Expression of macrophage elastase (MMP12) in rat tail intervertebral disc and growth plate after asymmetric loading.

OBJECTIVES: The aim of this study was to examine whether asymmetric loading influences macrophage elastase (MMP12) expression in different parts of a rat tail intervertebral disc and growth plate and if MMP12 expression is correlated with the severity of the deformity. METHODS: A wedge deformity between the ninth and tenth tail vertebrae was produced with an Ilizarov-type mini external fixator in 45 female Wistar rats, matched for their age and weight. Three groups were created according to the degree of deformity (10°, 30° and 50°). A total of 30 discs and vertebrae were evaluated immunohistochemically for immunolocalisation of MMP12 expression, and 15 discs were analysed by western blot and zymography in order to detect pro- and active MMP12. RESULTS: No MMP12 expression was detected in the nucleus pulposus. Expression of MMP12 in the annulus progressively increased from group I to groups II and III, mainly at the concave side. Many growth plate chondrocytes expressed MMP12 in the control group, less in group I and rare in groups II and III. Changes in cell phenotype and reduction of cell number were observed, together with disorganisation of matrix microstructure similar to disc degeneration. ProMMP12 was detected at the area of 54 kDa and active MMP12 at 22 kDa. CONCLUSIONS: Expression of MMP12 after application of asymmetric loading in a rat tail increased in the intervertebral disc but decreased in the growth plate and correlated with the degree of the deformity and the side of the wedged disc. Cite this article: Bone Joint Res 2014;3:273-9.

Subchondral cyst development and MMP-1 expression during progression of osteoarthritis: an immunohistochemical study.

BACKGROUND: Subchondral bone cyst (SBC) formation is often identified in patients with osteoarthritis. Furthermore, several studies have shown that expression of matrix metalloproteinases (MMPs) is elevated in patients with OA. OBJECTIVES: The aim of our study is to correlate the presence of SBCs and MMP-1 expression with the osteochondral alterations during OA progression. METHODS: We studied the cartilage and subchondral bone of 15 patients who had undergone total knee or hip replacement due to primary OA. As controls, we used the femoral heads of three patients without macroscopic OA changes. We evaluated three specimens per patient. RESULTS: Specimens were divided in four groups based on the Mankin histological severity score. Using immunohistochemistry, we noted SBCs at the site of greatest disease severity. Specifically, these were present more frequently in group III (Mankin score: 6-7) and IV (Mankin: ≥ 8), compared with group I (Mankin: 1-3) and II (Mankin: 4-5). Mild OA stages (Mankin: 1-6) were characterized by degeneration and thinning of the cartilage, followed by increased osteoblast and osteoclast activity of the subjacent bone and the subsequent appearance of SBCs. Simultaneously, we observed expression of MMP-1 in groups I and II in the cartilage and III and IV in both the cartilage and the subchondral bone. Moreover, osteoblast-like cells in the lining of the SBCs showed an increased expression of MMP-1 in stages III and IV. CONCLUSION: Our study provides immunohistological evidence that SBCs accumulate in advanced OA and contain activated cells, which express MMP-1, suggesting that they may thus participate in the osteochondral changes of OA. LEVEL OF EVIDENCE: Level III; prospective comparative study.

Calcitonin effect on Achilles tendon healing. An experimental study on rabbits.

OBJECTIVE: A positive potential effect of Calcitonin (CT) on Achilles tendon healing was investigated as well as the ability of MRI to follow the tendon healing process. MATERIALS AND METHODS: A standardized tenotomy of the Achilles tendon was performed on forty-two rabbits. Twenty-one animals received daily 21 IU /kg Calcitonin intramuscularly (treatment group CT) during the experiment and the remaining received saline solution (control group P). Seven animals from each group were killed at one, two and three weeks postoperatively. All animals had serial MRI scans and tendon samples underwent biomechanical and histological testing. RESULTS: For both groups, animals of the same subgroup showed statistically significant difference in signal intensity values of MRI between the 1st and 3rd week (p<0.001) and between the 2nd and 3rd week (p<0.001). Signal intensity values of MRI didn't show any differences between animals under treatment and controls measured at 1st (p=0.23), 2nd (p=0.23) and 3rd (p=0.53) postoperative week. Tendon samples from group CT showed statistically significant difference in ultimate tensile strength compared to controls at 2 (p<0,0005) and 3 (p<0,0005) weeks post-surgery. Histology showed a positive Calcitonin effect at all tendon healing stages. CONCLUSION: It is suggested that Calcitonin enhances Achilles tendon healing process.

