A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose

Abstract

IntroductionCurrent therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.MethodsStandardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.ResultsNon-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.ConclusionsThe present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.

Ex vivo culture platform for assessment of cartilage repair treatment strategies

“…Mature human cartilage discs (ø 5 mm) from OA femoral condyles (Wardale et al, 2015) 2. Mature bovine cartilage discs (ø 6 mm) from lateral facets of trochlea/patella groove (Pretzel et al, 2013) 3. Immature porcine chondral explants (ø 6mm) from femoropatellar joints (Vinardell et al, 2009) which is more representative of the in vivo situation than the models summarized in Table 1.…”

“…Changes in loading during everyday life, after treatments of knee injuries, but also age, obesity or chronic overloading due to sports are risk factors that can lead to the onset of OA (Felson et al, 2000;Abramson and Attur, 2009;Zhang and Jordan, 2010 sheep, goat, dog and pig), whose use is accompanied by high costs for animal caring and ethical issues (Cook et al, 2014;Hurtig et al, 2011). In order to refine and reduce the number of animal studies performed in the field of cartilage repair, standardized and representative ex vivo models that allow for a whole array of simultaneous tests are valuable tools (de Vries-van Melle et al, 2012;Vinardell et al, 2009;Pretzel et al, 2013). These ex vivo models need to fulfill the following requirements: The model has to be stable, without loss of physiological properties, viability and metabolic activity, for a relevant culture period, optimally 8 weeks, as this is the typical duration of in vivo animal studies on cartilage regeneration therapies (Sakata et al, 2015).…”

Nanocellulose and Proteins: Exploiting Their Interactions for Production, Immobilization, and Synthesis of Biocompatible Materials

“…The results indicated that cartilage cells still exhibited vital morphology after that period, with growth of chondrocytes on the surface of BC but not inside the pores. The chondrocytes at the nanocellulose surface showed successful re-differentiation [31].…”

Characterization of plasma fibronectin for migration, proliferation, and differentiation on human articular chondrocytes

“…There is some evidence that cartilage repair is further supported by the flow of cartilaginous matrix at the defect site into the defect (Bruns, Kersten, Silbermann, & Lierse, ). Additionally, endogenous chondrocytes from the native cartilage of the defect rim might aid in cartilage repair by migrating into the scaffold, as observed in an in vitro cartilage punch model (Pretzel et al, ). Therefore, pFN might further improve the regeneration by inducing the migration of chondrocytes near the defect area.…”