Clinical outcome is significantly better with spheroid-based autologous chondrocyte implantation manufactured with more stringent cell culture criteria.

Objective: Spherox (CO.DON AG) is an autologous chondrocyte implantation (ACI) product, consisting of spheroids of human autologous matrix-associated chondrocytes. The tendency of primary chondrocytes to dedifferentiate during cultivation and the high biologic variability caused by the autologous nature of the starting material makes it challenging to design a manufacturing process that performs consistently and delivers products that meet their intended function and the high quality criteria for cell-based ATMPs. The current study was submitted during the European authorization procedure, and addresses the requirement to justify the operational ranges of the manufacturing process using clinical data. Methods: In order to define the operational ranges, statistical correlation analyses were conducted between process parameters and clinical improvement data of 120 patients from Phase II and III treated with ACI (KOOS score, 1 year follow-up). Results: This approach identified cell culture time as a critical process parameter that negatively correlates with the product's efficacy. Subsequent analyses of the Phase III patients that were treated with chondrocyte spheroids that have been manufactured with shorter monolayer and spheroid cultivation times showed a higher average clinical improvement as well as a higher responder rate compared to the total group. In addition, retrospective analyses demonstrated superiority for the treatment with short-cultivated chondrocyte spheroids over micro-fracture treatment. Conclusion: These findings underscore the need to use clinical data to optimize the manufacturing process for autologous cell-based therapies. We expect that restricting the cultivation times during manufacturing minimizes the production of suboptimal batches, thus ensuring an efficacious product.

Are surface antigens suited to verify the redifferentiation potential and culture purity of human chondrocytes in cell-based implants.

Cell expansion in vitro is a prequisite to obtain a sufficient quantity of cells for cell-based cartilage repair of articular cartilage lesions. During this process verification of redifferentiation potential of highly expanded chondrocytes is required. Furthermore, cellular impurities of chondrocyte cultures have to be excluded. For this purpose, redifferentiation of expanded human chondrocytes in passage 3 or 5 was initiated in bioresorbable polyglycolic acid-fibrin (PGA-fibrin) scaffolds and selected potential markers were analysed during the process of cell expansion and redifferentiation. Chondrocyte expansion was accompanied by a decrease of collagen type II and COMP and an increase of collagen type I expression indicating cell dedifferentiation. Redifferentiation of chondrocytes in PGA-fibrin scaffolds was accompanied by an increase of collagen II/I ratio. Flow cytometric analyses revealed that in contrast to CD44 and CD49e, CD63 and CD166 showed significant changes in the number of positive cells during redifferentiation. CD14 and CD45 are not expressed by chondrocytes and are therefore possible candidates to detect specifically monocytes or haematopoetic cells in chondrocyte cultures. Characterization of surface antigen expression revealed two promising candidates (CD63 and CD166) to describe the process of redifferentiation, while CD14 and CD45 are suitable markers to exclude impurities by monocytes or haematopoetic cells.

Augmentation and repair tissue formation of the nucleus pulposus after partial nucleotomy in a rabbit model.

Disc degeneration alters disc height and mechanics of the spinal column and is associated with lower back pain. In preclinical studies gel-like materials or resorbable polymer-based implants are frequently used to rebuild the nucleus pulposus, aiming at tissue regeneration and restoration of tissue function. To compare the outcome of tissue repair, freeze-dried resorbable polyglycolic acid-hyaluronan (PGA/HA) implants without any bioactive components or bioactivated fibrin (fibrin-serum) was used in a degenerated disc disease model in New Zealand white rabbits. Animals with partial nucleotomy only served as controls. The T2-weighted/fat suppression sequence signal intensity in the nuclear region of operated discs as assessed by magnet resonance imaging was reduced in operated compared to healthy discs, indicating loss of water and did not change from week 1 to month 6 after surgery. Quantification of histological and immunohistochemical staining indicated that the implantation of PGA/HA leads to significantly more repair tissue compared to nucleotomy only. Type II collagen content of the repair tissue formed after PGA/HA or fibrin-serum treatment is significantly increased compared to controls with nucleotomy only. The data indicate that intervertebral disc augmentation after nucleotomy has a positive effect on repair tissue formation and type II collagen deposition as shown in the rabbit model.