Cortical bone response adjacent to applied light orthodontic forces in ovariectomized rats.

Cortical bone response to experimental orthodontic forces has not been adequately described in the available literature. The aim of this study was to investigate the application of light orthodontic forces on the cortical bone adjacent to the point of their application, in normal and ovariectomized rats. At the age of eight months, twenty-four female rats were divided equally into two groups: normal (group A) and ovariectomized (group B). A 20 gr* orthodontic force was applied to the maxillary right first molars in all animals for 14 days. Histological examination of the maxillary cortical bone, at the level of the first molar of the upper jaw, was performed on both sides of all animals. It was found that light orthodontic forces applied to the upper right first molars of normal rats result in thinner cortical bone with increased osteoblastic activity, normal lamellar orientation and normal distribution of osteocytes compared to the non-affected contralateral side. In ovariectomized rats it was found that light orthodontic forces applied to the upper right first molars result in thickening of the cortex, in comparison to the respective area of the opposite side where no orthodontic forces were applied. It seems that experimental light orthodontic forces, in rats, affect cortical bone remodeling differently, according to their hormonal status, in areas adjacent to the applied forces.

The effects of the short-term administration of low therapeutic doses of anti-COX-2 agents on the healing of fractures. An experimental study in rabbits.

We have evaluated the effect of the short-term administration of low therapeutic doses of modern COX-2 inhibitors on the healing of fractures. A total of 40 adult male New Zealand rabbits were divided into five groups. A mid-diaphyseal osteotomy of the right ulna was performed and either normal saline, prednisolone, indometacin, meloxicam or rofecoxib was administered for five days. Radiological, biomechanical and histomorphometric evaluation was performed at six weeks. In the group in which the highly selective anti-COX-2 agent, rofecoxib, was used the incidence of radiologically-incomplete union was similar to that in the control group. All the biomechanical parameters were statistically significantly lower in both the prednisolone and indometacin (p = 0.01) and in the meloxicam (p = 0.04) groups compared with the control group. Only the fracture load values were found to be statistically significantly lower (p = 0.05) in the rofecoxib group. Histomorphometric parameters were adversely affected in all groups with the specimens of the rofecoxib group showing the least negative effect. Our findings indicated that the short-term administration of low therapeutic doses of a highly selective COX-2 inhibitor had a minor negative effect on bone healing.

Primary mammary osteogenic sarcoma.

A 78 year-old female patient underwent a total mastectomy with axillary lymph node dissection for a primary breast osteosarcoma. Microscopically the tumor was identical to grade II skeletal osteosarcoma. Immunohistochemically no reactivity was detected, either for the epithelial markers EMA, AE1/AE3, CK8, 18, 19, or for HER-2/neu, estrogen and progesterone receptors, as well as fluorescent IN SITU hybridization for HER-2/neu. The diagnosis of this tumor fulfills certain clinicopathological criteria. Mammary osteosarcoma is usually developed in phyllodes tumors or carcinosarcomas of the breast as a result of metaplasia of the epithelial component. This rare tumor of the breast is occasionally associated with prior radiation therapy or well documented trauma. Mammary osteosarcoma is a biologically aggressive neoplasm with a 38% five-year survival rate. Surgical resection is the most effective therapy to date. Adjuvant treatment -chemotherapy or radiotherapy- has shown no clear benefit. An extensive review of the literature is also presented.

Effect of calcitonin in early and late stages of experimentally induced osteoarthritis. A histomorphometric study.

