Optimization of histologic grading schemes in spontaneous and surgically-induced murine models of osteoarthritis.

OBJECTIVE: To compare the Osteoarthritis Research Society International (OARSI) and Articular Cartilage Structure (ACS) grading schemes applied to multiple and single sections, along with additional histologic measures, in two mouse models of Osteoarthritis (OA). METHODS: Six coronal histologic stifle joint sections were collected from 40 C57BL/6J mice, including aged mice with spontaneous OA (approximately 18 months of age; n = 15) and young (12-week-old) mice that either underwent destabilization of the medial meniscus (DMM) surgery (n = 15) or sham surgery (n = 10). Sections were evaluated with the standard OARSI (0-6) scheme, a modified OARSI scheme, the ACS (0-12) scheme, histomorphometry of cartilage and bone, and scoring of osteophytes (0-3) and synovial hyperplasia (0-3). Principal components analysis (PCA) was used to determine the features explaining the greatest variability among the sections. RESULTS: The grading schemes performed similarly when applied to a single mid-coronal section or six total coronal sections per joint. OARSI grading produced similar results when applied to hematoxylin and eosin or toluidine blue-stained sections. Aged mice had higher severity scores in the LTP than DMM mice (mid-coronal OARSI grade aged = 2.3 and DMM = 1.1, p = 0.0006; ACS grade aged = 4.1 and DMM = 1.6, p = 0.0024). PCA resulted in retention of four factors that accounted for 78.4% of the total variance. Factor 1 (36.4%) included the OARSI grade, ACS grade, Toluidine blue grade, articular cartilage area and thickness and the osteophyte grade. CONCLUSIONS: Grading of a single mid-coronal section using either the OARSI or ACS schemes combined with osteophyte and histomorphometric measures can consistently define OA severity in mice.

Juvenile osteochondritis dissecans of the knee is a result of failure of the blood supply to growth cartilage and osteochondrosis.

OBJECTIVE: Juvenile osteochondritis dissecans (JOCD) is similar to osteochondrosis dissecans (OCD) in animals, which is the result of failure of the cartilage canal blood supply, ischemic chondronecrosis and delayed ossification, or osteochondrosis. The aim of the current study was to determine if osteochondrosis lesions occur at predilection sites for JOCD in children. METHOD: Computed tomographic (CT) scans of 23 knees (13 right, 10 left) from 13 children (9 male, 4 female; 1 month to 11 years old) were evaluated for lesions consisting of focal, sharply demarcated, uniformly hypodense defects in the ossification front. Histological validation was performed in 11 lesions from eight femurs. RESULTS: Thirty-two lesions consisting of focal, uniformly hypodense defects in the ossification front were identified in the CT scans of 14 human femurs (7 left, 7 right; male, 7-11 years old). Defects corresponded to areas of ischemic chondronecrosis in sections from all 11 histologically validated lesions. Intra-cartilaginous secondary responses comprising proliferation of adjacent chondrocytes and vessels were detected in six and two lesions, whereas intra-osseous responses including accumulation of chondroclasts and formation of granulation tissue occurred in 10 and six lesions, respectively. One CT cyst-like lesion contained both a pseudocyst and a true cyst in histological sections. CONCLUSION: Changes identical to osteochondrosis in animals were detected at predilection sites for JOCD in children, and confirmed to represent failure of the cartilage canal blood supply and ischemic chondronecrosis in histological sections.

Genetic ancestry in relation to the metabolic response to a US versus traditional Mexican diet: a randomized crossover feeding trial among women of Mexican descent.

