Genipin-Crosslinked Cartilage-Derived Matrix as a Scaffold for Human Adipose-Derived Stem Cell Chondrogenesis

Abstract

Autologous cell-based tissue engineering using three-dimensional scaffolds holds much promise for the repair of cartilage defects. Previously, we reported on the development of a porous scaffold derived solely from native articular cartilage, which can induce human adipose-derived stem cells (ASCs) to differentiate into a chondrogenic phenotype without exogenous growth factors. However, this ASC-seeded cartilage-derived matrix (CDM) contracts over time in culture, which may limit certain clinical applications. The present study aimed to investigate the ability of chemical crosslinking using a natural biologic crosslinker, genipin, to prevent scaffold contraction while preserving the chondrogenic potential of CDM. CDM scaffolds were crosslinked in various genipin concentrations, seeded with ASCs, and then cultured for 4 weeks to evaluate the influence of chemical crosslinking on scaffold contraction and ASC chondrogenesis. At the highest crosslinking degree of 89%, most cells failed to attach to the scaffolds and resulted in poor formation of a new extracellular matrix. Scaffolds with a low crosslinking density of 4% experienced cell-mediated contraction similar to our original report on noncrosslinked CDM. Using a 0.05% genipin solution, a crosslinking degree of 50% was achieved, and the ASC-seeded constructs exhibited no significant contraction during the culture period. Moreover, expression of cartilage-specific genes, synthesis, and accumulation of cartilage-related macromolecules and the development of mechanical properties were comparable to the original CDM. These findings support the potential use of a moderately (i.e., approximately one-half of the available lysine or hydroxylysine residues being crosslinked) crosslinked CDM as a contraction-free biomaterial for cartilage tissue engineering. IntroductionT rauma, congenital anomalies, and age-related degeneration may contribute to the loss of articular cartilage and, ultimately, to the onset of osteoarthritis. In these cases, tissue regeneration remains a challenging clinical problem because of the limited self-repair capacity of cartilage.1 Current therapies for cartilage injuries include physical procedures such as tissue debridement and microfracture of the subchondral bone, as well as transplantation of autologous or allogeneic osteochondral graftsorchondrocytes. 2-6These procedures can achieve a certain degree of success in some patients, but the clinical outcomes are inconsistent, and further cartilage degeneration is inevitable in most cases. 6,7 While significant advances have been made in tissueengineered repair of articular cartilage, a number of challenges remain in the development of functional cartilage replacements.8 For example, the selection of a cell source for cartilage tissue-engineering strategies is a complicated, yet, critical issue. 9 The use of autologous chondrocytes for regenerating or repairing cartilage lesions is available currently as a clinical therapy, but has certain important limitations. The isolation of autologous chond...

Bioengineering of Articular Cartilage: Past, Present and Future

“…Cheng et al showed acellular porcine cartilage-derived matrix was able to support the growth of neocartilage formation in the absence of exogenous growth factors [143]. Recently the same group was able to induce chondrogenic differentiation of human adipose-derived stem cells without exogenous growth factors on an acelluar cartilage matrix crosslinked with genipin to prevent scaffold contraction [145].Schwarz et al have shown the successful decellularisation and sterilization of porcine knee and nasal cartilage and human nasal cartilage. They also show the ability to remove proteoglycan content whilst maintaining the collagen structure.…”

Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells

“…heart valves, blood vessels, skin and cartilage [8,[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44]) scaffolds. In the case of articular cartilage, numerous studies have demonstrated that scaffolds derived from devitalized cartilage are chondroinductive and show great promise for regenerating damaged joints [8,[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47].…”

“…One of the factors that may limit ECM derived scaffolds for cartilage tissue engineering applications is cell-mediated contraction of the construct either during in vitro culture or following implantation into a defect [44]. To overcome such problems different crosslinking techniques can be used to minimise or prevent contraction such as dehydrothermal (DHT) [28,42,[48][49][50][51][52][53][54][55], UV light [42,50] and chemical crosslinking [42,54,56,57]. Chemical methods include the use of glutaraldehyde [54,[58][59][60], 1-Ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) [42,49,54,61,62] and genipin [57].…”

“…However it is unclear how these different crosslinking methods will influence the chondroinductive properties of cartilage ECM derived scaffolds, with recent studies demonstrating that high levels of crosslinking do not support new cartilaginous ECM accumulation [57].…”

“…Building on previous work in this field [42,57], the global aim of this study was to develop a cartilage ECM derived scaffold capable of promoting robust chondrogenesis of human IFP derived stem cells (FPSCs). To this end, we first sought to compare chondrogenesis of FPSCs within a cartilage ECM derived scaffold in the presence or absence of exogenously supplied TGF-β3.…”