Therapies with CCL25 require controlled release via microparticles to avoid strong inflammatory reactions.

BACKGROUND: Chemokine therapy with C-C motif chemokine ligand 25 (CCL25) is currently under investigation as a promising approach to treat articular cartilage degeneration. We developed a delayed release mechanism based on Poly (lactic-co-glycolic acid) (PLGA) microparticle encapsulation for intraarticular injections to ensure prolonged release of therapeutic dosages. However, CCL25 plays an important role in immune cell regulation and inflammatory processes like T-cell homing and chronic tissue inflammation. Therefore, the potential of CCL25 to activate immune cells must be assessed more thoroughly before further translation into clinical practice. The aim of this study was to evaluate the reaction of different immune cell subsets upon stimulation with different dosages of CCL25 in comparison to CCL25 released from PLGA particles. RESULTS: Immune cell subsets were treated for up to 5 days with CCL25 and subsequently analyzed regarding their cytokine secretion, surface marker expression, polarization, and migratory behavior. The CCL25 receptor C-C chemokine receptor type 9 (CCR9) was expressed to a different extent on all immune cell subsets. Direct stimulation of peripheral blood mononuclear cells (PBMCs) with high dosages of CCL25 resulted in strong increases in the secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-1ß (IL-1ß), tumor-necrosis-factor-α (TNF-α) and interferon-γ (IFN-γ), upregulation of human leukocyte antigen-DR (HLA-DR) on monocytes and CD4+ T-cells, as well as immune cell migration along a CCL25 gradient. Immune cell stimulation with the supernatants from CCL25 loaded PLGA microparticles caused moderate increases in MCP-1, IL-8, and IL-1ß levels, but no changes in surface marker expression or migration. Both CCL25-loaded and unloaded PLGA microparticles induced an increase in IL-8 and MCP-1 release in PBMCs and macrophages, and a slight shift of the surface marker profile towards the direction of M2-macrophage polarization. CONCLUSIONS: While supernatants of CCL25 loaded PLGA microparticles did not provoke strong inflammatory reactions, direct stimulation with CCL25 shows the critical potential to induce global inflammatory activation of human leukocytes at certain concentrations. These findings underline the importance of a safe and reliable release system in a therapeutic setup. Failure of the delivery system could result in strong local and systemic inflammatory reactions that could potentially negate the benefits of chemokine therapy.

Toward scaffold-based meniscus repair: effect of human serum, hyaluronic acid and TGF-ß3 on cell recruitment and re-differentiation.

OBJECTIVE: Repair approaches for the non-vascular meniscus are rarely developed. Recent strategies use scaffold-based techniques and inducing factors. The aim of the study was the investigation of cell recruitment and re-differentiation inducing factors for a scaffold-based meniscus repair approach. METHOD: 3D cultivation of in vitro expanded human meniscus-derived cells was performed in high-density cultures supplemented with 25% hyaluronic acid (HA), 10% human serum (HS) or 10 ng/ml transforming growth factor (TGF-ß3) compared to untreated controls. The in vitro cell recruitment potential of different HS concentrations was tested by chemotaxis assay. Analysis of chondrocytic markers (type I, II, IX collagen and proteoglycans) was performed on protein and gene expression level. RESULTS: Cells were attracted by 1-20% HS. 3D cultures supplemented with 10% HS and 25% HA showed meniscus-like gene expression profiles at day 7 with significantly increased cartilage oligomeric matrix protein (COMP) and aggrecan expression levels in the HS group and a slightly increased profile in the HA group compared to control. The TGF-ß3 group showed an additional induction of gene expression levels for type II and type IX collagen. Histological findings confirmed these results by proteoglycan and type I collagen staining in all groups and type II collagen staining only in the TGF-ß3 group. CONCLUSION: This study demonstrates that human meniscus cells are attracted by HS and allow for meniscal matrix formation in 3D culture in the presence of HA and HS, whereas TGF-ß3 additive does not initiate meniscal tissue. Regarding non-vascular meniscus repair, results of this study encourage scaffold-based repair approaches.

Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage.

OBJECTIVE: It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue. METHODS: Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes. RESULTS: Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca(2+)/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers. CONCLUSIONS: WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca(2+)/WNT pathway was activated in OA cartilage.

Mesenchymal stem cells improve murine acute coxsackievirus B3-induced myocarditis.

