Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

GSK-3ß inhibition suppresses instability-induced osteolysis by a dual action on osteoblast and osteoclast differentiation.

Currently, there are no medications available to treat aseptic loosening of orthopedic implants. Using osteoprotegerin fusion protein (OPG-Fc), we previously blocked instability-induced osteoclast differentiation and peri-prosthetic osteolysis. Wnt/ß-catenin signaling, which regulates OPG secretion from osteoblasts, also modulates the bone tissue response to mechanical loading. We hypothesized that activating Wnt/ß-catenin signaling by inhibiting glycogen synthase kinase-3ß (GSK-3ß) would reduce instability-induced bone loss through regulation of both osteoblast and osteoclast differentiation. We examined effects of GSK-3ß inhibition on regulation of RANKL and OPG in a rat model of mechanical instability-induced peri-implant osteolysis. The rats were treated daily with a GSK-3ß inhibitor, AR28 (20 mg/kg bw), for up to 5 days. Bone tissue and blood serum were assessed by qRT-PCR, immunohistochemistry, and ELISA on days 3 and 5, and by micro-CT on day 5. After 3 days of treatment with AR28, mRNA levels of ß-catenin, Runx2, Osterix, Col1α1, and ALP were increased leading to higher osteoblast numbers compared to vehicle-treated animals. BMP-2 and Wnt16 mRNA levels were downregulated by mechanical instability and this was rescued by GSK-3ß inhibition. Osteoclast numbers were decreased significantly after 3 days of GSK-3ß inhibition, which correlated with enhanced OPG mRNA expression. This was accompanied by decreased serum levels of TRAP5b on days 3 and 5. Treatment with AR28 upregulated osteoblast differentiation, while osteoclastogenesis was blunted, leading to increased bone mass by day 5. These data suggest that GSK-3ß inactivation suppresses osteolysis through regulating both osteoblast and osteoclast differentiation in a rat model of instability-induced osteolysis.

Evaluation of intraosseous sampling for measurements of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, creatinine kinase, gamma-glutamyl transferase and lactate dehydrogenase.

BACKGROUND: Intraosseous (IO) access can be established faster than a venous or arterial access when there is an urgent need for rapid initiation of treatment. The access can also be used to draw marrow samples. The aim of the present study was to evaluate the potential use of IO samples for enzyme determinations using a porcine model. MATERIALS AND METHODS: Bilateral tibial intraosseous cannulae and an arterial catheter were used for blood sampling from five healthy anesthetized pigs. Samples were collected at baseline and thereafter hourly for 6 h and analyzed for alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, creatinine kinase, gamma-glutamyl transferase and lactate dehydrogenase. RESULTS: Creatinine kinase, lactate dehydrogenase and alkaline phosphatase levels decreased over time. The differences between IO and arterial sampling were limited for all studied markers. CONCLUSION: The correlation between marrow and blood analysis for liver function tests and CK is sufficiently accurate in an emergency situation.

Prenatal Exposure to Continuous Constant Light Alters Endochondral Ossification of the Tibiae of Rat Pups.

UNLABELLED: Clinical and experimental studies suggest that prenatal exposure to stress can impact the growth and development of offspring. The effect of prenatal exposure to constant light, applied as a chronic stressor, on endochondral ossification of the tibiae of 3-day-old and 15-day-old pups was histomorphometrically evaluated. Pregnant rats were divided into 2 groups: mothers chronically exposed to a 12:12-hour light/light cycle (LL) and control mothers maintained on a 12:12-hour light/dark cycle on days 10-20 of pregnancy. On postnatal days 3 and 15, the pups were weighed and euthanized. The tibiae were resected and histologically processed to obtain sections for hematoxylin and eosin staining (HE) and tartrate-resistant acid phosphatase (TRAP) histochemistry, in order to perform histomorphometric determinations. The data were statistically analyzed. A significant decrease in hypertrophic cartilage thickness was observed in the tibiae of the 3-day-old (LL: 0.134 ± 0.02 vs. CONTROLS: 0.209 ± 0.023 mm; p < 0.01) and 15-day-old (LL: 23.32 ± 3.98 vs. CONTROLS: 22.96 ± 1.93 mm; p < 0.05) prenatally stressed pups. The subchondral bone volume was significantly lower in the tibiae of the 3-day-old LL pups (38.83 ± 6.14%) than in the controls (62.83 ± 10.67%; p < 0.01). The decrease in subchondral bone volume and hypertrophic cartilage thickness shows that the normal growth process of the tibia is impaired in prenatally stressed pups.