Regulation of osteoarthritis-associated key mediators by TNFα and IL-10: effects of IL-10 overexpression in human synovial fibroblasts and a synovial cell line.

Synovial fibroblasts (SF) contribute to the pathogenesis of osteoarthritis (OA), but the effects of intra-articular cytokines on SF are not completely understood. The aim of this study was to characterize the interplay between tumor necrosis factor (TNF)α and the anti-inflammatory interleukin (IL)-10. Non-immortalized human SF and SF of the human cell line K4IM were stimulated with recombinant TNFα, IL-10, or TNFα + IL-10 (10 ng/ml each) for 24 h or transduced with an adenoviral vector overexpressing human IL-10 (hIL-10) and subsequently treated with 10 ng/ml TNFα for 24 h. Effects on the gene expression and protein synthesis of IL-6, IL-10, matrix metalloproteinases (MMP)-1, -3, type I collagen, ß1-integrin, and CD44 were investigated via real-time detection polymerase chain reaction, immunofluorescence labeling, flow cytometry, and Western blotting. IL-10 release by transduced SF was confirmed with enzyme-linked immunosorbent assay. Both cell populations were activated by TNFα and by TNFα + IL-10, increasing their gene expression and protein synthesis of IL-6, IL-10, MMP-1, and MMP-3 and altering the synthesis of type I collagen, ß1-integrin, and CD44. hIL-10 overexpression greatly elevated the gene expression and protein synthesis of IL-10. However, transduction did not significantly affect the gene expression of IL-6, MMP-1, and MMP-3 in SF. The increased expression of pro-inflammatory and catabolic mediators in TNFα-activated SF indicates their role in OA pathogenesis, suggesting they are a potential therapeutic target. Although the vigorousness of the responses of non-immortalized SF and K4IM clearly differ, the K4IM cell line seems to be a suitable model for non-immortalized human SF.

Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage.

OBJECTIVE: Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. METHOD: Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. RESULTS: The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. CONCLUSION: These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP.

Effect of degeneration on gene expression of chondrogenic and inflammatory marker genes of intervertebral disc cells: a preliminary study.

AIM: New techniques for biological repair in the treatment of degenerative disc disease (DDD) have been developed recently. The question arises whether it is possible to find a predictive marker to identify a patient population which could benefit from this new treatment option. Standard magnetic resonance imaging (MRI) fails to differentiate between pathologic painful and asymptomatic aging discs. Neurological symptoms contribute to identifying the pathological level. In this preliminary translational research study we analysed the gene expression of structure proteins and inflammatory mediators as well as histological features of lumbar intervertebral discs in symptomatic patients with various signs of degeneration in the MRI. METHODS: Specimens of intervertebral disc tissue were obtained from 20 patients undergoing lumbar nucleotomy. Preoperatively, a group selection based on four pre-defined MRI-criteria was performed: Group 1 (mild signs of degeneration), group 2 (moderate), group 3 (moderate-severe), group 4 (severe). RESULTS: An increase of the expression of structural proteins and inflammatory markers could be observed in MRI-groups 2 and 3. Gene expression of collagen type I and II and aggrecan went along with levels of cyclooxygenase-2 (COX-2) and (fibroblast growth factor-2) FGF-2 expression. Histological examination showed signs of granulation tissue in only 35% of cases, but no differences between the groups. CONCLUSION: Our findings implicate that the gene expression of structural proteins might correlate with the appearance of inflammatory mediators in symptomatic patients with moderate disc changes in the MRI in this preliminary clinical subset. The assessment of cell activity and protein expression in a larger number of patients could be next step to support and supplement the present data.

