Collagen 1 gel may improve the regenerative capacity of minced adult and preosteoarthritic cartilage.

PURPOSE: Minced cartilage implantation (MCI) is an evolving technique for the treatment of osteochondral lesions. It was hypothesised that mincing of cartilage may affect chondrocyte viability and phenotype and that embedding in collagen 1 gel results in an improved outcome. The objective of this study was to evaluate the impact of cartilage mincing and whether collagen 1 gel mediates beneficial effects on the chondrocyte phenotype and viability. METHODS: Human cartilage samples from 11 patients undergoing total knee arthroplasty were collected and minced according to the MCI protocol. Minced cartilage was cultured for 1 week with and without embedding in collagen 1 gel and was compared with unminced cartilage flakes as control. Quantitative reverse transcription-PCR and immunohistochemical staining for the chondrocyte marker genes SOX9, COL2, ACAN, COL10 and MMP13 were used to examine the chondrocyte phenotype. Cell death was assessed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. RESULTS: Increased chondrocyte cell death of cultured cartilage after mincing was observed. Chondrocytes from minced cartilage exhibited significantly decreased expression and protein levels of homeostatic and hypertrophic chondrocyte markers. Embedding in collagen 1 gel showed no positive effect on viability. However, remarkable is the increased expression of ACAN and the preserved protein level of SOX9 in the collagen 1-embedded minced cartilage. CONCLUSIONS: This study shows that the mincing of cartilage leads to increased chondrocyte death and decreased expression of chondrocyte phenotypic marker genes after 7 days. The use of collagen 1 gel may improve the stability of the phenotype, which needs to be further elucidated. LEVEL OF EVIDENCE: Level III (therapeutic).

In vivo corrosion on retrieved hip endoprostheses and in vitro effects of corrosion products on bone mineralization.

In vivo corrosion of modular endoprostheses remains a great concern, as the release of heavy metal ions can impair the implant's service life and the wellbeing of the patient. The detailed corrosion mechanisms that occur in vivo are so far not completely understood. In this context, the effects of implant released cobalt (Co) and chromium (Cr) ions on osteoblast mineralization and gene expression have not been investigated extensively. This comprehensive study aimed at furthering the understanding of in vivo implant corrosion from the clinical signs via prosthesis retrievals and histology of the synovial membranes down to the molecular processes instigated by corrosion products and its effects on bone mineralization. A detailed in vivo failure analysis was performed investigating 22 retrieved hip endoprostheses from different manufacturers and taper material combinations. The aim was to find a correlation of taper damage and especially corrosion to susceptible biomedical alloys and its effect on periprosthetic tissue as well as the clinical implant performance with regard to revision diagnosis and presence of radiolucent lines (RLL). A second part investigated the effects of Co and Cr ions on the in vitro mineralization process of osteoblasts. Cell cultures were exposed to relevant concentrations of CoCl2 and CrCl3 (0 µM, 100 µM, 200 µM) with and without addition of phosphate. Mineralization behavior was analyzed with Alizarin Red assay and Von Kossa staining of calcium depots, alkaline phosphatase activity of osteoblasts and gene expression was analyzed with real time quantitative PCR. The retrieval study provides evidence of in vivo fretting and crevice corrosion on all metallic tapers combined with either ceramic or metal femoral heads. Within the modular taper junctions, selective dissolution of the α phase occurred in wrought TiAl6V4 alloys, and etching of the fine-grained wrought CoCr28Mo6 alloy implants was observed in formed crevices. In addition, significant amounts of wear particles and corrosion products were detected in retrieved synovial membranes. An increased risk for the occurrence of a RLL in the proximal zones was determined for patients with a corroded mixed metal taper. Whereas Co ions have hardly any effects on mineralization, Cr ions cause a significant concentration dependent decrease in mineralization rate of osteoblasts. However, this effect is alleviated by addition of a phosphate source. Our data reveal that Cr ions depleted dissolved phosphates by forming an insoluble complex (CrPO4), which inhibits the phosphate dependent mineralization process. No significant effect of the heavy metal ions on osteoblast activity by means of alkaline phosphate activity as well as on gene expression is determined. This study broadens the understanding of in vivo corrosion of metallic modular implants and its clinically relevant effects on mineralization. Based on these findings, in vivo corrosion of CoCr28Mo6 endoprostheses should be limited to avoid inhibitory effects of Cr3+ on bone mineralization which can contribute to premature implant failure.

Microglial CD74 Expression Is Regulated by TGFß Signaling.

Microglia play important roles during physiological and pathological situations in the CNS. Several reports have described the expression of Cd74 in disease-associated and aged microglia. Here, we demonstrated that TGFß1 controled the expression of Cd74 in microglia in vitro and in vivo. Using BV2 cells, primary microglia cultures as well as Cx3cr1CreERT2:R26-YFP:Tgfbr2fl/fl in combination with qPCR, flow cytometry, and immunohistochemistry, we were able to provide evidence that TGFß1 inhibited LPS-induced upregulation of Cd74 in microglia. Interestingly, TGFß1 alone was able to mediate downregulation of CD74 in vitro. Moreover, silencing of TGFß signaling in vivo resulted in marked upregulation of CD74, further underlining the importance of microglial TGFß signaling during regulation of microglia activation. Taken together, our data indicated that CD74 is a marker for activated microglia and further demonstrated that microglial TGFß signaling is important for regulation of Cd74 expression during microglia activation.

Characterization of the Leucocyte Immunoglobulin-like Receptor B4 (Lilrb4) Expression in Microglia.

As resident innate immune cells of the CNS, microglia play important essential roles during physiological and pathological situations. Recent reports have described the expression of Lilrb4 in disease-associated and aged microglia. Here, we characterized the expression of Lilrb4 in microglia in vitro and in vivo in comparison with bone marrow-derived monocytes and peritoneal macrophages in mice. Using BV2 cells, primary microglia cultures as well as ex vivo isolated microglia and myeloid cells in combination with qPCR and flow cytometry, we were able to provide a comprehensive characterization of Lilrb4 expression in distinct mouse myeloid cells. Whereas microglia in vivo display low expression of Lilrb4, primary microglia cultures present high levels of surface LILRB4. Among the analyzed peripheral myeloid cells, peritoneal macrophages showed the highest expression levels of Lilrb4. Moreover, LPS treatment and inhibition of microglial TGFß signaling resulted in significant increases of LILRB4 cell surface levels. Taken together, our data indicate that LILRB4 is a reliable surface marker for activated microglia and further demonstrate that microglial TGFß signaling is involved in the regulation of Lilrb4 expression during LPS-induced microglia activation.