Effect of dimethyl sulfoxide on inhibition of post-ovariectomy osteopenia in rats.

There is increasing evidence that oxidative stress, due to estrogen deficiency, leads to osteopenia. In this study, dimethyl sulfoxide (DMSO), an antioxidant solvent, was used against post-ovariectomy osteopenia (PO) in rats. Forty female rats were divided into 5 groups randomly as follows: Sham, control group; OVX, ovariectomized group; DMSO1, ovariectomized injected DMSO (0.5 ml/kg/d ip); DMSO2, ovariectomized injected DMSO (1 ml/kg/day ip) and DMSO3, ovariectomized injected DMSO (2 ml/kg/d ip). DMSO therapy started 1 week after ovariectomy and continued for 13 weeks. After 13th weeks, sera were prepared, and then L4 vertebrae and right tibial bones rinsed in fixative. Serum bone alkaline phosphatase (BALP), osteocalcin, pyridinoline, malondialdehyde (MDA) and glutathione (GSH) were measured. Trabecular volume density, trabecular and cortex thickness were estimated. Osteoclast and osteoblast numbers were counted morphometrically. The data were analyzed by ANOVA and then post hoc Tukey test at p < 0.05. The increase of pyridinoline and decrease of BALP in DMSO injected groups were inhibited compared with OVX group (p < 0.05). In DMSO injected groups, decrease of bone density, trabecular volume density, thickness of trabecular and tibial cortex were inhibited compared with OVX group (p < 0.05). MDA decreased significantly in DMSO injected groups compared with OVX group. Osteoclast number decreased in DMSO injected groups compared with OVX group (p < 0.05). Osteoblast number did not show significant change in DMSO groups compared with OVX group. In conclusion, DMSO ameliorates PO through decrease of osteoclast number, osteoclast inhibition and osteoblast activation. These effects may probably be mediated via antioxidant property of DMSO.

CD73-generated adenosine promotes osteoblast differentiation.

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.

Contributions of spinal D-amino acid oxidase to bone cancer pain.

D-Amino acid oxidase (DAAO), a FAD-dependent peroxisomal flavoenzyme that catalyzes oxidation of D-amino acids to hydrogen peroxide, is distributed in the spinal cord almost exclusively expressed within astrocytes. The present study aims to explore potential contributions of spinal DAAO to the development of bone cancer pain and morphine tolerance to analgesia. Tibia inoculation of carcinoma cells produced mechanical allodynia (but not heat hyperalgesia), in synchronous with induction of DAAO expression and DAAO enzymatic activity, as well as activation of spinal astrocytes marked by GFAP. Subcutaneous and intrathecal injection of the specific DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) blocked mechanical allodynia in a dose- and time-dependent manner in tumor-bearing rats, with maximum inhibition of 40-50 %. Multi-daily intrathecal injections of the DAAO gene silencer siRNA/DAAO also yielded anti-allodynic effects by approximately 40 % and the analgesia remained for at least 6 days. Subcutaneous injection of CBIO suppressed the production of spinal hydrogen peroxide and GFAP expression. 7-Day multiple bi-daily injections of CBIO produced anti-allodynia without inducing self-tolerance to analgesia or cross-tolerance to morphine, and concurrent injections of CBIO with morphine produced apparent additive anti-allodynia and completely prevented morphine tolerance in behaviors and spinal expression of µ-opioid receptors. Our results provide the first evidence that spinal DAAO contributes to the development of morphine tolerance to analgesia and bone cancer pain accounting for 40-50 % pain status, probably via production of hydrogen peroxide leading to activation of astrocytes. The unique characterizations of DAAO inhibitors make them a potential for the treatment of cancer pain when they are administered alone or in combination with morphine.

The role of α-zearalanol in reversing bone loss induced by ovarian hormone deficiency in rats.