AIMS: Coxsackievirus B3 (CVB3)-induced myocarditis, initially considered a sole immune-mediated disease, also results from a direct CVB3-mediated injury of the cardiomyocytes. Mesenchymal stem cells (MSCs) have, besides immunomodulatory, also anti-apoptotic features. In view of clinical translation, we first analysed whether MSCs can be infected by CVB3. Next, we explored whether and how MSCs could reduce the direct CVB3-mediated cardiomyocyte injury and viral progeny release, in vitro, in the absence of immune cells. Finally, we investigated whether MSC application could improve murine acute CVB3-induced myocarditis. METHODS AND RESULTS: Phase contrast pictures and MTS viability assay demonstrated that MSCs did not suffer from CVB3 infection 4-12-24-48 h after CVB3 infection. Coxsackievirus B3 RNA copy number decreased in this time frame, suggesting that no CVB3 replication took place. Co-culture of MSCs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis, oxidative stress, intracellular viral particle production, and viral progeny release in a nitric oxide (NO)-dependent manner. Moreover, MSCs required priming via interferon-γ (IFN-γ) to exert their protective effects. In vivo, MSC application improved the contractility and relaxation parameters in CVB3-induced myocarditis, which was paralleled with a reduction in cardiac apoptosis, cardiomyocyte damage, left ventricular tumour necrosis factor-α mRNA expression, and cardiac mononuclear cell activation. Mesenchymal stem cells reduced the CVB3-induced CD4- and CD8- T cell activation in an NO-dependent way and required IFN-γ priming. CONCLUSION: We conclude that MSCs improve murine acute CVB3-induced myocarditis via their anti-apoptotic and immunomodulatory properties, which occur in an NO-dependent manner and require priming via IFN-γ.

Chemokine profile of synovial fluid from normal, osteoarthritis and rheumatoid arthritis patients: CCL25, CXCL10 and XCL1 recruit human subchondral mesenchymal progenitor cells.

OBJECTIVE: The microfracture technique activates mesenchymal progenitors that enter the cartilage defect and form cartilage repair tissue. Synovial fluid (SF) has been shown to stimulate the migration of subchondral progenitors. The aim of our study was to determine the chemokine profile of SF from normal, rheumatoid arthritis (RA) and osteoarthritis (OA) donors and evaluate the chemotactic effect of selected chemokines on human subchondral progenitor cells. METHOD: Chemokine levels of SF were analyzed using human chemokine antibody membrane arrays. The chemotactic potential of selected chemokines on human mesenchymal progenitors derived from subchondral cortico-spongious bone was tested using 96-well chemotaxis assays. Chemokine receptor expression of subchondral progenitors was assessed by real-time gene expression analysis and immuno-histochemistry. RESULTS: Chemokine antibody array analysis showed that SF contains a broad range of chemokines. Ten chemokines that showed significantly reduced levels in RA or OA compared to normal SF or robustly high levels in all SF tested were used for further chemotactic analysis. Chemotaxis assays showed that the chemokines MDC/CCL22, CTACK/CCL27, ENA78/CXCL5 and SDF1α/CXCL12 significantly inhibited migration of progenitors, while TECK/CCL25, IP10/CXCL10 and Lymphotactin/XCL1 effectively stimulated cell migration. MCP1/CCL2, Eotaxin2/CCL24 and NAP2/CXCL7 showed no chemotactic effect on subchondral progenitors. Gene expression and immuno-histochemical analysis of corresponding chemokine receptors document presence of low levels of chemokine receptors in subchondral progenitors, with the CXCL10 receptor CXCR3 showing the highest expression level. CONCLUSION: These results suggest that SF contains chemokines that may contribute to the recruitment of human mesenchymal progenitors from the subchondral bone in microfracture.

Genome-wide expression profiling reveals new candidate genes associated with osteoarthritis.

INTRODUCTION: Although the extracellular matrix (ECM) is the functional element in articular cartilage and its degradation is central in the pathogenetic process in osteoarthritis (OA), increasing the knowledge about the cellular OA phenotype is essential. The aim of this study is therefore to provide a more complete picture of the cellular and molecular alterations detected in OA cartilage. MATERIAL AND METHODS: Human articular cartilage biopsies were collected from donors with macroscopical and microscopical signs of OA as well as donors with no previous history of OA and with microscopically intact cartilage. RNA was isolated from the biopsies and subjected to whole genome microarray analysis. Important results from the microarray analysis were verified using real-time PCR and immunohistochemistry. RESULTS: Our results reveal several new candidate genes not previously associated with OA to display significantly higher expression in OA cartilage than in normal donor cartilage, including genes involved in bone formation (CLEC3B, CDH11, GPNMB, CLEC3A, CHST11, MSX1, MSX2) and genes encoding collagens (COL13A1, COL14A1, COL15A1, COL8A2). DISCUSSION: This study is the first to report a comprehensive gene expression analysis of human OA cartilage compared to control cartilage from donors lacking macroscopical and microscopical signs of OA using recently developed microarrays containing the whole human genome. Our results could broadly confirm previously published data on many characteristic features of OA as well as adding a panel of genes to the list of genes known to be differentially expressed in OA. Elucidation of the phenotypical alterations occurring in OA chondrocytes is important for the development of effective treatments for OA.

