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.
Highlights
Current 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
transforming growth factor (TGF)-b1 seemed to slow down the process of superficial delamination throughout the entire culture period of eight weeks
Increased delamination in non-stimulated samples was accompanied by augmented migration of cells onto the surface of the cartilage and the bacterial nanocellulose (BNC) implant (Figure 2 A, C), suggesting that the matrix erosion leads to a loosened network around the chondrocytes and to active emigration of the cells
Summary
Current 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. Several surgical procedures have been introduced to address this problem, for example, lavage, shaving, debridement, abrasion, microfracturing techniques [3], osteochondral autologous transplantation systems [3] and, as the present gold standard, the matrixassisted, autologous chondrocyte transplantation (MACT) [4] These techniques, mostly do not stop the progression of cartilage degeneration. One reason for the failure is that the regenerated tissue mainly consists of fibrous or osseus cartilage with functional and biomechanical properties clearly inferior to those of hyaline cartilage. Bioresorbable polymers are usually degraded in the body within a few weeks, whereas the reconstruction of fully functional cartilage usually requires months or even years
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