To assess the ability of α-zearalanol (α-ZAL) to prevent bone loss in an ovariectomized (OVX) rat model of osteoporosis, α-ZAL was administered intragastrically to rats. After 35 days, the total body bone mineral density (BMD) was assessed in all rats. All sections were processed for immunohistochemistry and hematoxylin and eosin staining. One-way ANOVA and an LSD multiple-range test were used to determine the significant differences between groups. BMD was lower in the OVX and OVX + α-ZAL high-dose (OVX + High) groups compared to the sham-operated (Sham), OVX + 17ß-ethinylestradiol (OVX + E(2)), OVX + α-ZAL medium-dose (OVX + Medium) and OVX + α-ZAL low-dose (OVX + Low) groups (P < 0.05). Clear bone trabeculae arrangements were observed in the OVX + E(2,) OVX + Medium and OVX + Low groups. The expressions of bone morphogenetic proteins and basic fibroblast growth factor were up-regulated in the OVX + E(2), OVX + Medium and OVX + Low groups compared to the OVX and OVX + High groups (P < 0.05). The OVX + E(2), OVX + Medium and OVX + Low groups showed lower levels of bone Gla protein, bone alkaline phosphatase, tartrate-resistant acid phosphatase and tumor necrosis factor α expressions than the OVX and OVX + High groups (P < 0.05). The administration of α-ZAL to ovariectomized rats reverses bone loss and prevents osteoporosis.

The effect of Cox-2 specific inhibition on direct fracture healing in the rabbit tibia.

OBJECTIVE: The purpose of this study was to evaluate the effect of Cox-2 administration on direct (primary) fracture healing. METHODS: A transverse tibial osteotomy was created in adult male rabbits and rigidly fixed in compression using a 2.7-mm dynamic compression plate. Animals were randomized to receive either rofecoxib (12.5 mg orally per day) or placebo. Animals were killed at 4 weeks and fracture healing assessed by mechanical testing. RESULTS: There were no significant differences between the control and Cox-2 treated animals in terms of mechanical strength at 4 weeks. There was a high complication rate of peri-implant fractures during the daily medication administration. CONCLUSION: The immediate administration of a Cox-2 specific inhibitor did not impair primary (direct) bone healing at the dose administered in this rabbit tibial osteotomy model.

Colorectal cancer cells promote osteoclastogenesis and bone destruction through regulating EGF/ERK/CCL3 pathway.

Bone metastasis of colorectal cancer (CRC) cells leads to osteolysis. Aberrant activation of osteoclasts is responsible for bone resorption in tumor. In general, bone marrow-derived monocytes (BMMs) differentiate into osteoclasts, however, how CRC cells interact with BMMs and how to regulate the differentiation is elusive. We here report that CRC cells promote bone resorption in bone metastasis. Transcriptomic profiling revealed CCL3 up-regulated in MC-38 conditional medium treated BMMs. Further investigation demonstrated that CCL3 produced by BMMs facilitated cell infusion and thus promoted the osteoclastogenesis. In addition, CRC cells derived EGF stimulated the production of CCL3 in BMMs through activation of ERK/CREB pathway. Blockage of EGF or CCL3 can efficiently attenuate the osteolysis in bone metastasis of CRC.

Osteoblastic and osteolytic human osteosarcomas can be studied with a new xenograft mouse model producing spontaneous metastases.

There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.

Ablation of cathepsin k activity in the young mouse causes hypermineralization of long bone and growth plates.

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.

Effect of aluminum oxide-blasted implant surface on the bone healing around implants in rats submitted to continuous administration of selective cyclooxygenase-2 inhibitors.

PURPOSE: The continual use of selective cyclooxygenase-2 (COX-2) inhibitors may have a negative impact on bone repair around titanium implants. Because modified implant surfaces could be considered an important strategy to increase success rates in some conditions that interfere in bone healing, the aim of this study was to investigate whether an aluminum oxide (Al2O3)-blasted implant surface could reduce the negative action promoted by the continuous administration of selective COX-2 inhibitors on bone healing around implants. MATERIALS AND METHODS: Thirty Wistar rats received one titanium implant (machined or Al2O3-blasted surface) in each tibia and were randomly assigned to one of the following groups: saline (n = 14) or meloxicam (n = 16); each was administered daily for 60 days. Bone-to-implant contact (BIC), bone area (BA) within the limits of threads, and bone density (BD) in a zone lateral to the implant were examined in undecalcified sections. RESULTS: The Al2O3-blasted surface resulted in significantly increased BIC in both groups, and meloxicam significantly reduced bone healing around implants (P < .05). For the machined surface, significant differences were observed for BIC (39.48 +/- 10.18; 25.23 +/- 9.29), BA (60.62 +/- 4.09; 42.94 +/- 8.12), and BD (56.31 +/- 3.64; 49.30 +/- 3.15) in the saline and meloxicam groups, respectively. For the Al2O3-blasted surface, data analysis also demonstrated significant differences for BIC (45.92 +/- 11.34; 33.30 +/- 7.56), BA (61.04 +/- 4.39; 44.89 +/- 7.11), and BD (58.77 +/- 2.93; 50.04 +/- 3.94) for the saline and meloxicam groups, respectively. CONCLUSIONS: The Al2O3-blasted surface may increase BIC; however, it does not reverse the negative effects promoted by a selective COX-2 inhibitor on bone healing around implants.