Single-stage cartilage repair in the knee with microfracture covered with a resorbable polymer-based matrix and autologous bone marrow concentrate.

BACKGROUND: Different single-stage surgical approaches are currently under evaluation to repair focal cartilage lesions. This study aims to analyze the clinical and histological results after treatment of focal condylar articular lesions of the knee with microfracture and subsequent covering with a resorbable polyglycolic acid/hyaluronan (PGA -HA) matrix augmented with autologous bone marrow concentrate (BMC). METHODS: Nine patients with focal lesions of the condylar articular cartilage were consecutively treated with arthroscopic PGA -HA-covered microfracture and bone marrow concentrate (PGA -HA-CMBMC). Patients were retrospectively assessed using standardized assessment tools and magnetic resonance imaging (MRI). Five patients consented to undergo second look arthroscopy and 2 consented biopsy harvest. RESULTS: All the patients but one showed improvement in clinical scoring from the pre-operative situation to the latest follow-up (average 22±2months). The mean IKDC subjective score, Lysholm score, VAS and the median Tegner score significantly increased from baseline to the latest follow-up. Cartilage macroscopic assessment at 12months revealed that one repair appeared normal, three almost normal and one appeared abnormal. Histological analysis proofed hyaline-like cartilage repair tissue formation in one case. MRI at 8 to 12months follow-up showed complete defect filling. CONCLUSIONS: The first clinical experience with single-stage treatment of focal cartilage defects of the knee with microfracture and covering with the PGA -HA matrix augmented with autologous BMC (PGA -HA-CMBMC) suggests that it is safe, it improves knee function and has the potential to regenerate hyaline-like cartilage. LEVEL OF EVIDENCE: IV, case series.

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.

[The bovine cartilage punch model: a tool for the in vitro analysis of biomaterials and cartilage regeneration]. / Das bovine Knorpelstanzenmodell : Ein Werkzeug zur In-vitro-Analyse von Biomaterialien und Knorpelregeneration.

BACKGROUND: The limited regeneration capacity of hyaline articular cartilage requires detailed studies concerning the tissue integration of cartilage transplants with meaningful but time and/or resource-consuming and in part ethically problematic animal models or, alternatively, with in vitro test systems for implant materials. MATERIAL AND METHODS: The present study describes a regeneration model with bovine cartilage rings (outer Ø 6 mm, central defect Ø 2 mm) for insertion, cultivation and biomechanical or histological testing of cartilage replacement materials (HE and safranin O staining). In this study, resorbable polymers composed of polyglycolic acid (PGA) were analyzed. RESULTS: Biomechanical testing showed a continuous decrease of the push-out force for the PGA inserts from the cartilage rings, probably due to the resorbability of the material. Histologically, clear immigration of cells into cell-free PGA was observed even after 4 weeks of culture, but in particular after 10 weeks. In addition, storage of proteoglycans was interpreted as an initial sign of the formation of new matrix. CONCLUSION: Thus, the new regeneration model is in principle suitable for the testing of biomaterials, but shows limitations in assessing the "lateral bonding" of resorbable materials.

Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-ε-caprolactone scaffolds.

