Introduction: The chondrogenic differentiation of adult mesenchymal stem cells (MSCs) is a promising method for cartilage tissue engineering and repair of cartilage defects. The potential of MSCs to differentiate in chon-drocytes could be proved in many investigations, in matrix-dependent and matrix-free set-ups. A standard system for chondrogenic differentiation of MSCs utilizes alginat beads which allow a high cell density, will not generate a homogenious matrix. Therefore beads may not be optimal for regeneration of cartilage in articulating joints. Furthermore, after chondrogenic differentiation, cells in alginate beads may display signs of hypertrophy, including collagen X, alkaline phosphatase and MMP-13. The aim of our investigation was to explore the chondrogenic differentiation of MSC in a novel collagen-chondroitinsulphat-matrix.

Methods: Adult MSC were harvested from the bone marrow of donators who received a total hip replacement The cells were differentiated in a monolayer culture, on alginat beads and in a novel spongiform collagen-chondroitinsulphat-matrix. For differentiation, the medium was supplemented with dexamethason, ascorbic acid, and TGFβ. The total culture period was 21 days. Afterwards the expression of collagen-I, -II and -X, Interleukin (IL)-1β, IL-6, MMP-1, -3 and -13 was determined by quantitative RT-PCR. Histological analysis of the constructs were performed after 4 weeks of s.c. implantation in immunodeficient SCID-mice.

Results: Human MSC undergo chondrogenic differentiation in the novel collagen-chondroitinsulfat-matrix. In comparison to cells differentiated in alginat beads, a higher expression of collagen II but a comparable expression of collagen I, MMP-1, MMP-3 and IL-1β were observed. Collagen-X, MMP-13 or alkaline phosphatase were not detected in the cells differentiated in the new matrix, but could be found in cells the alginat beads. Furthermore, in comparison to the monolayer cultures, the collagen II expression was 100’000-fold raised, but no difference was found in the expression of collagen I, MMP-1, MMP-3 and IL-1β.

Discussion: The novel collagen-chondroitinsulphat-matrix supports an improved chondrogenic differentiation of MSCs with an elevated expression of collagen-II and very low expression of markers of hypertrophy in comparison to cells in alginate beads or in monolayer cultures. These results are a promising basis for improved tissue engineering of cartilage. The clinical application of these constructs seems to be possible, because the new matrix is approved for autologous chondrocyte transplantation and MSC can be expanded under GMP-compatible conditions.

A variety of scaffolds, including collagen-based membranes, fleeces and gels are seeded with osteoblasts and applied for the regeneration of bone defects. However, different materials yield different outcomes, despite the fact that they are generated from the same matrix protein, i.e. type I collagen. Recently we showed that in fibroblasts MMP-3 is induced upon attachment to matrix proteins in the presence of TGFbeta.

Aim: To investigate the regulation of matrix metalloproteinases (MMPs) and interleukins (IL) in osteoblasts upon attachment to type I collagen (col-1) in comparison to laminin -1 (LM-111) in the presence or absence of costimulatory signals provided by transforming growth factor beta (TGFbeta).

Methods: Osteoblasts were seeded in col-1–and LM-111-coated flasks and activated by the addition of TGFbeta. Mock-treated cells served as controls. The expression of genes was investigated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunocytochemistry and ELISA.

Results: Attachment of osteoblasts to col-1 or LM-111 failed to activate the expression of MMPs or ILs. In contrast, TGFbeta induced the expression of MMP-3, MMP-9, and MMP-13, IL-6 and IL-16 mRNAs. MMP-3 was found to be elevated in supernatants of activated cells. No difference was found in the expression of MMP-1, IL-8 and IL–18. Interestingly, the expression of IL-1beta mRNA was not activated by TGFbeta alone, but it was activated by attachment of osteoblasts to LM-111 in the presence of TGFbeta.

Conclusion: In contrast to fibroblasts, attachment of osteoblasts to col-1 or LM-111 had no effect on the induction of MMPs and ILs. TGFbeta induced the expression of MMPs and ILs in these cells but only MMP-3 was released. The results show significant differences between osteoblasts and fibroblasts in the effects of attachment to scaffold materials. This may have important consequences for tissue engineering of bone and for wound healing after surgery.

Introduction: After a meniscus trauma, preservation of the meniscus is the most important surgical goal. The use of scaffolds colonized with meniscus cells (fibrochondrocytes) to reconstruct meniscal defects seem to be a promising way for the treatment of a meniscus trauma. The goal of our investigations was the analysis of expression of different anabolic and catabolic factors in human fibrochondrocytes after seeding these cells onto a collagen I scaffold to investigate the regenerative potential of such a construct for the treatment of meniscus tears.