Ferulic acid, a natural polyphenol, protects against osteoporosis by activating SIRT1 and NF-κB in neonatal rats with glucocorticoid-induced osteoporosis.

Osteoporosis is a chronic disease whose symptoms include a reduction in bone strength, osteopenia, and damage to the bone microstructure. Ferulic acids are natural polyphenols present in various fruits that suppress the fusion and apoptosis of mature osteoclasts. Rats were divided into sham, control (osteoporosis), 10 mg/kg body weight ferulic acid, 20 mg/kg body weight ferulic acid, and 30 mg/kg body weight ferulic acid treatment groups. Osteoporosis was induced in neonatal by administration of dexamethasone (glucocorticoids). Bone mineral density (BMD), osteocalcin and alkaline phosphatase (ALP) levels, bone mechanical parameters, and mRNA and protein levels of sirtuin1 (SIRT1) and nuclear factor kappa-B (NF-κB) in the osteoporotic neonatal rats were assessed. Histopathological analysis was also conducted. Treatment with 20 and 30 mg/kg body weight ferulic acid increased BMD by 25% and 141.7%, respectively, but reduced ALP and osteocalcin levels. Furthermore, treatment with 20 or 30 mg/kg body weight ferulic acid significantly reduced the pixel intensity and significantly increased the peak load and ultimate stiffness. Ferulic acid significantly increased the mRNA and protein levels of SIRT1 and reduced those of NF-κB. Finally, the histopathological analysis showed that ferulic acid increased BMD. In summary, ferulic acid exhibited protective effects against osteoporosis in neonatal rats.

Daidzein but not other phytoestrogens preserves bone architecture in ovariectomized female rats in vivo.

Ovariectomy of immature female rats, results in significant decrease of trabecular bone volume and in cortical bone thickness. Previously, we found that estradiol-17beta (E(2)) restored bone structure of ovariectomized (Ovx) female rats to values obtained in intact sham-operated female rats. E(2) also selectively stimulated creatine kinase (CK) specific activity a hormonal-genomic activity marker. In the present study, we compared the effects of E(2) and the phytoestrogens: daidzein (D), biochainin A (BA), genistein (G), carboxy-derivative of BA (cBA), and the SERM raloxifene (Ral) in Ovx, on both histological changes of bones and CK, when administered in multiple daily injections for 2.5 months. Bone from Ovx rats, showed significant disrupted architecture of the growth plate, with fewer proliferative cells and less chondroblasts. The metaphysis underneath the growth plate, contained less trabeculae but a significant increased number of adipocytes in the bone marrow. D like E(2) and Ral but not G, BA, or cBA, restored the morphology of the tibiae, similar to that of control sham-operated animals; the bony trabeculeae observed in the primary spongiosa was thicker, with almost no adipocytes in bone marrow. Ovariectomy resulted also in reduced CK, which in both epiphysis and diaphysis was stimulated by all estrogenic compounds tested. In summary, only D stimulated skeletal tissues growth and differentiation as effectively as E(2) or Ral, suggesting that under our experimental conditions, D is more effective in reversing menopausal changes than any of the other isolated phytoestrogens which cannot be considered as one entity.

Regulation of the maturation of osteoblasts and osteoclastogenesis by glutamate.

Glutamate, an important central excitatory neurotransmitter, is also secreted by osteoblasts and may be involved in the regulation of bone metabolism. Glutamate receptors for N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) are demonstrated in bone cells. Here we investigated the in vivo effects of glutamate by local injection of AMPA, NMDA, and their antagonists into tibia as well as their in vitro effects on the maturation of osteoblasts and formation of osteoclasts. AMPA receptor antagonist CNQX and NMDA receptor antagonist MK-801 significantly inhibited the maturation and mineralization of osteoblasts in high-glutamate alpha-MEM. On the other hand, AMPA and NMDA up-regulated the mineralized deposition and osteocalcin mRNA expression of primary osteoblasts cultured in glutamate-free DMEM. AMPA and NMDA induced the phosphorylation of extracellular signal-related kinases (ERK) in osteoblasts within 15 min. In addition, NMDA but not AMPA up-regulated the number of osteoclasts while MK801 antagonized this potentiating effect. To explore the action of glutamate agonists on bone formation in animal model, AMPA was locally injected into tibia and it was found that the bone volume in secondary spongiosa significantly increased and co-treatment of CNQX antagonized the enhancing effect of AMPA. These results suggest that glutamate may play a physiological role in regulating the maturation of osteoblasts and osteoclastogenesis. Activation of both AMPA and NMDA receptors regulates the maturation of osteoblasts. NMDA but not AMPA affects receptor for activation of NF-kappaB ligand (RANKL)-induced osteoclastogenesis.