The objective of this study was to develop a scaffold for mesenchymal stromal cell (MSC) recruitment, proliferation, and chondrogenic differentiation. The concept behind the design is to mimic the cartilage matrix and contain stimulatory agents that make continuous supply of inductive factors redundant. Nanofibrous (N: ~400 nm) and microfibrous (M: ~10 µm) poly-ε-caprolactone (PCL) scaffolds were combined with 1% high-molecular-weight sodium hyaluronate (NHA/MHA), 1% hyaluronan (HA) and 200 ng transforming growth factor-beta 1 (TGF-ß1; NTGF/MTGF), or 0.1% bovine serum albumin (N/M). Scaffolds were seeded with MSCs from bone marrow and cultured without growth factors in vitro. Cultures with chondrogenic medium supplemented with TGF-ß1 served as controls. Proliferation, migration, and release of TGF-ß1 were investigated. Cell differentiation was evaluated by polymerase chain reaction (PCR) and real-time PCR. NTGF and MTGF exhibited primarily an initial release of TGF-ß1. None of the factors released by the scaffolds recruited MSCs. The expression of aggrecan was dependent on the scaffold ultrastructure with nanofibers promoting increasing and microfibers decreasing expression levels. Composites containing HA demonstrated elevated seeding efficiency and lower type I collagen expression. Expression of type II collagen was dependent on continuous or late supply of TGF-ß1, which was not provided by our scaffold design. The initial release of TGF-ß1 induced an expression of type I collagen and osteogenic marker genes. In conclusion, nanofibrous PCL scaffolds with or without augmentation are suitable for chondrogenic initiation of MSCs. Initial release of HA is sufficient in terms of directing the implanted MSCs toward a chondrogenic end, whereas a late release of TGF-ß1 is preferred to foster type II and avoid type I collagen expression.

Expanded human meniscus-derived cells in 3-D polymer-hyaluronan scaffolds for meniscus repair.

Treatment options for lesions of the avascular region of the meniscus using regenerative medicine approaches based on resorbable scaffolds are rare. Recent approaches using scaffold-based techniques for tissue regeneration known from cartilage repair may be a promising treatment option for meniscal tears. The aim of the study was the investigation of meniscus matrix formation of in vitro expanded human meniscus-derived cells in a three-dimensional (3-D) bioresorbable polymer graft for meniscal repair approaches. Cultivation of the human meniscus cells was performed in a resorbable scaffold material made of polyglycolic acid (PGA) and hyaluronic acid, stabilized with fibrin glue. Cell viability and distribution of human meniscus cells in PGA-hyaluronan scaffolds were evaluated by fluorescein diacetate and propidium iodide staining. Verification of typical meniscal extracellular matrix molecules like type I and type III collagen was performed histologically, immunohistochemically and by gene expression analysis. In results, 3-D scaffold-based meniscus cultures showed high cell viability over an observational period of 21 days in PGA-hyaluronan scaffolds. On the protein level, type I collagen and proteoglycans were evident. Gene expression analysis confirmed the re-expression of meniscus-specific markers in PGA-hyaluronan scaffolds. This study demonstrated that in vitro expanded human meniscus cells allow for formation of meniscal matrix components when cultured in 3-D PGA-hyaluronan scaffolds stabilized with fibrin. These results encourage scaffold-based approaches for the treatment of meniscal lesions.

Repair of a post-traumatic cartilage defect with a cell-free polymer-based cartilage implant: a follow-up at two years by MRI and histological review.

Microfracture is frequently used as the first line of treatment for the repair of traumatic cartilage defects. We present the clinical and histological results 18 months to two-years after treatment in a 26-year-old male with a post-traumatic chondral defect of the medial femoral condyle managed by microfracture covered with chondrotissue, a cell-free cartilage implant made of a resorbable polyglycolic acid felt and hyaluronic acid.

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.

Microencapsulation and chondrogenic differentiation of human mesenchymal progenitor cells from subchondral bone marrow in Ca-alginate for cell injection.

