Induction of original phenotype of human immortalized chondrocytes: a quantitative gene expression analysis.

We previously established a line of immortalized normal human articular chondrocytes, lbpva55, expressing the E6 and E7 transforming genes of the human papilloma virus type 16. With this study we investigated the phenotypic modulation ability of this cell line, cultured in different conditions, with the aim of validating its use for studies on cartilage metabolism and physiology. To this end, we performed a quantitative analysis, using real-time PCR technology, of the expression of the main structural components of the cartilage matrix (collagens I, II and aggrecan), of two transcription factors regulating chondrocyte differentiation (Sox-9 and Egr-1) and of some enzymes involved in matrix turnover (cathepsin B, MMP-1 and MMP-13). Results showed that, under defined conditions, lbpva55 cells were able to re-express the chondrocyte phenotype that was lost in a conventional monolayer condition, as demonstrated by an up-regulation of collagen II, the main marker of hyaline cartilage and Sox-9, a master gene regulator of chondrocytic differentiation. The gene expression profile of our immortalized cells compared with that of normal articular chondrocytes showed that this line could be used as a valid in vitro model for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases and for the cartilage engineering field.

Human chondrocytes and mesenchymal stem cells grown onto engineered scaffold.

The development of improved methods for treatment of chondral defects using autologous cells in combination with biomaterials leads to a new generation of implantable devices. Their association gives rise to a hybrid construct combining biological and material components that can be specifically committed. The comprehension of cellular and molecular mechanisms of cartilage repair and the use of biomaterials in combination with chondrocytes or mesenchymal stem cells in the treatment of cartilage defects has opened a new era of therapeutical strategies. Recently, their applicability in the treatment of early lesions in osteoarthritis is under investigation. To obtain new information on the behaviour of chondrocytes and mesenchymal stem cells grown on a hyaluronan derivative scaffold (Hyaff-11) already used in cartilage repair, we analysed a series of molecules expressed by these cells by Real-Time RT-PCR and immunohistochemical analyses. The data obtained with this work showed that this biomaterial is able to reduce the expression of some catabolic molecules by human chondrocytes and provide a good environment to support the differentiation of mesenchymal stem cells in chondrogenic sense. These observations confirm Hyaff-11 as a suitable scaffold both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects.

Down regulation of degenerative cartilage molecules in chondrocytes grown on a hyaluronan-based scaffold.

Hyaluronic-acid-based biomaterials used for cartilage repair allow the expression of specific extracellular matrix molecules by human chondrocytes grown onto them. We investigated whether these biomaterials could also create an environment in which the cells downregulate the expression of some catabolic factors. Chondrocytes were isolated from human articular cartilage obtained from the knees of patients with a history of trauma. First, the cells were expanded in monolayers and then they were seeded on a hyaluronic-acid derivative scaffold. Constructs and surnatants were collected and analysed at 1, 3, 7, 14 and 21 days after seeding. Immunohistochemical analysis for CD44 and caspase was carried out on paraffin-embedded sections. The Tunel method was used to identify chondrocyte apoptosis status. Secretion of MMP-1 and MMP-13 in the surnatants of the cells grown onto the biomaterial was measured by enzyme-linked immunosorbent assay. Nitric oxide (NO) production was evaluated by estimating the stable NO metabolite nitrite by the Griess method. A real-time RT-PCR analysis was performed on the constructs to evaluate the expression of type I and II collagens, aggrecan, Sox-9, MMP-1 and MMP-13 mRNAs at the different experimental times evaluated. Decreased levels of metalloproteinases and nitric oxide were observed in the surnatants of chondrocytes grown onto the hyaluronan-based scaffold. This was also confirmed by real-time PCR analysis which showed that the cells expressed the specific differentiated phenotype downregulating the expression of some catabolic molecules. Cells apoptosis decreased during the culture period, which further supported the biochemical data. The ability of the hyaluronan scaffold to reduce the expression and production of molecules involved in cartilage degenerative diseases indicates its use to treat early lesions of osteoarthritic patients.

Transplantation of chondrocytes seeded on collagen-based scaffold in cartilage defects in rabbits.