Expression and localization of plasma transglutaminase factor XIIIA in bone.

Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immunohistochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immunoreactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.

Expression of MMP-13 in osteoblast cells and rat tibia after exposure to gamma rays or accelerated carbon ions.

In past research, we found that carbon ion irradiation increased bone volume in rats, and a significant amount of cartilage remained inside the carbon ion-irradiated trabeculae. The amounts of matrix metalloproteinase 13 (MMP-13) mRNA in osteoblast-like MC3T3-E1 cells tended to decrease after carbon ion irradiation. The level of MMP-13 mRNA in non-irradiated cells was stable during the experimental period, but in gamma ray-irradiated cells it tended to increase. When localization of MMP-13 in locally irradiated experimental rats was investigated, it was found in the marginal trabeculae in both non-irradiated and gamma ray-irradiated animals. MMP-13 was detected in osteoid and neogenetic bone in the trabeculae surface. The trabeculae in carbon ion-irradiated bone remained cartilaginous. Carbon ion-irradiated rats exhibited weak expression of MMP-13 around the cartilage inside the trabeculae. We conclude that carbon ion irradiation reduced expression of MMP-13, thus suppressing both chondrocyte maturation and cartilage resorption. Increases in hyperplasia of the bone trabeculae and of bone volume were caused by ongoing bone addition and calcification in the absence of sufficient cartilage resorption.

The biomechanical and histological effects of posterior cruciate ligament rupture on the medial tibial plateau.

BACKGROUND: The objective of this study was to investigate the biomechanical and histological effects of the posterior cruciate ligament (PCL) on the medial tibial plateau. METHODS: A total of 12 cadaveric human knee specimens were collected and grouped as follows: the PCL intact group (n = 12), the anterolateral bundle rupture group (n = 6), the postmedial bundle rupture group (n = 6), and the PCL rupture group (n = 12). The strain on the anterior, middle, and posterior parts of the medial tibial plateau with an axial loading force at different flexion angles was measured and analyzed, respectively. Forty-eight rabbits were chosen for animal study: surgery was performed on the one side of each rabbit randomly (experimental group), while the other side was taken as control (control group). Every 12 rabbits were culled at each of the four selected time points to collect the medial tibial plateau for morphological and histological observation. RESULTS: The PCL rupture, either partial or complete, may generate an abnormal load on all the parts of the medial tibial plateau with axial loading at all positions. Noticeable time-dependent degenerative histological changes of the medial tibial plateau were observed in the rabbit models of PCL rupture. Compared with the control group, all the PCL rupture groups exhibited a higher expression of the matrix metalloproteinase-7 (MMP-7) and the tissue inhibitors of metalloproteinase-1 (TIMP-1) at all the time points. CONCLUSIONS: Either partial or complete PCL rupture may generate an abnormal load on all the parts of the medial tibial plateau with axial loading at all the positions and may cause cartilage degeneration on the medial tibial plateau.

Proteolytic enzymes in skeletal development: histochemical methods adapted to the study of matrix lysis during the transformation of a "cartilage model" into bone.

The replacement of a "cartilage model" by definitive bone is characterized by a series of localized excavations of the cartilage which are eventually followed by bone deposition. Each excavation requires lysis of cartilage components (defined here as the breakdown of a peptide bond) and their eventual resorption (defined here as microscopical visible cartilage loss). More precisely we have proposed that the lysis is affected by proteases capable of breaking down the main proteoglycan "aggrecan" and the main fibril element, "type II collagen". Four approaches combining biochemical, immunologic and microscopic techniques have been adapted to test this hypothesis. Each is applied to the rat tibial head's "cartilage model" where proteases have been shown to be major contributors to secondary ossification center formation. The approaches have been found both effective and distinct as cartilage resorbing enzymes have not only been identified but also detected in situ before and after activation. Achieved overall is an understanding of when, where and how specified proteases contribute to tissue component lyses. While the focus resides on the in situ proteolysis of cartilage, three of the approaches could be translated without change to other tissues, whereas one may require tissue specific adjustments before use.