Florian Halbwirth

A Novel Cross-Linked Hyaluronic Acid Porous Scaffold for Cartilage Repair: An In Vitro Study With Osteoarthritic Chondrocytes

Purpose An important feature of biomaterials used in cartilage regeneration is their influence on the establishment and stabilization of a chondrocytic phenotype of embedded cells. The purpose of this study was to examine the effects of a porous 3-dimensional scaffold made of cross-linked hyaluronic acid on the expression and synthesis performance of human articular chondrocytes. Materials and Methods Osteoarthritic chondrocytes from 5 patients with a mean age of 74 years were passaged twice and cultured within the cross-linked hyaluronic acid scaffolds for 2 weeks. Analyses were performed at 3 different time points. For estimation of cell content within the scaffold, DNA-content (CyQuant cell proliferation assay) was determined. The expression of chondrocyte-specific genes by embedded cells as well as the total amount of sulfated glycosaminoglycans produced during the culture period was analyzed in order to characterize the synthesis performance and differentiation status of the cells. Results Cells showed a homogenous distribution within the scaffold. DNA quantification revealed a reduction of the cell number. This might be attributed to loss of cells from the scaffold during media exchange connected with a stop in cell proliferation. Indeed, the expression of cartilage-specific genes and the production of sulfated glycosaminoglycans were increased and the differentiation index was clearly improved. Conclusions These results suggest that the attachment of osteoarthritic P2 chondrocytes to the investigated material enhanced the chondrogenic phenotype as well as promoted the retention.

Correlation Analysis of SOX9, -5, and -6 as well as COL2A1 and Aggrecan Gene Expression of Collagen I Implant–Derived and Osteoarthritic Chondrocytes

Objective Matrix-assisted autologous chondrocyte implantation is frequently applied to replace damaged cartilage in order to support tissue regeneration or repair and to prevent progressive cartilage degradation and osteoarthritis. Its application, however, is limited to primary defects and contraindicated in the case of osteoarthritis that is partially ascribed to dedifferentiation and phenotype alterations of chondrocytes obtainable from patients’ biopsies. The differentiation state of chondrocytes is reflected at the level of structural gene (COL2A1, ACAN, COL1A1) and transcription factor (SOX9, 5, 6) expression. Methods/Design We determined the mRNA abundances of COL2A1, ACAN, and COL1A1as well as SOX9, -5, and -6 of freshly isolated and passaged collagen I implant–derived and osteoarthritic chondrocytes via reverse transcription–polymerase chain reaction. Moreover, we analyzed the correlation of structural and transcription factor gene expression. Thus, we were able to evaluate the impact of the mRNA levels of transcription factors on the expression of cartilage-specific structural genes. Results Significant differences were obtained (1) for freshly isolated osteoarthritic versus collagen I implant–derived chondrocytes, (2) due to passaging of the respective cell sources, (3) for osteoarthritic versus nonosteoarthritic chondrocytes, and (4) for COL2A1 versus ACAN expression with respect to the coherence with SOX9, -5, and -6 transcript levels. Conclusion Our results might contribute to a better understanding of the transcriptional regulation of structural gene expression of chondrocytes with implications for their use in matrix-assisted autologous chondrocyte implantation.

Chondrogenic Gene Expression Differences between Chondrocytes from Osteoarthritic and Non-OA Trauma Joints in a 3D Collagen Type I Hydrogel:

Objective The purpose of the current study was to compare the donor age variation of chondrocytes from non-OA (osteoarthritic) trauma joints in patients of young to middle age (20.5 ± 3.7, 31.8 ± 1.9, 41.9 ± 4.1 years) embedded in matrix-associated autologous chondrocyte transplantation (MACT) grafts (CaReS). The chondrocyte-specific gene expression of CaReS grafts were then compared to chondrocytes from OA joints (in patients aged 63.8 ± 10 years) embedded in a collagen type I hydrogel. Design OA chondrocytes and articular chondrocyte-laden grafts were cultured over 14 days in chondrogenic growth medium. We performed reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate the mRNA expression levels of chondrocyte-specific and hypertrophic markers. Results Gene expression analysis with RT-qPCR revealed no significant difference in chondrocyte-specific genes (COL2A1, ACAN, SOX9, SOX5, SOX6) among 3 different age group of patients with CaReS grafts. In a comparative analysis of OA chondrocytes to articular chondrocytes, chondrogenic markers (COL2A1, SOX6) exhibited higher expression in OA chondrocytes (P < 0.05). Hypertrophic or OA cartilage pathogenesis marker (MMP3, MMP13) expression was higher and COL1A1 had significantly lower expression (P < 0.05) in OA chondrocytes than articular chondrocytes when cultivated in collagen type I hydrogels. Conclusion In summary, we identify that donor age variation does not influence the chondrogenic gene expression of the CaReS system. We also identified that freshly isolated OA chondrocytes embedded in collagen type I hydrogels can exhibit chondrogenic gene expression as observed in articular chondrocytes on the CaReS grafts. Transforming OA chondrocytes to articular chondrocytes can be regarded as an alternative option in the MACT technique.

CaReS®, Cartilage Regeneration System: Autologous Chondrocyte Transplantation in a Collagen Gel

Articular cartilage is a white, shiny, moisture tissue comprising less than 5 % cells, about 35 % extracellular matrix of mostly collagen type II and proteoglycans and about 60 % water, and provides outstanding biomechanics. Hence the tissue looks simple in its structure; the biomechanical properties are linked to the complex nanostructured architecture of the tissue, which partly relates to the high water content bound to macromolecules [26]. Since the composition of articular cartilage is not restored by natural healing, many attempts have been made to improve the quality of the repair tissue including microfracture [13] or osteochondral autografting; both methods are limited due to tissue quality or resources of grafts [3, 8, 10, 16]. Autologous chondrocyte transplantation has changed the paradigm of the treatment of cartilage defects from repair to regeneration, and this has been demonstrated in randomised trials proving the concept of regenerating tissue in a cell-based therapy approach [21]. However, the limitations of the periosteal flap concerning size and thickness and surgical demands with suturing and the variability of biological reaction including hypertrophy, calcification and delamination [2, 16, 17, 20, 27], as well as the uncontrolled cell transplantation in a cell suspension [25], have supported the introduction of the use biomaterials as a scaffold. The first attempt was to replace the periosteal flap by a collagen membrane which were sutured or glued to the defect combined with the cell suspension injected or seeded onto the membrane during the surgery before implantation [2, 4, 9, 15, 19]. Concerns about the phenotype of the cultured cells led to preculturing techniques on biomaterials in sponges or gels to maintain the chondrocytic function of the cells and allow a more controlled dispersion of the chondrocytes throughout the defect [22]. The matrix characteristics concerning biochemical composition, biophysical appearance in gel, foams or sponges, degradation dynamics and products, toxicity, immunological reactions and general biocompatibility are important parameters of biomaterial development [1, 5, 9, 15, 18]. The cell-biomaterial interaction in a biological environment is the decisive process of successful cartilage regeneration.

Mechanische Belastung und Bindegewebe

Anpassungen des Bindegewebes an mechanische Belastungen in Muskeln, Sehnen, Bandern oder Knochen fuhren zu einer gesteigerten Synthese und zum Umsatz von Matrixproteinen, einschlieslich des Kollagens. Regelmasiges Belasten des Gewebes, wie zum Beispiel durch korperliches Training, fuhrt zu einem gesteigerten Umsatz von Kollagen und einer Netzkollagensynthese und steht im Zusammenhang mit einer Anpassung der mechanischen Eigenschaften des Gewebes, die potenziell zu einem belastungsresistenteren Gewebe fuhrt. Die Anpassungszeit des Bindegewebes an chronische Belastung ist verglichen mit jener von kontraktilen Elementen der Skelettmuskulatur oder des Myokards langer.