The application of stem cells is a promising therapeutic approach for cartilage regeneration. For cell therapies, a biocompatible injectable carrier, which improves retention and cell distribution and enables cell differentiation, is a prerequisite. In this study, Ca-alginate microcapsules containing human subchondral cortico-spongious progenitor cells were prepared and the chondrogenic differentiation potential was verified by real-time reverse transcription-polymerase chain reaction analysis of typical chondrogenic marker genes. The results confirmed that these cells were able to differentiate along the chondrogenic lineage when encapsulated in Ca-alginate microcapsules with a mean diameter of 600-700microm and stimulated with TGF-beta3. Chondrogenic marker genes type II collagen, aggrecan and cartilage oligomeric matrix protein were induced together with type I collagen, whereas adipogenic and osteogenic marker genes showed no induction over 14 days. After 28 days, proteoglycans and type II collagen were evident histochemically and immunohistochemically. Mechanical stability as well as permeability of Ca-alginate capsules were analysed over the course of cultivation and found to be qualified for stable cell immobilization and sufficient exchange of solutes. Therefore, from the cell biology point of view, Ca-alginate, an established hydrogel scaffold material is suited for regenerative therapies of cartilage defects based on the injection of progenitor cells.

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.

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.

Modulatory effects of inflammation and therapy on GDF-5 expression in rheumatoid arthritis synovium.

OBJECTIVE: Growth differentiation factor-5 (GDF-5), a member of the transforming growth factor (TGF)-beta family, is involved in joint development during embryogenesis and has the potential to regenerate cartilage in adult animals. As progression of chronic joint diseases is influenced by cytokines of the synovial tissue, we examined the expression and effects of GDF-5 in this tissue. METHODS: Microarray experiments were investigated for differential expression of GDF-5 in synovial tissues, synovial fibroblasts, and peripheral blood cells. GDF-5 expression was validated by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry, double immunofluorescence, and in situ hybridization in synovial tissue of normal donors (ND) and patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Effects of inflammation and therapy were investigated in RA and OA fibroblasts after stimulation with interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, methotrexate (MTX), and prednisolone. The influence of GDF-5 on macrophages was studied by chemotaxis assay. RESULTS: Microarray analysis and immunostaining revealed expression predominantly in synovial fibroblasts. Compared to patients without immunomodulating drugs, expression of GDF-5 was decreased significantly in patients receiving glucocorticoids and/or disease-modifying antirheumatic drugs (DMARDs) (p = 0.007), but did not differ between the total group of ND, OA, and RA. Stimulation with prednisolone and TNFalpha reduced GDF-5 expression in OA and RA fibroblasts, whereas MTX and IL-1beta revealed minor or no relevant change. GDF-5 also reduced cell migration of macrophages (p<0.001). CONCLUSION: GDF-5 is expressed in synovial fibroblasts and may counteract macrophage infiltration. Its modulation by inflammation and therapy suggests that glucocorticoids play a conflicting role by suppressing not only inflammation but also putative mechanisms of repair.

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.

Gene expression in endoprosthesis loosening: chitinase activity for early diagnosis?

The aim of the study was to identify markers for the early diagnosis of endoprosthesis loosening, for the differentiation between wear particle-induced and septic loosening and to gather new insights into the pathogenesis of endoprosthesis loosening. Gene expression profiles were generated from five periprosthetic membranes of wear particle-induced and five of infectious (septic) type using Affymetrix HG U133A oligonucleotide microarrays. The results of selected differentially expressed genes were validated by RT-PCR (n = 30). The enzyme activity and the genotype of chitinase-1 were assessed in serum samples from 313 consecutive patients hospitalized for endoprosthesis loosening (n = 54) or for other reasons, serving as control subjects (n = 259). Eight hundred twenty-four genes were differentially expressed with a fold change greater than 2 (data sets on http://www.ncbi.nlm.nih.gov/geo/ GSE 7103). Among these were chitinase 1, CD52, calpain 3, apolipoprotein, CD18, lysyl oxidase, cathepsin D, E-cadherin, VE-cadherin, nidogen, angiopoietin 1, and thrombospondin 2. Their differential expression levels were validated by RT-PCR. The chitinase activity was significantly higher in the blood from patients with wear particle-induced prosthesis loosening (p = 0.001). However, chitinase activity as a marker for early diagnosis has a specificity of 83% and a sensitivity of 52%, due to a high variability both in the disease and in the control group.

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.