LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa ß (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Transmission Electron Microscopy of Bone.

Electron microscopic analysis of mineralized tissues like bone and dentin is essential for understanding of cell-cell/cell-matrix interactions, and the three-dimensional organization of these tissues. This chapter describes a few methods to process mineralized tissues obtained from different sources for ultrastructural analysis by transmission electron microscopy.

Adaptation of the dermal collagen structure of human skin and scar tissue in response to stretch: an experimental study.

Surgeons are often faced with large defects that are difficult to close. Stretching adjacent skin can facilitate wound closure. In clinical practice, intraoperative stretching is performed in a cyclical or continuous fashion. However, exact mechanisms of tissue adaptation to stretch remain unclear. Therefore, we investigated collagen and elastin orientation and morphology of stretched and nonstretched healthy skin and scars. Tissue samples were stretched, fixed in stretched-out position, and processed for histology. Objective methods were used to quantify the collagen orientation index (COI), bundle thickness, and bundle spacing. Also sections were analyzed for elastin orientation and quantity. Significantly more parallel aligned collagen bundles were found after cyclical (COI = 0.57) and continuous stretch (COI = 0.57) compared with nonstretched skin (COI = 0.40). Similarly, more parallel aligned elastin was found after stretch. Also, significantly thicker collagen bundles and more bundle spacing were found after stretch. For stretched scars, significantly more parallel aligned collagen was found (COI = 0.61) compared with nonstretched scars (COI = 0.49). In conclusion, both elastin and collagen realign in a parallel fashion in response to stretch. For healthy skin, thicker bundles and more space between the bundles were found. Rapid changes in extension, alignment, and collagen morphology appear to be the underlying mechanisms of adaptation to stretching.

Transmission electron microscopy of bone.

This chapter describes procedures to process mineralized tissues obtained from different sources for transmission electron microscopy (TEM). Methods for fixation, resin embedding, staining of semi-thin sections and ultrathin sections are presented. In addition, attention will be paid to processing of cultured bone explants for TEM analysis.

Plasminogen activators are involved in the degradation of bone by osteoclasts.

Osteoclastic bone degradation depends on the activity of several proteolytic enzymes, in particular to those belonging to the classes of cysteine proteinases and matrix metalloproteinases (MMPs). Yet, several findings suggest that the two types of plasminogen activators (PA), the tissue- and urokinase-type PA (tPA and uPA, respectively) are also involved in this process. To investigate the involvement of these enzymes in osteoclast-mediated bone matrix digestion, we analyzed bone explants of mice that were deficient for both tPA and uPA and compared them to wild type mice. The number of osteoclasts as well as their ultrastructural appearance was similar for both genotypes. Next, calvarial and metatarsal bone explants were cultured for 6 or 24 h in the presence of selective inhibitors of cysteine proteinases or MMPs and the effect on osteoclast-mediated bone matrix degradation was assessed. Inhibition of the activity of cysteine proteinases in explants of control mice resulted in massive areas of non-digested demineralized bone matrix adjacent to the ruffled border of osteoclasts, an effect already maximal after 6 h. However, at that time point these demineralized areas were not observed in bone explants from uPA/tPA deficient mice. After prolonged culturing (24 h), a comparable amount of demineralized bone matrix adjacent to actively resorbing osteoclasts was observed in the two genotypes, suggesting that degradation was delayed in uPA/tPA deficient bones. The activity of cysteine proteinases as assessed in bone extracts, proved to be higher in extracts from uPA/tPA(-/-) bones. Immunolocalization of the integrin alpha(v)beta(3) of in vitro generated osteoclasts demonstrated a more diffuse labeling of osteoclasts derived from uPA/tPA(-/-) mice. Taken together, our data indicate that the PAs play a hitherto unrecognized role in osteoclast-mediated bone digestion. The present findings suggest that the PAs are involved in the initial steps of bone degradation, probably by a proper integrin-dependent attachment to bone.

Structure of the periodontium in cathepsin C-deficient mice.

Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency leads to changes in the structure of the periodontal tissues as a result of which its barrier function to pathogens is compromised. We studied the structure of the periodontium in 9-month-old cathepsin C-deficient mice (cathepsin C(-/-)) and compared this with age-matched wild-type mice. Our observations showed that the overall structure of the gingiva, periodontal ligament, alveolar process, and cementum layer are normal in cathepsin C(-/-) mice, with one exception, namely that epithelial rests of Malassez in the periodontal ligament of the cathepsin C(-/-) mice are slightly enlarged. In both experimental and control animals, we noted cyst formation in rests of Malassez. No signs of periodontal infection were observed. It is concluded that cathepsin C deficiency does not lead to major changes in the structure of the periodontium.

Functional changes in articular cartilage after meniscal allograft transplantation: a quantitative histochemical evaluation in rabbits.

PURPOSE: To evaluate quantitatively functional changes in articular cartilage after immediate and delayed meniscus transplantation in rabbits. TYPE OF STUDY: Experimental study. METHODS: Thirty rabbits were divided into 5 groups: groups A and C were subjected to meniscectomy only, groups B and D underwent meniscal transplantation immediately after meniscectomy, and group E had delayed transplantation 6 weeks after meniscectomy. Six nonoperated knees served as controls. Functional changes in articular cartilage were examined at 6 weeks (groups A, B) and 1 year (groups C, D, E, controls) after surgery by measuring proteoglycan content of the extracellular matrix as a measure of its quality and lactate dehydrogenase (LDH) activity in chondrocytes as a measure of their vitality. RESULTS: At 6-week and 1-year follow-up, no significant differences were found between the immediate transplant group and postmeniscectomy group. The delayed transplant group showed a significantly decreased proteoglycan content compared with the postmeniscectomy group. No significant differences in cellular LDH activity were found between the immediate transplant group and postmeniscectomy group at 6 weeks and 1 year. However, the delayed transplant group showed a significant decrease in LDH activity compared with the postmeniscectomy group. CONCLUSIONS: Immediate meniscal transplantation in rabbits did not significantly reduce degenerative changes of articular cartilage in comparison with meniscectomy on a short-term and long-term basis, whereas delayed transplantation led to more degenerative changes than meniscectomy. CLINICAL RELEVANCE: Before meniscus transplantation can be considered as an alternative to meniscectomy in clinical practice, it has to be determined whether this procedure has any protective effect on articular cartilage on the long term.

Histologic changes in articular cartilage after medial meniscus replacement in rabbits.

PURPOSE: Meniscal allografts show capsular ingrowth, but it remains to be established whether meniscal transplantation can prevent long-term degeneration of articular cartilage. This study examined whether immediate or delayed transplantation of the medial meniscus can protect the knee from degenerative changes. TYPE OF STUDY: Experimental study. METHODS: Thirty-five rabbits were divided into 5 groups. Three rabbits developed infective arthritis and were excluded from the study. Group A (6 animals) and group C (6 animals) had meniscectomy only; group B (7 animals) and group D (6 animals) underwent meniscal transplantation immediately after meniscectomy; group E (7 animals) had delayed transplantation 6 weeks after meniscectomy. Six nonoperated knees served as controls. Histologic changes of the articular cartilage were examined 6 weeks (groups A, B) and 1 year (groups C, D, E, controls) after surgery. RESULTS: All operated groups showed more histologic changes than the control group (P < .00001 for both the medial tibial plateau and medial femoral condyle in all groups). At 6 weeks follow-up, no differences were found between the postmeniscectomy group and the transplanted group. At 1 year, immediately transplanted knees showed less degenerative changes than meniscectomized knees (P < .0001 for medial tibial plateau and P < .005 for medial femoral condyle). Delayed transplantation resulted in more degenerative changes than both meniscectomy only and immediate transplantation (for both comparisons, P < .00001 for both medial tibial plateau and medial femoral condyle). CONCLUSIONS: Immediate meniscal transplantation in rabbits has a protecting effect on articular cartilage for the long-term whereas delayed transplantation leads to even more degenerative changes than meniscectomy only. CLINICAL RELEVANCE: If these results are applicable to humans, they would support immediate, not delayed, transplantation after removal of a meniscus.