BACKGROUND/OBJECTIVES: Certain populations with a large proportion of indigenous American (IA) genetic ancestry may be evolutionarily adapted to traditional diets high in legumes and complex carbohydrates, and may have a detrimental metabolic response to US diets high in refined carbohydrates and added sugars. We tested whether IA ancestry modified the metabolic response to a US versus traditional Mexican diet in a controlled dietary intervention. SUBJECTS/METHODS: First and second generation Mexican immigrant women (n=53) completed a randomized crossover feeding trial testing the effects of a US versus traditional Mexican diet. The metabolic response to the diets was measured by fasting serum concentrations of glucose, insulin, insulin-like growth factor-1 (IGF-1), IGF-binding protein-3 (IGFBP-3), adiponectin, C-reactive protein, interleukin-6 and computed homeostasis model assessment for insulin resistance (HOMAIR). Blood collected at baseline was used for genotyping, and estimation of African, European and IA ancestries with the use of 214 ancestry informative markers. RESULTS: The genetic ancestral background was 56% IA, 38% European and 6% African. Women in the highest IA ancestry tertile (>62%) were shorter in height, less educated and less acculturated to the US lifestyle, and tended to have higher waist-to-hip ratio compared with women in the middle and lowest IA ancestry tertiles, respectively. Compared with the US diet, the traditional Mexican diet tended to reduce glucose, insulin, IGF-1, IGFBP-3 and HOMAIR among women in the middle IA ancestry group (IA ancestry ⩽45-62%), whereas having no effect on biomarkers related to inflammation. CONCLUSIONS: We observed modest interactions between IA ancestry and the metabolic response to a US versus traditional Mexican diet among Mexican immigrant women.

Osteochondrosis Can Lead to Formation of Pseudocysts and True Cysts in the Subchondral Bone of Horses.

Osteochondrosis arises as a result of focal failure of the blood supply to growth cartilage. The current aim was to examine the pathogenesis of pseudocysts and true cysts in subchondral bone following failure of the blood supply to the articular-epiphyseal cartilage complex in horses. Cases were recruited based on identification of lesions (n = 17) that were considered likely to progress to or to represent pseudocysts or true cysts in epiphyseal bone in histological sections and included 10 horses ranging in age from 48 days to 5 years old. Cases comprised 3 warmbloods, 3 Standardbreds, 1 Quarter horse and 1 Arabian with spontaneous lesions and 2 Fjord ponies with experimentally induced lesions. Seven lesions consisted of areas of ischemic chondronecrosis and were compatible with pseudocysts. Two lesions were located at intermediate depth in epiphyseal growth cartilage, 2 lesions were located in the ossification front, 2 lesions were located in epiphyseal bone and 1 lesion was located in the metaphyseal growth plate (physis). Ten lesions contained dilated blood vessels and were compatible with true cysts. In 2 lesions the dilated blood vessels were located within the lumina of failed cartilage canals. In the 8 remaining lesions areas of ischemic chondronecrosis were associated with granulation tissue in the subjacent bone and dilated vessels were located within this granulation tissue. Failure of the blood supply and ischemic chondronecrosis can lead to formation of pseudocysts or dilatation of blood vessels and formation of true cysts in the epiphyseal bone of horses.

Replication of genetic loci for ages at menarche and menopause in the multi-ethnic Population Architecture using Genomics and Epidemiology (PAGE) study.

STUDY QUESTION: Do genetic associations identified in genome-wide association studies (GWAS) of age at menarche (AM) and age at natural menopause (ANM) replicate in women of diverse race/ancestry from the Population Architecture using Genomics and Epidemiology (PAGE) Study? SUMMARY ANSWER: We replicated GWAS reproductive trait single nucleotide polymorphisms (SNPs) in our European descent population and found that many SNPs were also associated with AM and ANM in populations of diverse ancestry. WHAT IS KNOWN ALREADY: Menarche and menopause mark the reproductive lifespan in women and are important risk factors for chronic diseases including obesity, cardiovascular disease and cancer. Both events are believed to be influenced by environmental and genetic factors, and vary in populations differing by genetic ancestry and geography. Most genetic variants associated with these traits have been identified in GWAS of European-descent populations. STUDY DESIGN, SIZE, DURATION: A total of 42 251 women of diverse ancestry from PAGE were included in cross-sectional analyses of AM and ANM. MATERIALS, SETTING, METHODS: SNPs previously associated with ANM (n = 5 SNPs) and AM (n = 3 SNPs) in GWAS were genotyped in American Indians, African Americans, Asians, European Americans, Hispanics and Native Hawaiians. To test SNP associations with ANM or AM, we used linear regression models stratified by race/ethnicity and PAGE sub-study. Results were then combined in race-specific fixed effect meta-analyses for each outcome. For replication and generalization analyses, significance was defined at P < 0.01 for ANM analyses and P < 0.017 for AM analyses. MAIN RESULTS AND THE ROLE OF CHANCE: We replicated findings for AM SNPs in the LIN28B locus and an intergenic region on 9q31 in European Americans. The LIN28B SNPs (rs314277 and rs314280) were also significantly associated with AM in Asians, but not in other race/ethnicity groups. Linkage disequilibrium (LD) patterns at this locus varied widely among the ancestral groups. With the exception of an intergenic SNP at 13q34, all ANM SNPs replicated in European Americans. Three were significantly associated with ANM in other race/ethnicity populations: rs2153157 (6p24.2/SYCP2L), rs365132 (5q35/UIMC1) and rs16991615 (20p12.3/MCM8). While rs1172822 (19q13/BRSK1) was not significant in the populations of non-European descent, effect sizes showed similar trends. LIMITATIONS, REASONS FOR CAUTION: Lack of association for the GWAS SNPs in the non-European American groups may be due to differences in locus LD patterns between these groups and the European-descent populations included in the GWAS discovery studies; and in some cases, lower power may also contribute to non-significant findings. WIDER IMPLICATIONS OF THE FINDINGS: The discovery of genetic variants associated with the reproductive traits provides an important opportunity to elucidate the biological mechanisms involved with normal variation and disorders of menarche and menopause. In this study we replicated most, but not all reported SNPs in European descent populations and examined the epidemiologic architecture of these early reported variants, describing their generalizability and effect size across differing ancestral populations. Such data will be increasingly important for prioritizing GWAS SNPs for follow-up in fine-mapping and resequencing studies, as well as in translational research.