Human intervertebral disc-derived cells are recruited by human serum and form nucleus pulposus-like tissue upon stimulation with TGF-beta3 or hyaluronan in vitro.

The aims of this work were to test whether human intervertebral disc-derived nucleus pulposus cells (hNP-cells) are attracted by human serum and to analyze if matrix generation from hNP-cells is promoted under the influence of transforming growth factor-beta3 (TGF-beta3) or hyaluronan (HA) in vitro. Using the multi-well chemotaxis assay to determine cell migration under the influence of different concentrations of human serum, it was demonstrated that dedifferentiated hNP-cells are able to migrate towards a serum fraction gradient in a concentration-dependent manner. Re-differentiation capacity of hNP-cells in 3D micro-masses under the influence of TGF-beta3 or hyaluronan was also tested. Gene expression analysis of types I, II, III and IX collagen, as well as aggrecan, COMP and LINK of hNP-cells in 3D-micro-mass cell-culture revealed a strong increase of these markers in TGF-beta3 treated cells. Furthermore, histochemical and immuno-histochemical staining after 28d showed proteoglycan and type II collagen-rich matrix for both, the TGF-beta3 and the hyaluronan treated cells. These findings show that TGF-beta3 or hyaluronan are able to induce the differentiation and that human serum stimulates the migration of hNP-cells in vitro. Therefore, hyaluronan and serum are suited for cell-free biomaterials as cell migration and differentiation inducing factors intended for biological treatment strategies of the intervertebral disc.

[Regenerative potential of human adult precursor cells: cell therapy--an option for treating cartilage defects?]. / Regeneratives Potenzial humaner adulter Vorläuferzellen: Zelltherapie--eine Option bei der Behandlung von Knorpeldefekten?

Cell-based therapeutical approaches are already in clinical use and are attracting growing interest for the treatment of joint defects. Mesenchymal stem and precursor cells (MSC) cover a wide range of properties that are useful for the regeneration process of bone and cartilage defects. The following article is an overview of the regenerative potential of MSC and discusses how the properties of these cells can be used for the development of new strategies in regenerative medicine.

Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures.

Articular cartilage has only very limited potential for self-repair and regeneration. For this reason, various tissue engineering approaches have been developed to generate cartilage tissue in vitro. Usually, most strategies require ascorbate supplementation to promote matrix formation by isolated chondrocytes. In this study, we evaluate and compare the effect of different ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate, sodium l-ascorbate, and l-ascorbate-2-phosphate were administered in 100 microM, 200 microM, and 400 microM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation were evaluated histologically and could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied by real-time RT-PCR and compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. Again, all supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes. Besides that, we could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations.

Human mastoid periosteum-derived stem cells: promising candidates for skeletal tissue engineering.

Currently, mesenchymal stem cells (MSCs) are considered as the most eligible cells for skeletal tissue engineering. However, factors such as difficult stimulation and control of differentiation in vivo hamper their clinical use. In contrast, periosteum or periosteum-derived cells (PCs) are routinely clinically applied for bone and cartilage repair. PCs have often been named MSCs but, although cells of osteochondrogenic lineages arise from MSCs, it is unclear whether periosteum really contains MSCs. Our aim was to investigate the MSC-like character of PCs derived from the periosteum of mastoid bone. Harvesting of periosteum from mastoid bone is easy, so mastoid represents a good source for the isolation of PCs. Therefore, we analysed the MSC-like growth behaviour and the expression of embryonic, ectodermal, endodermal and mesodermal markers by microarray and FACS technology, and the multilineage developmental capacity of human PCs. Regarding clinical relevance, experiments were performed in human serum-supplemented medium. We show that PCs do not express early embryonic stem cell markers such as Oct4 and Nanog, or the marker of haematopoietic stem cells CD34, but express some other MSC markers. Osteogenesis resulted in the formation of calcified matrix, increased alkaline phosphatase activity, and induction of the osteogenic marker gene osteocalcin. Staining of proteoglycans and deposition of type II collagen documented chondrogenic development. As shown for the first time, adipogenic stimulation of mastoid-derived PCs resulted in the formation of lipid droplets and expression of the adipogenic marker genes aP2 and APM1. These results suggest MSC-like PCs from mastoid as candidates for therapy of complex skeletal defects.

