Purpose: Today, for regenerative treatment of injured or early osteoarthritic cartilage, ACI and MACI are clinically applied. Also mesenchymal stem cell based approaches have reached the clinic. Here, AMIC® combines microfracture with implantation of a collagen membrane, and Chondrotissue® microfracture with a PGA/hyaluronan scaffold. AMIC® is a passive approach; endogenous MSC flow into the membrane. Chondrotissue®, in whose development we were involved, is between passive and active; MSC flow into the scaffold is enhanced by the addition of MSC recruiting serum. Our aim is to develop an active in situ approach, wherein the implantation of scaffolds, loaded with MSC recruiting chemokines and differentiation factors, combined with microfracture, allows the use of endogenous MSC to promote OA cartilage regeneration. Methods: MSC were isolated from bone marrow of normal donor and OA hip joint cancellous bone and characterized (morphology, growth, surface marker, multilineage potential). Also their Ck receptor profile was elucidated (qPCR, immunohistochemistry). Cartilage and chondrocytes were harvested from ND and OA femoral heads, and supernatants were analyzed for their Ck profile (protein array). Then, the recruitment potential of supernatants and Ck on MSC was examined (chemotaxis assay), and Pertussis toxin, siRNA and antibodies were applied to block Ck receptors. Moreover, expression profiles of Ck stimulated ND and OA MSC were generated and compared (microarrays). Selected Ck were also encapsulated in a PLGA release system using the w/o/w emulsion and solvent evaporation technique, and in vitro and in vivo applied to chemoattract superparamagnetic iron oxide nanoparticle (SPION) labeled MSC (rat model, MRI). Finally, comparative gene expression profiling was performed for ND and OA MSC fibrinogen/PLGA transplants after chondrogenic stimulation with TGFß3. Results: ND and OA MSC have a fibroblast-like morphology, a similar doubling time, differentiate to fat, bone and cartilage, are CD73, CD105 positive, and CD45 negative. Moreover, both express most Ck receptors. Proteomics revealed similar Ck profiles of ND and OA cartilage supernatants. In contrast, ND and OA chondrocyte supernatants had different Ck spectra. Only OA supernatants were CXCL12 (SDF1) positive. In chemotaxis assays, cartilage supernatants recruited more MSC than chondrocyte supernatants, and OA cartilage supernatants more MSC than ND supernatants. Based on protein arrays, CCL25 (TECK) and was analyzed in more detail. CCL25 recruited a high number of ND and OA MSC and represents a candidate for in situ approaches. MSC treatment with Pertussis toxin could block supernatant induced chemotaxis, whereas treatment with CCR9-siRNA or CCR9-antibodies (the CCL25 receptor) had only a small effect. Subsequent expression profiling of CCL25 induced ND and OA MSC gave us a deep insight into the Ck dependent mobilization of these cells. For example, 22 genes were differentially expressed in both ND and OA MSC. Most of them are involved in pathways related to homing (PDE4B), cytoskeletal and membrane reorganization (IGFBP1) and movement (CXCL8, PTGS2). In an ongoing study, CCL25 loaded PLGA particles are tested in a rat model. Here, for in vivo MRI monitoring of MSC migration towards CK releasing particles, SPION labeled MSC are used. Finally, the regenerative potential of OA and ND MSC was studied in fibrinogen/PLGA transplants. Chondrogenesis resulted in fibro- and joint cartilage. Moreover, ND and OA expression profiles showed a similar expression of marker genes known in context of OA (COL10A1, MMP1 and -3). Conclusions: Here we have shown that end-stage OA MSC behave like ND MSC, and that we have established the key knowledge and tools for an active, MSC-based in situ therapy of injured or OA joint cartilage. In future, addition of anti-inflammatory drugs may allow the use of such approaches during inflammation and thus, also for other arthritic diseases like RA.

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.

Developmental hypothyroidism causes brain retardation due to impairment of neuronal migration and oligodendroglial myelination. Brominated flame retardants (BFRs), potential environmental pollutants to cause bioaccumulation, usually exert low or no toxicity in conventional toxicity studies, but some of them are known to induce mild hypothyroidism. To search for target genes responsible for possible brain retardation due to developmental exposure to BFRs, we performed a global gene expression profiling specific to the cerebral white matter of rat pups exposed to decebromodiphenyl ether (DBDE) during development. Dams were given DBDE at 10, 100, or 1000 ppm in diet during the period from gestation day 10 to postnatal day 21 (weaning). As a positive control for hypothyroidism, methimazole (MMI) at 200 ppm or propylthiouracil (PTU) at 3 or 12 ppm was given via drinking water. At weaning, bilateral cerebral white matter and corpus callosum (CC) were selectively microdissected from male pups and subjected to microarray analysis. As a result, 10% of genes showing altered expression by DBDE were identical to those induced by anti-thyroid agents (MMI and PTU) including glial cell differentiation, axon guidance, myelination, or cellular migration. Also, DBDE-alone induced expression alteration of genes related to similar functions as with anti-thyroid agents. At 11 weeks of age, offspring were subjected to brain morphometry regarding white matter components. As a result, as well as anti-thyroid agents, DBDE reduced the CC area and the density of CNPase-positive oligodendrocytes suggestive of reduced white matter development. Results thus suggest that glial development-related genes obtained here with DBDE may be linked to or independent of developmental hypothyroidism that is shown here for the first time with DBDE as white matter hypoplasia.