Non-terminal animal model of post-traumatic osteoarthritis induced by acute joint injury.

OBJECTIVE: Develop a non-terminal animal model of acute joint injury that demonstrates clinical and morphological evidence of early post-traumatic osteoarthritis (PTOA). METHODS: An osteochondral (OC) fragment was created arthroscopically in one metacarpophalangeal (MCP) joint of 11 horses and the contralateral joint was sham operated. Eleven additional horses served as unoperated controls. Every 2 weeks, force plate analysis, flexion response, joint circumference, and synovial effusion scores were recorded. At weeks 0 and 16, radiographs (all horses) and arthroscopic videos (OC injured and sham joints) were graded. At week 16, synovium and cartilage biopsies were taken arthroscopically from OC injured and sham joints for histologic evaluation and the OC fragment was removed. RESULTS: OC fragments were successfully created and horses were free of clinical lameness after fragment removal. Forelimb gait asymmetry was observed at week 2 (P = 0.0012), while joint circumference (P < 0.0001) and effusion scores (P < 0.0001) were increased in injured limbs compared to baseline from weeks 2 to 16. Positive flexion response of injured limbs was noted at multiple time points. Capsular enthesophytes were seen radiographically in injured limbs. Articular cartilage damage was demonstrated arthroscopically as mild wear-lines and histologically as superficial zone chondrocyte death accompanied by mild proliferation. Synovial hyperemia and fibrosis were present at the site of OC injury. CONCLUSION: Acute OC injury to the MCP joint resulted in clinical, imaging, and histologic changes in cartilage and synovium characteristic of early PTOA. This model will be useful for defining biomarkers of early osteoarthritis and for monitoring response to therapy and surgery.

Early lesions of articular osteochondrosis in the distal femur of foals.

Failure of the cartilage canal blood supply to epiphyseal growth cartilage has been implicated in the pathogenesis of articular osteochondrosis in horses and other animal species. In a previous study of the developmental pattern of the blood supply in the tarsus of foals, early lesions of osteochondrosis were consistently found in regions where the cartilage canal vessels traversed the chondro-osseous junction. The developmental pattern of blood vessels has also been described in the distal femoral epiphysis; however, the group of foals examined in that study did not have lesions of osteochondrosis in this location. Therefore, the relationship between the occurrence of early lesions of osteochondrosis and the developmental pattern of the blood supply to epiphyseal growth cartilage in this site in foals has not been examined. Distal femora were collected from 30 fetuses and foals (up to 11 months old) submitted for postmortem examination. Sections from the lateral trochlear ridge and medial femoral condyle of both hind limbs were examined histologically. Sixteen cartilage lesions were found in 7 of the 30 fetuses and foals. All lesions contained evidence of cartilage canal necrosis and ischemic chondronecrosis. The lesions were located in regions where cartilage canal vessels traversed the chondro-osseous junction, as previously observed in the tarsus. The location and morphology of lesions indicated that a subclinical stage of ischemic chondronecrosis existed that preceded and predisposed to the development of osteochondrosis dissecans and subchondral bone cysts.