Human polymer-based cartilage grafts for the regeneration of articular cartilage defects.

The use of autologous chondrocyte implantation (ACI) and its further development combining autologous chondrocytes with bioresorbable matrices may represent a promising new technology for cartilage regeneration in orthopaedic research. Aim of our study was to evaluate the applicability of a resorbable three-dimensional polymer of pure polyglycolic acid (PGA) for the use in human cartilage tissue engineering under autologous conditions. Adult human chondrocytes were expanded in vitro using human serum and were rearranged three-dimensionally in human fibrin and PGA. The capacity of dedifferentiated chondrocytes to re-differentiate was evaluated after two weeks of tissue culture in vitro and after subcutaneous transplantation into nude mice by propidium iodide/fluorescein diacetate (PI/FDA) staining, scanning electron microscopy (SEM), gene expression analysis of typical chondrocyte marker genes and histological staining of proteoglycans and type II collagen. PI/FDA staining and SEM documented that vital human chondrocytes are evenly distributed within the polymer-based cartilage tissue engineering graft. The induction of the typical chondrocyte marker genes including cartilage oligomeric matrix protein (COMP) and cartilage link protein after two weeks of tissue culture indicates the initiation of chondrocyte re-differentiation by three-dimensional assembly in fibrin and PGA. Histological analysis of human cartilage tissue engineering grafts after 6 weeks of subcutaneous transplantation demonstrates the development of the graft towards hyaline cartilage with formation of a cartilaginous matrix comprising type II collagen and proteoglycan. These results suggest that human polymer-based cartilage tissue engineering grafts made of human chondrocytes, human fibrin and PGA are clinically suited for the regeneration of articular cartilage defects.

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study.

Mesenchymal stem cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, which suggest these cells as an attractive cell source for cartilage tissue engineering approaches. Our objective was to study the effects of TGF-beta1, hyaluronic acid and synovial fluid on chondrogenic differentiation of equine MSC. For that, bone marrow was aspirated from the tibia of one 18-month-old horse (Haflinger) and MSC were isolated using percoll-density centrifugation. To promote chondrogenesis, MSC were centrifuged to form a micromass and were cultured in a medium containing 10 ng/ml TGF-beta1 or 0.1mg/ml hyaluronic acid (Hylartil, Ostenil) or either 5%, 10% or 50% autologous synovial fluid as the chondrogenesis inducing factor. Differentiation along the chondrogenic lineage was documented by type II collagen and proteoglycan expression. MSC induced by TGF-beta1 alone showed the highest proteoglycan expression. Combining TGF-beta1 with hyaluronic acid could not increase the proteoglycan expression. Cultures stimulated by autologous synovial fluid (independent of concentration) and hyaluronic acid demonstrated a pronounced, but lower proteoglycan expression than cultures stimulated by TGF-beta1. The expression of cartilage-specific type II collagen was high and about the same in all stimulated cultures. In summary, hyaluronic acid and autologous synovial fluid induces chondrogenesis of equine mesenchymal stem cells, which encourage tissue engineering applications of MSC in chondral defects, as the natural environment in the joint is favorable for chondrogenic differentiation.

[Molecular characterization of tissue-engineered articular chondrocyte transplants based on resorbable polymer fleece]. / Molekulare Charakterisierung von gezüchteten humanen dreidimensionalen Chondrozytentransplantaten.