OBJECTIVE: To investigate both prophylactic and therapeutic roles of salmon calcitonin on the articular cartilage of rabbit's knees. METHODS: Right knee instability was produced in 30 New Zealand white rabbits by sectioning the cranial cruciate ligament (CCL). Animals were separated into four groups: placebo prophylactic-stage group (n=6), killed 8 weeks post surgery, calcitonin prophylactic-stage group (n=6), treated immediately after surgery with salmon calcitonin and killed at 8 weeks, placebo therapeutic-stage group (n=9) killed at 16 weeks post surgery and calcitonin therapeutic-stage group (n=9), treated with salmon calcitonin from 8th to 16th week and killed at 16 weeks post surgery. A histomorphometric study was based on the morphological changes of the articular cartilage and subchondral bone (degeneration indexes), as well as the articular cartilage thickness, chondrocytes' arrangement and their metabolic activity (regeneration indexes). RESULTS: Calcitonin groups showed smoother articular surface, no or minimal signs of ulceration, smaller osteophytes, and less subchondral cystic formation than placebo groups. Normal distribution of chondrocytes or hypercellularity was noticed in areas of mild osteoarthritic (OA) changes in the calcitonin groups indicating regeneration activity. Periodic Acid Schiff's and Alcian blue staining were negative in the placebo groups while increased absorption in the calcitonin groups revealed high anabolic activity. CONCLUSIONS: In prophylactic stages salmon calcitonin seemed to inhibit the progression of osteoarthritis by increasing the layers of hyaline cartilage, restoring the cellular metabolism, and decreasing the volume of osteophytes. In therapeutic stages, the hormone had a healing effect by decreasing the subchondral cysts, regenerating the hyaline cartilage and restoring cellular metabolism. Both macroscopic and histological findings of this study supported the biochemical results of previous studies showing the therapeutic effect of calcitonin on osteoarthritis.

Protective effect of plant extract from Onobrychis ebenoides on ovariectomy-induced bone loss in rats.

OBJECTIVE: Certain plant extracts have been the object of recent studies due to their mild estrogenic action and their possible potential role in osteoporosis prevention and/or treatment. The present study was undertaken to investigate the possible protective effect of the aqueous solution of the plant Onobrychis ebenoides, with proven in vitro mild estrogenic action, on bone mass loss of the ovariectomized (Ovx) rat experimental model of osteoporosis. METHODS: Forty intact female mature (10-month-old) Wistar rats were separated into three groups: Ovx, Ovx plus plant extract (Ph) and sham-operated (control). Ph administration in the drinking water at a dose of 300 mg/kg body weight/day commenced immediately after Ovx. Bone mineral density (BMD) values, percentage change from the baseline measurement and histomorphometry of the tibia, as well as body and uterine weight, were examined and compared between groups. RESULTS: Comparison of BMD absolute values of the whole tibia of Ovx + Ph and Ovx animals at both 3 and 6 months post-Ovx were highly significant (p < 0.0005), showing a protective effect on treated animals. The extract did not appear to have such a beneficial effect on BMD of the proximal tibia of the treated animals compared to the Ovx animals after 3 months; however, a significant protective effect was observed at 6 months post-Ovx in treated animals compared to the Ovx (p = 0.015). When the % changes from baseline measurement of the whole tibia of Ovx + Ph and controls were compared, there was no significant difference at 3 or 6 months, demonstrating a highly protective effect; the respective comparisons of proximal tibia % changes did not display such protection. Body and uterine weight comparisons showed no significant difference between Ovx and treated rats, whereas, the level of significance for each group compared to controls was p < 0.0005. CONCLUSIONS: The Ph studied showed a highly significant protective effect on BMD of the whole tibia of Ovx rats after 3 and 6 months of treatment, compared to the non-treated animals. Its effect on the proximal tibia was less pronounced, but also statistically significant compared to non-treated rats after 6 months. The lack of significant effect on body and uterine weight is in favor of its selective estrogen receptor modulator-like activity, and merits further studies.

Pulmonary dirofilariasis mimicking lung tumor with chest wall and mediastinal invasion.

We present a case of human pulmonary dirofilariasis, diagnosed in a 55-year-old heavy smoker (male) by wedge resection of the pulmonary lesion and histologic examination. The dirofilaria lesion had the clinical and radiographic appearance of a peripheral pulmonary lesion invading the anterior chest wall and mediastinum. The reported radiographic image of the disease is different from the usually described picture of a pulmonary coin lesion.