The novel carbohydrate tumor antigen C2-O-sLe x is upregulated in canine gastric carcinomas.

Sialyl Lewis x-modified core 2 branched O-glycans (C2-O-sLe(x)) on human leukocytes mediate much higher-affinity adhesion to selectins on activated vascular endothelium than does sialyl Lewis x on other structures. In some canine and human carcinomas, high expression of sLe(x)-decorated carbohydrates has been associated with metastasis and, in humans, a poor prognosis, but detection in canine gastric carcinomas is unreported. The authors hypothesized that these carbohydrates are highly expressed in more malignant types of canine gastric carcinomas, they promote metastasis, and they are associated with a poorer prognosis for dogs. The objectives were to determine the presence and importance of C2-O-sLe(x) expression in canine gastric carcinomas. Routine histological sections of 16 canine gastric carcinomas were categorized on the basis of 3 classification schemes: World Health Organization, Lauren, and Goseki. Serial sections were stained with antibodies directed against C2-O-sLe(x) (CHO-131 monoclonal antibody), cytokeratin (Lu-5 monoclonal antibody), and stains to detect neutral and acid mucins (periodic acid-Schiff and alcian blue). Whereas normal gastric mucosal epithelial cells were negative for C2-O-sLe(x), 56% of the tumors examined were positive for C2-O-sLe(x). Importantly, the majority of more poorly differentiated tumor types had more numerous and larger intensely stained areas of C2-O-sLe(x) expression compared with moderate to well-differentiated tumor types. Signet ring-type carcinomas had markedly higher distribution and intensity of periodic acid-Schiff and alcian blue staining than did tubular carcinomas. These findings suggest that C2-O-sLe(x) is a tumor-associated antigen that may play a role in the invasiveness and metastatic potential of certain types of canine gastric carcinomas.

Cbfa1-dependent expression of an interferon-inducible p204 protein is required for chondrocyte differentiation.

We reported earlier that an interferon-inducible p204 protein serves as a cofactor of Cbfa1 and promotes osteogenesis. Here we establish that p204 demonstrates prominent expression in growth plate chondrocytes. It is differentially expressed in the course of bone morphogenetic protein-2-triggered chondrocyte differentiation of pluripotent C3H10T1/2 cells. This expression is probably due to the activation of p204 gene by Cbfa1 and repression by Sox5 transcription factor. Cbfa1 and Sox5 bind to the 5'-flanking regulatory region of p204 gene at their consensus binding elements. Overexpression of p204 accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type X Collagen and matrix metalloproteinase-13; however, knockdown of p204 via an siRNA approach abolishes hypertrophic chondrocyte differentiation. p204 acts as a cofactor of Cbfa1 in chondrocyte hypertrophy: (1) overexpression of p204 augments, whereas suppression of p204 decreases, the Cbfa1-dependent transactivation of a Collagen X-specific reporter gene; (2) p204 enhances Cbfa1-mediated chondrocyte hypertrophy; and (3) p204 associates with Cbfa1 in chondrocyte differentiation. In addition, altered expression of p204 in chondrocyte hypertrophy was accompanied by altered levels of Indian hedgehog (IHH) and parathyroid hormone/parathyroid hormone-related peptide receptor-1 (PTHR1). Collectively, p204 is a novel regulator of chondrocyte differentiation by (1) acting as a coactivator of Cbfa1 and (2) affecting IHH/PTPrP signaling.

Uterine infarctions in cynomolgus monkeys (Macaca fascicularis).