Three-dimensional arrangement and subsequent transplantation of chondrocytic cells in resorbable polymers has been shown to be a promising technique for the treatment of cartilaginous defects. Engineering of artificial cartilage tissue includes dedifferentiation of chondrocytes in monolayer culture, the use of biodegradable matrices and polymer scaffolds, and re-expression of chondrocytic marker genes in three-dimensional culture. The aim of this study was to characterize molecularly the phenotypic changes occurring with autologous cartilage tissue engineering. Human articular chondrocytes were isolated, cultured in medium containing human serum, and expanded up to passage 3. Chondrocytes were embedded in human fibrinogen and in polyglactin-polydioxanon fleeces and cultured three-dimensionally up to 4 weeks. Dedifferentiation of chondrocytes in monolayers and formation of cartilage tissue in vitro or after subcutaneous transplantation into nude mice was assessed by gene expression analysis of typical chondrocytic genes, histology, and immunohistochemistry. The expansion of chondrocytes with human serum resulted in the induction of type I and type III collagens, whereas cartilage-specific type II collagen, cartilage oligomeric matrix protein, cartilage link protein, and aggrecan were repressed and induced again after three-dimensional arrangement of chondrocytes in polyglactin-polydioxanon. Transplantation experiments documented the synthesis of proteoglycan and cartilage-specific type II collagen in vivo. Three-dimensional arrangement of human articular chondrocytes in resorbable polyglactin-polydioxanon fleeces supports chondrogenic differentiation and the formation of a hyaline-like cartilaginous matrix in vitro and in vivo.

[Tissue engineering: chances and challenges for application in rheumatic diseases]. / Tissue EngineeringChancen und Herausforderungen für die Anwendung in der Rheumatologie.

Current technologies of tissue engineering offer new strategies for the treatment of cartilage and bone defects. Beyond implantation of cell suspensions, second generation products of biomaterial enforced with in vitro preformed tissues are clinically applied. Ongoing research and development focus on differentiation factors and tissue protection. In search for sources of autologous cells which are easier to collect and which may serve for more complex tissues like osteochondral implants, mesenchymal stem cells are investigated. The design of in vitro experiments, which are required for these investigations, has produced tissue engineering technologies, which may serve for pathophysiology research in inflammatory joint diseases and for exploration of treatment strategies. These together with the advances in biological therapies of rheumatic diseases are the basis of new concepts, which promise application of tissue engineering also in inflammatory joint diseases.

Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices.

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

[Tissue engineering: new treatment of cartilage alterations in degenerative joint diseases in horses--preliminary results of a long term study]. / Tissue Engineering: Neue Behandlungsansätze bei Knorpelveränderungen bei degenerativen Gelenkerkrankungen des Pferdes--erste Ergebnisse einer Langzeitstudie.

Degenerative alterations in fetlock joints of the forelimb are common diagnoses for horses. The hyaline cartilage has a low capacity to regenerate and the treatment by veterinarians is often insufficient. As a final result, horses with articular cartilage defects are often not able to take part in competitions anymore. To establish an autologous cartilage repair method, we set artificial lesions (8 mm in diameter) into the fetlock joints of the forelimb of three horses. These defects were closed with autologous chondrocyte implants, which were fixed with titan-suture-anchors. After 3, 12 and 24 months, biopsies were taken by arthroscopy. One horse was euthanized after 9, another one after 24 months. The repair tissue was examined histologically and by biochemical analysis of hydroxyproline and glycosaminoglycan, which are typical cartilage related substances. After 9 months, the integration of the implant into native cartilage was demonstrated by electron microscopy. After 24 months, histological staining showed a similar morphology of the cartilage repair tissue compared with the surrounding native cartilage. Biochemical analysis of typical cartilage matrix molecules revealed formation of hyaline-like cartilage within tissue engineered autologous chondrocyte transplants.

Human platelet supernatant promotes proliferation but not differentiation of articular chondrocytes.

The objective of the study was to evaluate the growth-promoting activity of human platelet supernatant on primary chondrocytes in comparison with fetal calf serum (FCS) supplemented cell culture medium. Furthermore, the differentiation potential of platelet supernatant was determined in three-dimensional artificial cartilage tissues of bovine articular chondrocytes. Proliferation of articular and nasal septal chondrocytes was assayed by incorporation of BrdU upon stimulation with ten different batches of human platelet supernatant. On bovine articular chondrocytes, all these batches were at least as growth-promoting as FCS. On nasal septal chondrocytes, nine out of ten batches revealed increased or equivalent mitogenic stimulation compared with medium supplemented with FCS. Three-dimensional culture and subsequent histological analysis of matrix formation were used to determine the differentiation properties of platelet supernatant on articular chondrocytes. Human platelet supernatant failed to induce the deposition of typical cartilage matrix components, whereas differentiation and matrix formation were apparent upon cultivation of articular chondrocytes with FCS. Proliferation assays demonstrated that human platelet supernatant stimulates growth of articular and nasal septal chondrocytes; however, platelet supernatant failed to stimulate articular chondrocytes to redifferentiate in three-dimensional chondrocyte cultures. Therefore platelet lysate may be suitable for chondrocyte expansion, but not for maturation of tissue-engineered cartilage.