The findings of this study show that a porous biomaterial scaffold derived entirely from native cartilage ECM has the ability to induce the chondrogenic differentiation of ASCs without the use of exogenous growth factors. By measures of gene expression, protein synthesis, histology, and biomechanical testing, ASCs produced significant amounts of cartilaginous tissue within the interstitial space of the porous cartilage matrix without additional exogenous growth factors, leading to significant increases in the functional properties of the scaffolds after 4 and 6 weeks of in vitro culture. Although the mechanisms leading to the induction of this chondrogenic differentiation remain to be determined, the results suggest that direct cell–matrix interactions significantly influenced the phenotype of ASCs.
An important characteristic of the scaffolds used in this study is the relative simplicity of the processing method (i.e., homogenization and lyophilization) used to fabricate highly porous structures from native cartilage ECM. Recently, decellularized scaffolds derived from a variety of intact tissues have been applied in engineering different tissues or organs, including tendons, ligaments, blood vessels, skin, nerves, skeletal muscle, small intestinal submucosa, urinary bladder, heart, and liver.75–81
Decellularized and lyophilized osteochondral grafts have also been used successfully to treat full-thickness cartilage lesions in a rabbit model.82
In the present study, we adopted a relatively simple yet effective method to reconstitute the cartilage tissue into a three-dimensional porous structure with a porosity of ~95% and an interconnected network of pores with an average pore size of 221
μm (). The fact that the scaffold was manufactured using purely physical processing techniques suggests that their native biochemical makeup is likely to be unchanged, which could alleviate safety concerns associated with man-made biomaterial scaffolds. However, the effects of scaffold processing on the matrix biochemistry have yet to be characterized. The porous nature of the scaffolds distinguishes them from traditional cartilage allografts because it facilitates cell seeding. Further, due to the endogenous content of organic ECM molecules and cartilage-derived matrix proteins (CDMPs), we believe that the cartilage-derived ECM scaffolds would be analogous to DBM and similar bone derivatives currently used in bone repair.
The scaffold induced temporal increases in critical cartilage-specific gene transcripts over the first 14 days of culture, providing evidence of the influence of the scaffold on cell differentiation. In particular, mRNA transcript levels for aggrecan, the large aggregating proteoglycan found in articular cartilage, and type II collagen, the principal collagen found in articular cartilage, were significantly upregulated. These results are comparable to the effects of growth factors that would otherwise be required to induce significant levels of AGC1 and COL2A1 transcripts in these cells.59,60
Specifically for COL2A1 transcript, ASCs produce little to none of this cartilage-specific transcript in their native environment or even during monolayer expansion, providing further evidence that the scaffold is cueing the cells to produce type II collagen. The paucity of COL2A1 transcript after monolayer expansion combined with significant increases in COL2A1 production directly lead to the 1600-fold increase at day 14 that was observed in this study. The scaffold also inhibited expression of the chondrocyte hypertrophy marker, COL10A1, as has been shown with exogenous BMP-6 treatment.59
It is important to note that by histological and immunohistochemical measures, the ASC-seeded scaffolds produced a fibrocartilaginous phenotype as noted by coexpression of type I and II collagen (), though type II collagen dominated the composition. A link between early expression of COL2A1 and type II collagen production was observed ( and ). However, it should also be noted that ASCs express a high level of COL1A1 at isolation, in the order of 1000-fold higher than that of COL2A1. This level of COL1A1 typically persists through monolayer expansion and increases during differentiation culture.56,60
Conversely, in this study, a trend toward decreased COL1A1 expression over the first 14 days was observed, which corresponded to significant decreases in type I collagen accumulation in the matrix at days 28 and 42 of culture ( and ). Type I collagen was primarily located in regions between the larger masses within the neotissue and was associated with a fibroblast-like morphology () as opposed to the rounded chondrocyte–like morphology observed in the other larger areas of the neotissue. Earlier immunohistochemical time points could elucidate whether a type I collagen phenotype precedes type II collagen deposition within these ECM-derived scaffolds or whether other factors in the scaffold/ASC matrix are implicated in this morphology and collagen phenotype shift. Nonetheless, the fact that the scaffold minimized type I collagen synthesis within each construct as a whole and induced such large quantities of type II collagen indicates that the ASCs are responding to signals and conditions generated from the cartilage-derived scaffold to differentiate to a cartilage phenotype. This significant production of type II collagen at the protein level was encouraging, as the synthesis and assembly of significant amounts of collagen is often considered to be a limiting step in cartilage formation, prompting the search for other means to induce collagen synthesis.83
Noting the difficulty in synthesizing collagen and the fact that matrix biosynthetic rates are upregulated in injured cartilage,84
Ng et al
. suggested that the limiting factor in collagen synthesis is related to the lack of appropriate stimuli to induce rapid tissue remodeling.83
As exhibited in this study, under appropriate stimulation from the ECM-derived scaffold to the ASCs embedded within the scaffolds, collagen biosynthesis and accumulation was significant in a relatively short culture period.