Uterine infarctions have not been reported in domestic animals, and there are few reports in the human medical literature. In a retrospective study, uterine infarctions were identified in 9 of 323 (2.8%) female cynomolgus monkeys (Macaca fascicularis) necropsied over a 13-year period. The infarctions were grossly visible, after fixation, on the serosal surface of the uterus in 2 monkeys; the remainder were first recognized in histologic sections. Histologically, the lesions consisted of well-demarcated regions of endometrial and myometrial necrosis and of hemorrhage. All affected monkeys had histologic evidence of a previous pregnancy, which included enlarged myometrial vessels with an expanded perivascular matrix. In all monkeys with uterine infarctions, there was clinical evidence of severe systemic illness, which included trauma, diarrhea, hypovolemia, or septicemia. The major pathologic findings in affected monkeys included cutaneous or skeletal muscle necrosis (n = 5), enterocolitis (n = 4), pulmonary edema or diffuse alveolar damage (n = 3), and intestinal amyloidosis (n = 1). Histopathologic evidence of intravascular fibrin thrombi in multiple organs of 5 monkeys was consistent with a diagnosis of disseminated intravascular coagulopathy (DIC). Based on these findings, it appears that uterine infarction is an uncommon finding in cynomolgus monkeys and may occur secondary to a severe systemic illness, predisposing to DIC.

Characterization of bone mineral composition in the proximal tibia of cynomolgus monkeys: effect of ovariectomy and nandrolone decanoate treatment.

Life postmenopausal women, ovariectomized cynomolgus monkeys (Macaca fascicularis) experience accelerated loss of bone mass. Treatment of ovariectomized monkeys with nandrolone decanoate results in an increase in bone mass to levels comparable to those of intact animals. The changes in bone composition that occur with these treatments, however, are less well characterized. In the present study, we used synchrotron Fourier-transform infrared microspectroscopy (FT-IRM) and curve-fitting methods to monitor specific changes at cortical, subchondral, and trabecular bone regions in the proximal tibia. Four groups were studied: (1) sham-operated (sham); (2) ovariectomized and treated with placebo for 2 years (ovx); (3) ovx + nandrolone decanoate for 2 years (NAN); and (4) ovx + nandrolone decanoate beginning 1 year after ovx (dNAN). The results demonstrate that ovariectomy and nandrolone treatment did not affect the degree of mineralization as defined by the phosphate/protein ratio, but acid phosphate content (HPO(4)(2-)) in cortical and subchondral bone was increased by ovariectomy, suggesting this bone to be less mature due to increased remodeling that occurs after ovariectomy. In the subchondral and cortical bone regions, ovariectomized monkeys showed a lower total carbonate content (CO(3)(2-)/matrix ratio) than sham controls, specifically due to the decrease in labile carbonate content. In the trabecular region, no change of carbonate content was observed. Treatment with nandrolone decanoate was found to restore the loss in carbonate, where the resulting mineral had a larger quantity of type B carbonate. Finally, we correlated carbonate content with dual-energy X-ray absorptiometry measurements, and found a positive correlation between bone mineral density and type A carbonate in bone, which is stoichiometrically related to the amount of calcium in bone. Therefore, the results presented herein identify significant differences in bone chemistry after ovariectomy and nandrolone treatment, which may help explain previous findings that, although nandrolone decanoate treatment increased bone mass, it could not reverse the decrease in bone strength due to ovariectomy.

Tissue distribution and measurement of cartilage oligomeric matrix protein in patients with magnetic resonance imaging-detected bone bruises after acute anterior cruciate ligament tears.

Histologic and immunostaining analyses were performed on articular cartilage/subchondral bone biopsy specimens overlying MRI-detected bone bruises in 12 patients with anterior cruciate ligament (ACL) tears. Staining with toluidine blue for proteoglycan revealed loss of staining from the superficial portion of the articular cartilage. Immunostaining for cartilage oligomeric matrix protein (COMP) showed an increased staining in the superficial matrix of the articular cartilage. Using polyclonal antisera against COMP, the authors performed a competitive enzyme-linked immunosorbent assay (ELISA) on the synovial fluid from the injured and uninjured knees. There was an approximately 10-fold higher synovial fluid COMP levels in injured knees. The COMP levels were greater in those patients who had synovial fluid samples harvested closer to the date of initial injury. Western blot analysis of the synovial fluid showed an increased presence of COMP degradation fragments from injured knees. These results are indicative of a significant injury to the articular cartilage, and may represent preclinical posttraumatic osteoarthritic lesions.

Colonic adenocarcinoma in rhesus macaques.

