Eugenia Niculescu-Morzsa

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.

Investigation of Collagen Transplants Seeded with Human Autologous Chondrocytes at the Time of Transplantation

Objective: The treatment of cartilage defects with matrix-embedded autologous chondrocytes is a promising method to support the repair process. In this study we gathered quality parameters of collagen I matrices and embedded autologous chondrocytes at the time of transplantation We determined number, morphology, and distribution of matrix-embedded chondrocytes as well as their synthesis performance concerning sulphated glycosaminoglycans (sGAG) and collagen 1A1 and 2A1 mRNA levels. Results: Chondrocytes were equidistantly distributed in the collagen matrices, and cell numbers ranged from 6 to 34 × 104 cells/g wet weight. Significant amounts of sGAG were detected in all of the investigated transplants but did not correlate with the number of cells within the respective transplants. Moreover, collagen I mRNA levels exceeded that of collagen II up to 17-fold. Collagen I and II ratio and sGAG amounts indicated significant interindividual differences of chondrocytes. The variation of transplant-associated sGAG levels could be attributed to the differential biosynthesis performance of chondrocytes. Conclusions: These results confirm the vitality and the chondrocytic phenotype of matrix-embedded cells (CaRes®) with respect to sGAG synthesis. However, chondrocytes showed collagen I mRNA expression partially far exceeding that of collagen II, indicating a rather dedifferentiated cellular status. In addition, sGAG synthesis performance of different patients’ chondrocytes varied significantly. Nevertheless, a 2-year clinical study of chondrocyte-seeded collagen matrices as investigated in this work delivered promising results. However, future studies are planned to determine markers for the regenerative potential of donor chondrocytes.

Platelet-Rich Plasma Supports Proliferation and Redifferentiation of Chondrocytes during In Vitro Expansion

Articular cartilage regeneration is insufficient to restore sports injuries or defects that can occur from trauma. Treatment options for cartilage repair include autologous chondrocyte implantation (ACI) by isolation, expansion, and reimplantation of healthy donor chondrocytes. Chondrocyte expansion onto 2D substrates leads to dedifferentiation and loss of the cellular phenotype. We aimed to overcome the state of dedifferentiation by biochemical stimuli with platelet derivatives such as platelet-rich plasma (PRP) and hyperacute serum (HAS) to achieve sufficient cell numbers in combination with variable oxygen tension. Human articular chondrocytes from osteoarthritic (OA) cartilage chondrocytes were switched from 10% FCS supplementation to either 10% PRP or 10% HAS after initial passaging for further experiments under normoxic (20% O2) or hypoxic (1% O2) conditions. An XTT assay measured the effect of PRP or HAS on the cell proliferation at 3, 6, and 9 days. The chondrogenic redifferentiation potential of dedifferentiated chondrocytes was determined with reverse transcriptase quantitative real-time PCR for markers of expression for type II collagen (COL2A1), type I collagen (COL1A1), and matrix metalloproteinases MMP3, matrix metalloproteinase 13 (MMP13) at 24 and 72 h. Measured protein levels of 100% PRP or HAS by multiplex quantification revealed basic fibroblast growth factor, G-CSF, and PDGF were significantly higher in PRP than in HAS (p < 0.05) but LEPTIN levels did not differ. The quantified protein levels did not differ when isolated from same donors at a different time. Chondrocyte proliferation indicated that supplementation of 10% HAS enhanced the proliferation rate compared to 10% PRP or 10% FCS at 6 and 9 days significantly (p < 0.05). mRNA levels for expression of COL1A1 were significantly downregulated (p < 0.05) when cultured with 10% PRP than 10% HAS or 10% FCS under normoxic/hypoxic conditions. COL2A1 was significantly upregulated (p < 0.05) in PRP than 10% HAS or 10% FCS. MMP3 expression was downregulated after 72 h under all conditions. MMP13 was upregulated with 10% PRP at both 24 and 72 h but significantly downregulated under hypoxia (1% O2) for all circumstances. While HAS has its effect on chondrocyte proliferation, PRP enhances both proliferation and redifferentiation of dedifferentiated chondrocytes. PRP can replace standard usage of FCS for chondrogenic priming and expansion as implications for clinical use such as ACI procedures.

A protocol for gene expression analysis of chondrocytes from bovine osteochondral plugs used for biotribological applications

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