Recent success in tissue engineering by restoring cartilage defects by transplanting autologous chondrocyte cells on a three-dimensional scaffold has prompted the improvement of this therapeutic strategy. Here we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a biomaterial made of an equine collagen type I-based scaffold. Full-thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired by using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histological examination of the reconstructed tissues at 1, 3, 6, and 12 months after transplantation showed that at 1 and 3 months there was no formation of reconstructed tissue in any of the groups evaluated; after 6 months there was evidence of a newly regenerated tissue with some fibrocartilaginous features only in the group treated with biomaterial-seeded cells, and at 12 months a more organized tissue was evident in the same group. With regards to the group transplanted with biomaterial alone and the untreated control group, there was no evidence of new tissue production. These results advocate the use of this collagen-based scaffold for further in vivo studies on large size animals and, finally, in human clinical trials for the treatment of knee cartilage defects.

Molecular and immunohistological characterization of human cartilage two years following autologous cell transplantation.

BACKGROUND: There are only a few studies concerning the cellular, biochemical, and genetic processes that occur during the remodeling of graft tissue after autologous chondrocyte transplantation. The purpose of the present study was to quantify the expression of genes encoding extracellular matrix proteins and regulatory factors that are essential for cell differentiation in cartilage biopsy specimens from patients who had this treatment two years previously. METHODS: Two cartilage biopsy specimens from each of four patients who had been treated with autologous chondrocyte transplantation and from two multiorgan donors were used. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression of types I, II, and X collagen; aggrecan; cathepsin B; and early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for matrix proteins and regulatory proteins was carried out on paraffin-embedded sections. RESULTS: Type-I collagen mRNA was expressed in all of the samples evaluated. Type-II collagen was present in autologous chondrocyte transplantation samples but at lower levels than in the controls. Type-X collagen messenger was undetectable. Aggrecan mRNA was present in all of the samples at lower levels than in the controls, while cathepsin-B messenger levels were higher and Egr-1 and Sox-9 mRNAs were expressed at lower levels. The immunohistochemical analysis showed slight positivity for type-I collagen in all of the sections. Type-II collagen was found in all of the samples with positivity confined inside the cells, while the controls displayed a positivity that was diffuse in the extracellular matrix. Cathepsin B was slightly positive in all of the samples, while the controls were negative. Egr-1 protein was particularly evident in the areas negative for type-II collagen. Sox-9 was positive in all samples, with evident localization in the superficial and middle layers. CONCLUSIONS: In biopsy specimens from autologous chondrocyte transplantation tissue at two years, there is evidence of the formation of new tissue, which displays varying degrees of organization with some fibrous and fibrocartilaginous features. Long-term follow-up investigations are needed to verify whether, once all of the remodeling processes are completed, the newly formed tissue will acquire the more typical features of articular cartilage.

Cathepsin B as a soluble marker to monitor the phenotypic stability of engineered cartilage.

The clinical need for improved human autologous chondrocyte transplantation has motivated the use of different biomaterials, which are aimed at fixing the cells in the defect area and permit their proliferation and differentiation. The maintenance of the original phenotype by isolated chondrocytes grown in vitro is an important requisite for their use in repairing damaged articular cartilage. The methods to verify the expression of cartilage-specific molecules usually involve destructive procedures to recover the cells from the scaffolds for tests. The aim of our study was to find a soluble marker able to attest the occurrence of a differentiation process by chondrocytes grown onto a biomaterial used for cell transplantation. We turned our attention to cathepsin B which is known to be abnormally synthesized in de-differentiated chondrocytes and scarcely produced in the differentiated ones. The production of cathepsin B by human articular chondrocytes expanded in vitro and then grown onto a hyaluronan-based polymer derivative (Hyaff-11) three-dimensional scaffold was evaluated with a specific enzyme-immunoassay at different experimental times together with the expression of mRNA by real-time PCR. We showed that cathepsin B, which is abundantly produced by chondrocytes grown in a monolayer culture, decreases significantly after the cells are seeded onto the scaffold, giving further evidence of a re-differentiation process. This result suggests cathepsin B a practical soluble marker to evaluate the "good" quality of transplantable constructs.