Coupling Freshly Isolated CD44⁺Infrapatellar Fat Pad‐Derived Stromal Cells with a TGF‐β3 Eluting Cartilage ECM‐Derived Scaffold as a Single‐Stage Strategy for Promoting Chondrogenesis

Abstract

An alternative strategy to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold is used to provide an environment conductive to proliferation and tissue-specific differentiation in vivo.The objective of this study was to develop a cartilage extracellular matrix (ECM) derived scaffold that could facilitate the rapid proliferation and chondrogenic differentiation of freshly isolated stromal cells. By freeze-drying cryomilled cartilage ECM of differing concentrations, it was possible to produce scaffolds with a range of pore sizes. The migration, proliferation and chondrogenic differentiation of infrapatellar fat pad derived stem cells (FPSCs) depended on the concentration/porosity of these scaffolds, with greater sGAG accumulation observed in scaffolds with larger sized pores. We then sought to determine if freshly isolated fat pad derived stromal cells, seeded onto a TGF-β3 eluting ECM-derived scaffold, could promote chondrogenesis in vivo. While a more cartilage-like tissue could be generated using culture expanded FPSCs compared to non-enriched freshly isolated cells, fresh CD44 + stromal cells were capable of producing a tissue in vivo that stained strongly for sGAGs and type II collagen. These findings open up new possibilities for in-theatre cell based therapies for joint regeneration.3

Scaffolds Derived from ECM Produced by Chondrogenically Induced Human MSC Condensates Support Human MSC Chondrogenesis

“…Morphologically, they resembled scaffolds fabricated in this manner from native porcine tissue. 18 However, scaffolds derived from engineered tissues contained less DNA (4.1× less in −MS and 5.2× less in +MS scaffolds; Figure 3A) compared to that in scaffolds made from native porcine tissues using the same protocol. Less nucleic material in scaffolds fabricated from engineered cartilage-derived ECM scaffolds described in this work may be beneficial in reducing their immunogenicity in an allogeneic therapy.…”

“…Cell-seeded scaffolds (N = 3−4) were evaluated in unconfined compression based on previously described methods. 18 Briefly, frozen constructs were thawed in PBS at RT containing 0.1% protease inhibitor (Sigma-Aldrich) for 30 min. A preload of 0.03 N was applied for 500 s using an R Series Controller mechanical testing device (Test Resources Inc., Shakopee, MN).…”

“…These results support our previous findings that showed that native cartilage-derived ECM scaffolds soak loaded with TGF-β3 can successfully induce chondrogenesis of infrapatellar fat pad-derived stem cells at least as well as media-supplemented scaffolds. 17,18 In addition, in a different a system, particles of decellularized tissue-engineered cartilage ECM incorporated within hMSC aggregates do not induce chondrogenesis without TGF-β1 supplementation (unpublished data), so TGF-β1-delivery from the scaffolds formed using this material may be critical to generating cartilaginous tissue without exogenous growth factor supplementation in the culture media. The chondrogenesis inducing TGF-β1-loaded ECM scaffolds make in vitro culture potentially less labor intensive and expensive by removing the need for growth factor supplementation at each media change.…”

“…15 In addition to their inherent properties, scaffolds composed of cartilaginous ECM may be altered to enhance their mechanical properties 16 and to serve as bioactive factor delivery depots. 17,18 While native cartilage tissue ECM has been extensively explored in its ability to drive chondrogenesis, in vitro cell-derived ECM presents certain advantages. 19 First, autologous extracellular matrix-based scaffolds can only be fabricated from cultured cell-derived tissues as there are currently no tissue banks for autologous cartilage.…”

Growth plate extracellular matrix-derived scaffolds for large bone defect healing

“…However, it should be noted that there was some variability observed within scaffolds, due to the different sizes of GP particles used to form the 3D scaffolds. This limitation can be avoided in future studies through the use of a cryo-mill which can generate a more homogeneous GP particle size range, which would lead to less variation from batch to batch (Almeida et al, 2015). In addition, more stringent decellularisation protocols will be developed, which will be particularly important when translating this scaffold material towards clinical applications (Badylak, 2004); however, no evidence of a chronic immune response was detected at either time point in the scaffold treated groups.…”