the factors that cause this phenomenon.142 After assessing genes known to maintain pluripotency of ESCs, such as OCT3/4, c-Myc, SOX2, and KLF4, they successfully generated pluripotent cells from murine embryonic and adult fibroblasts, termed iPSCs. Soon after, they, along with Yu et al., reproduced these results in adult human fibroblasts.141,173 However, Yu et al.173 were the first to report pluripotency in cells reprogrammed to express OCT4, SOX2, NANOG, and LIN28. These results supported the potential of these cells to be used for regenerative medicine applications, and confirmed similar surface marker expression to ESCs. Unfortunately, this method was burdened by a low success rate, prompting others to explore alternative routes, such as using microRNAs and creating new vectors.39 One of the largest obstacles that still remains with iPSCs is the tumorogenic tendencies linked to the factors employed, further stressing the need for alternative techniques. Few groups have induced chondrogenesis of iPSCs, though Wei et al.160 cited chondrogenic differentiation of induced osteoarthritic chondrocytes following in vitro and in vivo assessment. In vitro, cells were transfected with TGF-β1 and embedded in alginate with chondrocytes. RT-PCR determined type II collagen and GAG levels to be comparable to chondrocytes and two-fold greater than iPSCs cultured without chondrocytes. Following 2 weeks of in vitro differentiation, constructs were implanted subcutaneously in a murine model. Post-operative assessment 6 weeks later confirmed chondrogenesis had been achieved, as seen by type II collagen production. Teramura et al.143 differentiated murine iPSCs along the chondrogenic lineage by treating cells in a micromass culture with STEMPRO® chondrogenic medium. Following 2 weeks of treatment, type II collagen expression was increased 20-fold from non-induced iPSC cultures. While these groups have cited successful chondrogenic differentiation, the relative novelty of the field restricts assessment of their potential, though future exploration remains promising.
Conclusion
Since their discovery, stem cells have been considered propitious candidates for many tissue engineering applications, especially cartilage regeneration. Currently, there is a great deal of research supporting this favoritism. However, further investigation is necessary to improve upon current methods of differentiation and, ultimately, regeneration. This literature review confirms great variability in results from stem cell studies (Fig. 2). Individual findings are influenced by how the cells were handled, including isolation procedures, surface marker analyses, and differentiation assessments, as well as the use of multiple growth factors to stimulate chondrogenesis (Table 5). Some differences can be attributed to the influence of distinct, stem cell niches prior to isolation. While MSCs from various tissues have been shown to play similar roles, such as repair and immunosuppression, the inherent differences among microenvironments could influence the properties of resident stem cells.83 MSCs (e.g., BMSCs and ASCs) are not all the same cell type, as demonstrated by the widely differing responses observed when treated with the same growth factors. MSCs are not stem cells by the classical definition, due to their finite proliferative capacities. Instead, they can be more accurately described as multipotent, progenitor cells. This description is supported by a study showing that cells from adipose tissue differentiate through different molecular mechanisms than those from skin, while still reaching the same desired cell type.66 Results depict contrasting stem cell properties, potentially caused by microenvironments or idiosyncrasies of these cells aligning to their roles in the tissue. The goal of this review was to provide a comprehensive summary of stem cell sources for chondrogenesis while addressing these characteristic differences. However, new tissues are continuously being investigated as promising cell depots. Likewise, previously established stem cell sources, such as skin, are showing promise in the area of cartilage regeneration.129
Many modifications should be considered in order to enable standardization and reliable comparisons within the field. This is a difficult proposition, however, since investigators have distinct goals for their projects, and allowing direct comparisons to other stem cell sources may not be one of them. With respect to 3D environments for chondrogenesis, employing basic pellet cultures, rather than scaffolds, could provide standardization that allows greater insight into chondrogenic abilities of each cell type. This would eliminate interactions between cells and scaffold materials, permitting assessment of baseline differentiation. Additionally, consistency among reported parameters should be improved upon. Though mechanical properties are considered supremely important for determining tissue functionality, type II collagen and sGAG quantification should be reported as well, potentially using a quantitative, enzyme-linked immunosorbent assay (ELISA) or DMMB assay, respectively. These can be further normalized by quantifying data on a per cell basis, rather that total amounts, which can vary based on number of cells and size of the pellet. True control samples, where stem cells are not induced, would also enhance standardization, enabling examination of fold-increases that can be used for cross-study comparisons. Furthermore, wet weight and dry weight should be considered, since the hydration of cartilage tissue is an informative parameter. Though current studies comparing stem cells have provided great insight, treatment with universal cocktails hinders the ability to identify optimal stem cell sources for cartilage repair, as direct comparisons are difficult to make. Additionally, many in vivo studies fail to assess regeneration using previously determined optimal conditions, specifically growth factors. This suggests that greater effort to adapt in vitro results to in vivo studies could improve upon cartilage regeneration, potentially revealing an optimal stem cell source for future therapies.
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This work was supported in part by National Institutes of Health grant AR054673.
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Beane, O.S., Darling, E.M. Isolation, Characterization, and Differentiation of Stem Cells for Cartilage Regeneration. Ann Biomed Eng 40, 2079–2097 (2012). https://doi.org/10.1007/s10439-012-0639-8
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DOI: https://doi.org/10.1007/s10439-012-0639-8