Morphometric changes in the epiphyseal plate of the growing and young adult male rat after long-term salmon calcitonin administration.

The function of the epiphyseal plate is related to the differentiation and maturation of the chondrocytes, especially of the hypertrophic zone. Salmon calcitonin exerts a positive effect on chondrocytes of different types of cartilage, e.g., articular cartilage, osteochondral callus formation, and the epiphyseal plate. In the present study, the effect of long-term daily salmon calcitonin treatment upon epiphyseal plate function was examined in 80 male Wistar rats aged 12 weeks at the beginning of the experiment. A daily dose of 6 IU of salmon calcitonin enhanced the number of the chondrocytes of the hypertrophic zone of the upper tibial epiphyseal plate, increased the mean thickness of the epiphyseal plate, and accelerated the longitudinal growth of long bones. It was found that the peripheral growth of the epiphyseal plate was delayed after calcitonin treatment in comparison with the placebo-treated animals. The most effective period for calcitonin treatment on epiphyseal plate function seems to be the late accelerated period of growth, i.e., puberty. In conclusion, long-term salmon calcitonin treatment has a beneficial effect on longitudinal skeletal growth and this effect remains throughout the adult life of the animal. Salmon calcitonin does not enlarge the surface of the epiphyseal plate.

[Bilateral choroidal metastasis revealing disseminated cancer: a case report]. / Métastases choroïdiennes bilatérales révélatrices d'un cancer disséminé. A propos d'un cas.

We report the case of a 46-year-old woman, with no prior medical history, who developed a metamorphopsia of the right eye, followed by visual impairment. Ophthalmological examination revealed bilateral choroidal metastasis. Further investigations led to the conclusion of multifocal metastasis of bronchial adenocarcinoma.

Influence of orthotopic placement on the incorporation and mechanical strength of a loaded structural cortical graft: an experimental study in rabbits.

The biological and mechanical behavior of loaded cortical autografts were evaluated in an experimental study using 40 adult male New Zealand rabbits divided into 4 groups. A double osteotomy was performed on all animals at the mid-diaphysis of the right ulna. The bone segment (5 mm) in between the osteotomies was considered a cortical autograft and fixed with an intramedullary Kirschner wire. The graft was fixed in an orthotopic manner in groups A and C, and rotated 180 degrees in groups B and D. After the animals were sacrificed (at 2 months for groups A and B and at 4 months for groups C and D), bone mineral density, graft cross-sectional geometry, and SSI index were assessed. Mechanical testing of the grafted area was carried out using a three-point bending configuration, and the parameter fracture load was assessed. Graft union and incorporation also was studied in histologic sections. In group C (orthotopic graft placement--4 months), bone specimens showed statistically significantly higher values for fracture load, total cross-sectional area, volumetric total bone mass and density, and polar SSI (P values < or = .05) compared to the other groups. In the same group, union and incorporation of the graft was complete, the cortex showed low porosity, and the collagen fibers were mature and properly oriented. Structural cortical bone autografts placed in an orthotopic manner recognize the new mechanical environment as optimal and thus their biological and mechanical behavior are enhanced. Loaded structural cortical grafts should be placed in an orthotopic manner when used for the management of cortical bone defects.

Mechanical stimulation in the form of vibration prevents postmenopausal bone loss in ovariectomized rats.

Physical exercise is recommended for the prevention and treatment of osteoporosis. However, its exact role and effectiveness in adulthood is unclear. While vigorous exercise of long duration enhances bone density, few adult individuals comply with such training programs. The present study evaluates the influence of nonphysiological mechanical stimulation, in the form of low intensity vibration (frequency: 50 Hz, acceleration: 2 g, 30 min/day for 5 days/week), on the prevention of bone loss in an animal model of postmenopausal osteoporosis. In the ovariectomised groups of rats a statistically significant (p < 0.05) decrease of bone density (femur and tibia) was recorded at 5 weeks postovariectomy. This effect was maintained for the 12 week duration of the study. Vibration prevented early bone loss after ovariectomy. Vibrated ovariectomised rats showed statistically significantly higher (p < 0.05) BMD values compared to those of their ovariectomised controls at 5 weeks. Vibration did not influence the bone density of the SHAM-operated rats. Although vibration increased ultimate strength (fracture load of the rat femur) in the ovariectomised rats, this finding was not statistically significant. Our data indicate that this method of safe and easily applicable vibration, in the form of a vibrating platform, is effective in preventing early postovariectomy bone loss in an animal model.