Material and Methods: Human meniscus tissue was digested in collagenase and dispase and cells were characterized by immunohistochemistry. To test scaffolds, we used a commercially available bovine collagen I matrix approved for surgical purposes. The scaffold was colonized with human fibrochondrocytes in a density of 106 cells per cm2. Cells expanded at the same ínoculation density w/o scaffold served as mock-controls. After 14 and 28 days in culture, the cells were extracted from the scaffold by aid of collagenase (Sigma, Deisenhofen, FGR) and analyzed for the expression of different factors, including IL-1β, IL-6, TGF-β, TIMP-1, TIMP-3, MMP-1, and MMP-3 using a quantitative RT-PCR-technology.

Results: Bovine collagen I matrices could be colonized with human fibrochondrocytes. After 14 and 28 days of incubation on the scaffolds, the cells show the same mRNA expression levels of IL-1β, TIMP-1, TIMP-3, and TGF-β when compared to controls. In contrast, after 14 days IL-6 (12.7-fold ± 4.4, p< 0.001), MMP-1 (11.3-fold ± 2.4, p< 0.001), and MMP-3 (13.7-fold ± 6.8, p< 0.031) were upregulated on transcription levels in the scaffold when compared to controls after the same period of culture. After 28 days of culture in scaffold the expression of MMP-3 was upregulated 78.2-fold (± 7.4, p< 0.0001), MMP-1 (71.3-fold ± 5.9, p< 0.0001) and IL-6 was elevated 98.9-fold (± 9.1, p< 0.0001) compared to controls.

Discussion/Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint colonized onto a bovine collagen I matrix. We could show that meniscus cells revealed a significantly increased expression of MMP-1 and MMP-3, and also a significant elevation of IL-6 mRNA after 14 and 28 days of culture. No changes were found in the expression levels of IL-1β, TGF-β, and the TIMPs. This suggests that the meniscus cells colonized onto a bovine collagen I scaffold produce a considerable amount of catabolic or inflammatory factors. This may lead to a destruction of the scaffold-matrix itself and the extracellular matrix of the meniscus. Secondly, IL-6 could induce a global inflammation around the scaffold by activating the IL-6 inflammation cascade.

The meniscus of the human knee joint has an outstanding function for stability, shock absorption and power transmission of the thigh on the shank. After a meniscus trauma so far often only the partial or complete removal of the meniscus has to be performed. Only with injuries in the outside third a primary suture of a tear leads to the healing due to the existing vascularisation in a high number of cases in younger patients. After partial or total meniscektomie cartilage degeneration and resulting osteoarthrosis of the knee joint often is the consequence.

A goal of our investigations was the establishment of meniscus cell cultures as well as their characterisation regarding the expression of different growth factors, cytokines and proteins and the influence by adding different recombinant growth factors. We are able to cultivate human fibrochondrocytes, which originate from menisci of the knee from patient undergoing total knee replacement. Investigations were performed by immune-histochemistry and RT PCR. We could show the expression of collagen I, II, III and VI, the matrix-metalloproteinases 1, -2, -3 and -8 in the human meniscus. In Addition the expression of TGFβ1, BMP II, AS.02, Thy 1, TGFβ1, iNOS and interleukin (IL) -1, -6 and -18, ECGF and VEGF was proved. PDGF-1 and collagen X could not be found in the meniscus investigated. Same expression analysis was performed in same patients’ synovial cells and chondrocytes from knee joint. Differences were found in the collagen expression. Synovial cells do not synthesise collagen II but collagen I. Investigated chon-drocytes show a high level of collagen I an II expression, but fibrochondrocytes a low level of collagen II and high of collagen I, too. After stimulation of meniscus cells with IL-1, TGFβ1 and TNF-α no difference was found in the expression of TGFβ1, BMP II and IL-18, but a total inhibition of IL-6. TGFβ1 suppressed IL-1 expression totally compared to not stimulated fibrochondrocytes.

We were able to cultivate, characterize and stimulate human fibrochondrocytes from meniscus of the knee. We could show that meniscus cells express a huge amount of different growth factors, cytokines and proteins and can be distinguished from synovial cells and joint chondrocytes by the low level expression of collagen II. We also investigated first time the reaction of human meniscus cells after stimulation by recombinant growth factors. These results are a basis for the tissue engineering of meniscus tissue.