Colon cancer is the second most common cause of cancer mortality in human beings, but relatively few cases have been described in macaques. The present report documents two fatal cases of colon cancer in aged rhesus macaques. Case 1 was a 20-year-old female in which extensive invasion of the caecum by a scirrhous adenocarcinoma led to perforation and a severe fibrinopurulent peritonitis. Case 2 was a 32-year-old male with a stricture at the ileocaecal junction, also caused by a scirrhous adenocarcinoma, which had metastasized to a regional lymph node. Both neoplasms showed aggressive local involvement of the proximal large bowel, which appears to be a predilection site in rhesus monkeys. Descriptions of spontaneous cases of colon cancer in non-human primates may lead to the development of models for certain aspects of the disease in man.

Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts.

Cartilage oligomeric matrix protein has been implicated as an important component of endochondral ossification because of its direct effects on chondrocytes. The importance of this protein for skeletal development and growth has been recently illustrated by the identification of mutations in cartilage oligomeric protein genes in two types of inherited chondrodysplasias and osteoarthritic phenotypes: multiple epiphyseal dysplasia and pseudoachondroplasia. In the present study, we report the presence of cartilage oligomeric protein in embryonic and adult osteoblasts. A foot from a 21-week-old human fetus, subchondral bone obtained from knee replacement surgery in an adult patient, and a limb from a 19-day-postcoital mouse embryo were analyzed with immunostaining and in situ hybridization. In the human fetal foot, cartilage oligomeric protein was localized to osteoblasts of the bone collar and at the newly formed bone at the growth plate and bone diaphyses. Immunostaining was performed on the adult subchondral bone and showed positive intracellular staining for cartilage oligomeric protein of the osteoblasts lining the trabecular bone. There was no staining of the osteocytes. Immunostaining of the mouse limb showed the most intense staining for cartilage oligomeric protein in the hypertrophic chondrocytes and in the surrounding osteoblast cells of the developing bone. Cartilage oligomeric protein mRNA and protein were detected in an osteoblast cell line (MG-63), and cartilage oligomeric protein mRNA was detected from human cancellous bone RNA. These results suggest that the altered structure of cartilage oligomeric protein by the mutations seen in pseudoachondroplasia and multiple epiphyseal dysplasia may have direct effects on osteoblasts, contributing to the pathogenesis of these genetic disorders.

Functional estrogen receptors in adult articular cartilage: estrogen replacement therapy increases chondrocyte synthesis of proteoglycans and insulin-like growth factor binding protein 2.

OBJECTIVE: Epidemiologic studies suggest a protective effect of estrogen replacement therapy (ERT) against the development of knee and hip osteoarthritis, but a potential mechanism for this effect is not known. The present study was done to determine if functional estrogen receptors (ERs) are present in adult articular cartilage and to determine if ERT in vivo affects the production of insulin-like growth factor binding proteins (IGFBPs). METHODS: Reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry were used to measure messenger RNA (mRNA) and protein for ERs in adult monkey articular cartilage. Cultured chondrocytes transfected with a reporter construct containing the estrogen response element (ERE/luciferase) were stimulated with estrogen in vitro to determine functional activity of the ERs. IGFBP production was measured by ligand and immunoblotting of conditioned media of cells cultured from control and estrogen-treated surgically menopausal monkeys. Proteoglycan (PG) synthesis was estimated by measurement of 35SO4 incorporation. RESULTS: ERa and ERbeta mRNA were present in adult monkey articular cartilage, and ER protein was demonstrated by immunoblotting and immunohistochemistry. Estrogen treatment in vitro of cells transfected with the ERE/luciferase construct resulted in a 2.87-fold increase (P = 0.0163) in reporter production over that of untreated cells. Compared with untreated controls, IGFBP-2 production was significantly increased (P < 0.008) in conditioned media of chondrocytes cultured from monkeys that had received ERT in vivo. Increased IGFBP-2 in these cultures was associated with a 1.41-fold increase (P = 0.02) in the level of sulfate incorporation. CONCLUSION: Transcriptionally functional ER are present in adult articular cartilage, and ERT increases the production of IGFBP-2 and the synthesis of PGs by chondrocytes from surgically menopausal monkeys. These results indicate that estrogen can have a direct effect on adult articular cartilage.

Examination of bone chemical composition in osteoporosis using fluorescence-assisted synchrotron infrared microspectroscopy.