Reconstruction of anterior cruciate ligament using the doubled tendon graft technique: an experimental study in rabbits.

In an attempt to assess reconstruction of the anterior cruciate ligament (ACL) by a new method, 18 New Zealand white rabbits underwent ACL replacement using the medial one-third of the patellar tendon and the semitendinosus tendon, thus partly reproducing the anatomical configuration of the ACL, with the semitendinosus tendon replacing the posterolateral bundle of the ACL and the patellar tendon replacing the anteromedial bundle. The Noulis-Trillat-Lachman test was performed before and after transection of the ACL, after reconstruction and before sacrifice. The animals were divided into four groups and were killed at 3, 6, 12 and 22 weeks after surgery. Femur-ligament-tibia complexes were evaluated postoperatively for gross morphology and histological appearance. The tendons of the first group showed compact parallel fibres with no definitive separation of their bundles and areas of disorganized collagen matrix. Tendons were surrounded by trabecular lamellar bone haphazardly arranged. The tendons of the second, third and fourth groups looked more like normal tendon. The trabecular bone surrounding the tendons formed a tunnel. The Noulis-Trillat-Lachman test result was negative before the procedure, 6.5 +/- 0.5 mm on average after transection of the ACL, 1.5 +/- 0.6 mm after the procedure, and negative again before sacrifice. The joints of the animals killed at 12 and 22 weeks showed signs of osteoarthritic lesions.

Soft tissue and cancellous bone reaction to the implantation of novel biodegradable pins and plates in rabbits.

Biodegradable implants for osteosynthesis have had insufficient strength and a fast degradation rate. We produced a polymer based lactide to increase the molecular weight and strength of the implant and delay the degradation rate. This novel fiber-reinforced material in the form of plates and pins was tested in vitro for mechanical performance and in vivo in a neutral mechanical environment to assess biocompatibility. 21 rabbits with implants in the tibia and femoral condyles were killed at 1, 6, 12 months after implantation. Only a mild foreign-body reaction was observed and the implant was encapsulated by a thin 0.8-1.6 mm layer of connective tissue. Only a few lymphocytes and giant cells were detected in the early stages, dense fibroblasts in the middle stage, and giant cells clearing material particles in the late stage. In conclusion, the novel fiber-reinforced polymer based on polylactides was found mechanically superior to former degradable implants and biocompatible, showing no late inflammatory tissue response.

Immunohistochemical expression of subunit beta HCG in breast cancer.

The ectopic production of HCG by non-trophoblastic tumors is well documented. Adenocarcinomas arising in the mammary gland have been shown to stain positively for the beta subunit of HCG using immunoperoxidase technique. In the present study we used the Monoclonal antibodies (M. abs) H6, H34 and H54 directed against the subunits of this hormone. A total of 31 breast tissue sections from formalin fixed, paraffin embedded unselected material was examined, out of which 22 concerned different types of primary breast cancer (Ca), 5 local recurrences, 3 cystic disease (CD) and one was an axillary lymph node metastasis (LNM). All cases were evaluated for cytoplasmic localization of the beta subunit HCG using the Mab H36 and among the 27 breast Ca 9 were studied with Mab H34 and 6 with Mab H54. Our results with the Mab H6 showed the following: 1) 55.5% of the Ca including the recurrences, were strongly positive (+) and 18.5% weakly positive (+/-); 2) 63.6% of the Grade III tumors were negative (-), whereas 81.2% of the Grade II were +, 18.7% +/- and none was -; 3) It seems that by increasing infiltration of the tumor size positivity is increased; 4) Lymphocytic infiltration of the tumor stroma and LNM did not appear related; 5) From the FU data of 14 cases collected up to now, the 10-year-survival seems to be inconclusive; 6) The 3 cases of CD were negative except for the apocrine cells which were positive; 7) The unique axillary LNM was positive.(ABSTRACT TRUNCATED AT 250 WORDS)