The biomechanical properties of tissue-engineered cartilage and how they compare to native articular cartilage are among the most important outcome measures in functional tissue engineering.85
In this study, the biomechanical properties improved progressively over time. The aggregate modulus reached 150
kPa after 42 days in culture (). These values are on the same order of magnitude as those of native articular cartilage (500–900
These values are also similar to those reported previously for chondrocytes and mesenchymal stem cells (MSCs) encapsulated in agarose.33,34,87,88
Using articular chondrocytes, these authors obtained aggregate moduli of 100 and 15
kPa after 4 weeks in mechanically stimulated and static culture, respectively,34
and after 70 days in static culture, the yield equilibrium modulus of chondrocyte-embedded agarose reached 140
kPa compared to MSCs embedded in agarose with a value of 48
ECM accumulation in the scaffold also decreased the permeability of the constructs by approximately 10-fold from initial values to a value of 0.05
/(N·s) at 42 days, which is approximately 10 times greater than that of native articular cartilage.89
Interestingly, the improvement in the biomechanical properties was qualitatively associated with the increase in normalized collagen content over time, but seemed to be independent of the temporal changes in GAG content. Because diffusion is considered to be the primary mechanism for macromolecular transport in tissue-engineered cartilage and a rate-limiting factor in scaling up tissue-engineered constructs, providing an adequate nutrient supply may be necessary for enhanced cell proliferation and ECM production,90
which is likely to further improve the biomechanical properties of these scaffolds. The use of bioreactors to improve nutrient transport, therefore, may be beneficial for further development of the ASC-seeded scaffolds.
We have previously demonstrated that ASCs encapsulated (or suspended) in hydrogel scaffolds (alginate and agarose) produce relatively small amounts of pericelluar and ECM after 28 days in culture under established chondrogenic conditions.56,58,59,91
These previous results are in contrast to the abundant matrix that we observed with the ECM-derived scaffolds. Further, the chondrogenic differentiation of the ASCs was induced without exogenous growth factors as has previously been required.56,58,59,91
One possible explanation is that direct anchorage of the cells to the ECM-derived scaffold may provide cues for cell proliferation and differentiation. Failure to anchor may result in a state of “anoikis” or “homelessness,” which, in turn, has been shown to invoke apoptosis in anchorage-dependent cells.92,93
Not only can failure to provide proper anchorage points result in apoptosis, but failure to provide the proper type of substrate for anchorage can also inhibit differentiation. As evidence of this, chondrogenesis of mouse limb bud MSCs can be inhibited by blocking specific integrins, providing further support of the need of certain types of stem cells to be anchored before differentiation can occur.94
Larson et al
. demonstrated that culturing chondrocytes with a native pericellular matrix greatly enhanced the production of ECM macromolecules compared to chondrocytes without a pericellular matrix.95
Chondrogenesis of MSCs has also been enhanced by culture in type II collagen–based hydrogels, further suggesting that native ECM components, at least in part, guide the differentiation process.96
Another potential mechanism for the upregulation of ECM production by ASCs may be through the production of “matrikines,” or partially broken down matrix macromolecules, as a byproduct of the manufacturing process. Matrikines have been shown to have profound effects on cell activity and consequently on matrix synthesis and degradation (reviewed by Maquart et al
). The residual, endogenous amounts of active growth factors that remain in the scaffold after processing might also influence the differentiation of the ASCs. Indeed, the potential for scaffolds derived from various native biological tissues to release bioactive factors for both cell proliferation and differentiation has been demonstrated in several recent studies.77,98
Analogous to the work of Urist,68
it is possible that growth factors retained in the scaffolds include CDMPs, which may be released by proteolytic degradation of the ECM-derived scaffold or through diffusion and stimulate ASCs down the chondrogenic lineage.99
Future studies quantifying the biochemical profile and endogenous content of CDMPs in the ECM-derived scaffold, blocking integrin-mediated attachment, and/or investigating specific collagen or peptide sequences may yield additional data on ASC chondrogenesis.
In summary, this study reports the development of a devitalized, cartilage ECM-derived porous scaffold to promote chondrogenesis in ASCs. Our findings support the hypothesis that a scaffold derived from reconstituted cartilage ECM can influence the growth and chondrogenic differentiation of adult stem cells, even in the absence of exogenous growth factors in the culture medium.