Although it is clear that osteoporosis is associated with a reduction in bone mass and a fragile skeleton, it is not understood whether the chemical composition of osteoporotic bone is different from normal bone. In this study, cynomolgus monkeys (Macaca fascicularis) were administered fluorochrome labels at one and two years after ovariectomy (Ovx) or Sham ovariectomy (intact), that were taken up into newly remodeled bone. Using fluorescence-assisted synchrotron infrared microspectroscopy, the chemical composition of bone from intact versus Ovx monkeys has been compared. Results from overall composition distributions (labeled + non-labeled bone) reveal similar carbonate/protein and phosphate/protein ratios, but increased acid phosphate content and different collagen structure in the Ovx animals. Analysis of the fluorochrome-labeled bone indicates similar degrees of mineralization in bone remodeled after one year, but decreased mineralization in Ovx bone remodeled two years after surgery. Thus, bone from monkeys with osteoporosis can be characterized as having abnormal collagen structure and reduced rates of mineralization. Coupled with factors such as trabecular architecture and bone shape and size, these ultrastructural factors may play a contributing role in the increased bone fragility in osteoporosis.

Estrogen replacement therapy modulation of the insulin-like growth factor system in monkey knee joints.

OBJECTIVE: Epidemiologic studies have suggested that estrogen replacement therapy may lower the risk of osteoarthritis in women, but the mechanism of this effect is unknown. Since estrogen acts in other tissues in part through regulation of the insulin-like growth factor (IGF) system as well as cytokines including interleukin-6 (IL-6), we determined whether estrogen replacement regulates the levels of these factors in synovial fluid (SF). METHODS: Levels of IGF-1, IGF-2, IGF binding proteins (IGFBP) 1-3, and IL-6 were measured in SF samples obtained from 67 female adult cynomolgus monkeys that had been ovariectomized and treated for 30 months in 1 of 3 groups. Group 1 (n = 24) had no estrogen replacement (control), group 2 (n = 22) received estrogen (Premarin) at the human equivalent of 0.625 mg/day, and group 3 (n = 21) received estrogen at the same dose as group 2, plus progesterone (Provera) at the equivalent of 2.5 mg/day. RESULTS: Compared with controls, estrogen-treated monkeys had 2-fold higher SF levels of IGF-1 (P < 0.001), 1.7-fold higher IGF-2 (P < 0.006), 5.9-fold higher IGFBP-1 (P < 0.02), and 2.5-fold higher IGFBP-3 (P < 0.001). Estrogen plus progesterone-treated monkeys had SF levels of IGF-1, IGF-2, IGFBP-1, and IGFBP-3 that were intermediate between the levels in the control and estrogen groups, except that the level of IGFBP-3 was significantly greater than that in the control group (P < 0.001). SF levels of IGFBP-2 and IL-6 did not differ by treatment group. Treatment group did not affect the serum levels of IGF-1 and IL-6, but IGF-2 and IGFBP-3 were increased by 1.6- and 1.8-fold, respectively, in the estrogen group (P < 0.001). There was no correlation between changes in serum and SF levels of IGF components, except for a weak correlation for IGFBP-3 levels from control (r = 0.464, P = 0.04) and estrogen-treated (r = 0.577, P = 0.008) animals. CONCLUSION: This study demonstrates a significant effect of estrogen replacement on IGF system components in synovial fluid, of which at least some are distinct from any systemic changes observed. The results indicate a potential stimulatory effect of estrogen on joint tissues in vivo.

Articular cartilage changes seen with magnetic resonance imaging-detected bone bruises associated with acute anterior cruciate ligament rupture.

