Meniscal samples were processed for histology, immunohistochemistry and in situ hybridization, for assessment of cell density, cells actively dividing as well as apoptotic cells, distribution of COMP and estimate the proteoglycan content.
Meniscus injury is one of the causes of secondary osteoarthritis (OA). Cartilage oligomeric matrix protein (COMP) is a major component of the extracellular matrix of the musculoskeletal system. This study was undertaken to evaluate the changes occurring in meniscus from the knees of anterior cruciate ligament (ACL) transected rabbits during the early stages of OA development, especially regarding COMP changes. Ten skeletally mature white New Zealand male rabbits underwent ACL transaction of the right knee joint. Left knee joints were used as controls. Animals were sacrificed at 4 and 12 weeks post-surgery. Meniscal tissues were processed for histology and immunohistochemistry. The number of cells and positive cells were counted per high-power field (HPF). Anti-COMP antiserum was obtained according to Hauser et al. with minor modifications. Monoclonal Ki67 antibody was used to find out cells undergoing active division. TUNEL reaction was used for the study of apoptosis. Alcian blue staining was used to study glycosaminoglycans. At 4 weeks post-ACL section 2/5 of the medial menisci presented with incomplete vertical posterior tears, while all lateral menisci were no altered. At 12 weeks post-ACL section 5/5 of the medial menisci and 2/5 of lateral menisci presented tears. At 4 weeks postsurgery menisci showed: a weak increase of cells with a significant increase of cells undergoing active division; an increase in the number of apoptotic cells; glycosaminoglycans staining was increased and COMP staining was weakly increased. At 12 weeks postsurgery cells per HPF reverted to normal number; the number of cells undergoing active division decrease below normal; whereas the number of apoptotic cells was still elevated; glycosaminoglycans staining was more elevated than at 4 weeks postsurgery and COMP staining of extracellular matrix remain elevated. Areas of large and abundant cell clusters were seen post-ACL around menisci tears. We concluded that after ACL transaction, extracellular matrix changes and altered cell distribution occur early in the meniscus. Cellular division as well as apoptosis occur early too. Elevated concentrations of COMP after ACL transection might indicate meniscus changes early in osteoarthritis process.
Type 1 diabetes mellitus (DM) is associated with a decreased bone formation. Osteoblastic expression of parathyroid hormone-related protein(PTHrP) -an important modulator of osteoblast differentiation- decreases in age-related osteopenia. We here examined the putative role of PTHrP on the decreased osteoblastic function in DM. We performed marrow ablation in the tibiae of diabetic mice after streptozotocin injection (glycemia >
300mg/dl). Some mice were treated with PTHrP(1–36) (100 ng/g/every other day, s.c.) or vehicle for 2 weeks. Both tibiae were then removed for histological evaluation or total RNA isolation. In vitro, MC3T3-E1 cells were grown in differentiation medium (a-MEM), with or without high glucose(HG) (25 mM) (or mannitol, as osmotic control), supplemented (or not) with PTHrP(1–36) (100 nM). In some experiments, anti-PTHrP N-terminal antibody C13 (1:100) or PTHrP(7–34) (1 μM) were added to normal-glucose medium. RANKL secretion was measured in the cell-conditioned medium by ELISA. Gene expression was analyzed by real-time PCR. DM induced a 10–15% weight loss and a decrease (20–40%;p<
0.05) in the gene expression of the following osteoblastic factors in the regenerating tibia for 6 days: PTHrP, the PTH/PTHrP type1 receptor (PTH1R), osteocalcin, VEGF and its receptors 1 and 2; and in the OPG/RANKL ratio, related to an increased PPAR-γ mRNA expression. Compared to control mice, the regenerating tibia of DM mice showed a 5-fold increase in adipocyte number, and a decreased osteoblast number and osteoid surface. In MC3T3-E1 cells, HG decreased (20–40%) the OPG/RANKL ratio and the gene expression of both PTHrP/PTH1R and VEGF systems. PTHrP(1–36) reversed these HG-related effects in vivo and in vitro. Similar inhibitory effects were induced by a neutralizing PTHrP antibody or the antagonist PTHrP(7–34) in these cells in normal glucose. In conclusion, a deficit in PTHrP production by osteoblasts seems to be at least in part responsible for the DM-related decreased bone formation in mice.
SBA-15 is a siliceous mesoporous ordered material with hexagonal arrangement of 9-nm tubular pores connected by micropores, high pore volume and abundance of silanol groups. This functionalised material could thus tailor the release kinetics of specific biomolecules to the clinical needs. Non-functionalized SBA-15 and its C8- or C3-alkyl-derivatives were coated with parathyroid hormone–related protein (PTHrP)(107–111) to assess their relative effects on osteoblastic cell growth and function. SBA-15 was functionalized with either octyl or propyl trimethoxysilane (C8 or C3 precursor, respectively) in ACN for 24h and then were coated (or not) by dipping in 10 nM PTHrP (107–111) solution for 24 h at 4°C. After air drying, biomaterials were transferred to culture dishes. MC3T3-E1 cells were cultured in differentiation medium with SBA-15, C3-SBA-15 and C8-SBA-15, loaded or not with the peptide. Cell viability and proliferation were evaluated by trypan blue exclusion and a proliferation kit (Promega), respectively. Alkaline phosphatase (ALP) activity and collagen secretion were determined by colorimetric methods. Gene expression was analyzed by real-time PCR. Mineralization was assessed by alizarin red staining. PTHrP(107–111)-coated SBA-15 increased cell proliferation (50%), cell viability (20%), and ALP activity (15%) over control values within 2–4 days. At day 2, collagen secretion increased (20%), and also the gene expression of ALP, PTHrP, and VEGF, which normalized at day 8, in these cells. An increase (by 30–40%) in all of these parameters was induced by peptide-coated C3-SBA-15 at day 4. Similar stimulatory effects were also observed with PTHrP(107–111)-coated C8-SBA-15 but only at day 8. At day 10, collagen secretion slightly increased (10–15%), and also mineralization (30–40%) with both functionalized materials coated with the PTHrP peptide. In conclusion, PTHrP(107–111)-coated SBA-15 stimulates osteoblastic function in vitro; an effect delayed by C3- or C8-functionalization. These data further support the clinical impact of this bioceramic as functionalized implants in vivo.