Introduction: The highest goal after meniscus damage is the preservation of the meniscus, which is often not possible due to the bad healing of meniscus lesions in the avascular zone. Therefore, the goal of our investigations was the analysis of expression of different angiogenic factors, growth hormones and cytokines in human meniscus cells (fibrochondrocytes). The mutual influence of the fibrochondrocytes by endothelial cell cocultures was analyzed, in order to examine the molecular bases of the healing of meniscus tears in vascularized zones more exactly. For this purpose, commercially available HUVEC [human umbilical vein endothelial cells] were used as well established and stable endothelial cell model.

Material and Methods: Meniscal fibrochondrocytes were expanded in DMEM medium enriched with antibiotics and 10 % FCS. Cocultures of mensical cells and HUVEC were incubated in transwells over four and twelve days, separated by a semipermeable membrane. The expression of Angiopoietin-1, Angiopoietin-2, End-ostatin, VEGF, SMAD-4, Thrombospondin-1, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, IL-1β, iNOS, MMP-1, MMP-3, MMP-13, collagen-I, -II, -III, -VI, X, and -XVIII were examined by RT-PCR and immunhistochemistry in fibrochondrocytes in the comparison to cultures without endothelial coculture. A proliferation assay was used to investigate the mitotic activity in the coculture compared to the control culture after 4 and 12 days.

Results: In presence of HUVEC, meniscal fibrochon-drocytes expressed the following factors at rates comparable to cells w/o HUVECS: Angiopoietin-1, Angiopoietin-2, VEGF, SMAD-4, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, iNOS, MMP-1, MMP-3, MMP-13, Thrombostatin-1, collagen-I, -II, -III, -VI, X, and -XVIII. In contrast, expression of end-ostatin (5.1-fold ± 1.2, p< 0.01) and IL-1β (10.3-fold ± 2.3, p< 0.003) were expressed significantly higher in the coculture when compared to the individual cell cultures. The proliferation rate of HUVEC was significantly decreased in coculture when compared to controls: 22 % after 7 days and 35 % after 14 days (p< 0.001).

Discussion/ Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint. We could show that coculture of meniscus cells with endothelial cells revealed an increased expression of the anti-angiogenetic factor endostatin and the pro-inflammatory IL-1β. This suggests that meniscus cells are trying to inhibit proliferation of endothelial cells in their neigbourhood, which implicates huge problems in the research field of neoangiogenisis and tissue engineering in meniscus tissue for new healing methods after meniscus trauma.

Aim: The healing capacity of human articular cartilage is very limited in the adult. Therefore tissue engineering techniques were developed to treat cartilage lesions. To it, autologous chondrocytes are harvested from the affected joint and expanded in vitro. During expansion chondrocytes may dedifferentiate, characterized by an increase in type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of cartilage after transplantation, we investigated if the human platelet supernatants (hPS) containing PDGF and TGF-b or recombinant human bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures (2D) and in three-dimensional culture (3D) systems.

Methods: Chondrocytes from articular knee cartilage of 14 individuals (mean age 36.5 6.5 years) with no history of inflammatory joint disease were isolated and expanded under GMP conditions suitable for clinical purposes. The hPS was prepared from blood of 3 donors and pooled. Cells were seeded either in 2D cultures or embedded in alginate beads (3D) in presence or absence of hPS or recombinant human BMP-2 (generous gift of Dr. Hortschansky, Jena, FRG). After two weeks in culture, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA methods.

Results: Expansion of chondrocytes in primary culture (P0) did not yield populations of dedifferentiated or hypertrophic cells. Expanding cells in first subculture (P1) resulted in spontaneous reduction of type II collagen expression and increase in type I collagen mRNA amounts. Seeding P1 chondrocytes in 3D culture significantly reduced type I collagen, BMP-4 and IL-18 and maintained high type II collagen and BMP-2 encoding mRNA (p < 0.05). Reduction of IL-1b and elevation of IL-10 mRNA were noted but were statistically not significant. Addition of BMP-2 to 2D chondrocytes had no effect on type II collagen or IL-1b mRNA amounts (p < 0.05). In alginate cultures BMP-2 induced type II collagen and reduced IL-1b mRNA amounts. In contrast, addition of hPS containing PDGF and TGF-b, promoted mitotic activity in 2D and alginate cultures. The hPS reduced in 2D cultures type II and induced type I collagen expression. Even in alginate beads induction of type I collagen was detected.

Conclusions: We conclude that the chondrogenic phenotype is stabilized by BMP-2 more effectively in alginate beads but not in monolayer cultures. The hPS promotes proliferation of chondrocytes in vitro but induces elevated type I expression, an indicator of chondrocyte dedifferentiation.