Occult osteochondral lesions (bone bruises) have been documented on magnetic resonance images in more than 80% of patients sustaining acute anterior cruciate ligament ruptures. Despite the high prevalence of these lesions, little is known about the histologic changes in the adjacent articular cartilage. Ten patients with acute anterior cruciate ligament ruptures who had a preoperatively documented (by magnetic resonance imaging) geographic bone bruise at the sulcus terminalis on the lateral femoral condyle underwent a 3-mm diameter trephine biopsy of the articular cartilage and subchondral bone overlying the bone bruise at the time of anterior cruciate ligament reconstruction. Biopsy samples of the articular cartilage and subchondral bone were stained with hematoxylin and eosin and toluidine blue. All patients had significant arthroscopic and histologic articular cartilage irregularity in the area overlying the bone bruise. Arthroscopic findings of the articular cartilage included softening (dimpling), fissuring, or overt chondral fracture. Histologic examination revealed degeneration of the chondrocytes and loss of toluidine blue staining in the articular cartilage (loss of proteoglycan). There was necrosis of osteocytes in the subchondral bone, and empty lacuna were visible. This study defines the exact histologic changes of the articular cartilage overlying a geographic bone bruise secondary to an acute anterior cruciate ligament tear. Our findings suggest that a geographic bone bruise found on magnetic resonance imaging indicates substantial damage to normal articular cartilage homeostasis.

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma.

Production of nitric oxide by solid tumors may have important ramifications regarding tumor growth and potential metastasis. This study demonstrated that the chondrosarcoma of the Swarm rat has upregulated mRNA for inducible nitric oxide synthase and produces nitric oxide. These results were confirmed by (a) the presence of a 4.4-kb band of mRNA detected by Northern blot using a probe for inducible nitric oxide synthase, (b) a 133-kDa band of protein that was detected with either a polyclonal or monoclonal antibody to the inducible nitric oxide synthase of the murine macrophage, and (c) the detection of nitrites from the culture medium of freshly cultured, isolated chondrosarcoma cells. This study showed that the expression of inducible nitric oxide synthase and the production of nitric oxide by the tumor can be increased by stimulation with endotoxin lipopolysaccharide and can be inhibited by inducible nitric oxide synthase inhibitors (L-N(g)-monomethyl arginine and aminoguanidine). Immunostaining confirmed the presence of inducible nitric oxide synthase within the tumor cells and appeared to localize the enzyme to the cytoplasm of the cells. A human chondrosarcoma was also shown to have an upregulated inducible nitric oxide synthase by both the detection of mRNA for inducible nitric oxide synthase and the presence of nitrites from the culture medium of the tumor in organ culture. Because the chondrosarcoma of the Swarm rat is a well differentiated solid tumor that rarely metastasizes, nitric oxide may be produced by the tumor to promote local growth by effects on vascular supply.

Human chondrocyte expression of growth-arrest-specific gene 6 and the tyrosine kinase receptor axl: potential role in autocrine signaling in cartilage.

OBJECTIVE: To determine if human articular chondrocytes express the axl tyrosine kinase receptor and its ligand Gas-6, a protein product of growth-arrest-specific gene 6, and to determine if Gas-6 and axl function in the regulation of chondrocyte growth and survival. METHODS: The presence of Gas-6 and axl was examined in situ in human articular cartilage by immunohistochemistry and in vitro in cell culture studies using primary human chondrocytes and immortalized human chondrocytes. The ability of recombinant Gas-6 to mediate adhesion of chondrocytes and to stimulate chondrocyte axl phosphorylation was determined. Studies of the role of Gas-6 and axl in cell proliferation and survival were also performed. RESULTS: Both Gas-6 and axl were detected in cartilage by immunohistochemical staining. Gas-6 and axl messenger RNA (mRNA) and protein were also detected in cultures of primary and immortalized human chondrocytes. Compared with cells cultured in medium containing 10% serum, Gas-6 mRNA levels were increased in immortalized chondrocytes cultured in serum-free medium, while axl expression decreased. Chondrocytes attached to Gas-6-coated plastic, and the attachment was blocked by a soluble Ig fusion protein containing the axl extracellular domain. Recombinant human Gas-6 and serum-free conditioned medium from primary and immortalized human chondrocyte cultures stimulated chondrocyte axl tyrosine phosphorylation. A mitogenic effect was noted both when immortalized chondrocytes were stimulated with recombinant Gas-6 or when they were made to overexpress axl by transfection. Addition of recombinant Gas-6 to serum-free medium resulted in increased survival of primary chondrocytes cultured at low density in agarose. CONCLUSION: These findings present evidence for an autocrine signaling pathway in cartilage involving Gas-6 and the axl tyrosine kinase adhesion receptor. Stimulation of axl by Gas-6 may play an important role in the control of chondrocyte growth and survival.