The differentiation of prehypertrophic into hypertrophic chondrocytes drives an OA-remodeling program and IL-34 expression.

OBJECTIVES: We hypothesize that chondrocytes from the deepest articular cartilage layer are pivotal in maintaining cartilage integrity and that the modification of their prehypertrophic phenotype to a hypertrophic phenotype will drive cartilage degradation in osteoarthritis. DESIGN: Murine immature articular chondrocytes (iMACs) were successively cultured into three different culture media to induce a progressive hypertrophic differentiation. Chondrocyte were phenotypically characterized by whole-genome microarray analysis. The expression of IL-34 and its receptors PTPRZ1 and CSF1R in chondrocytes and in human osteoarthritis tissues was assessed by RT-qPCR, ELISA and immunohistochemistry. The expression of bone remodeling and angiogenesis factors and the cell response to IL-1ß and IL-34 were investigated by RT-qPCR and ELISA. RESULTS: Whole-genome microarray analysis showed that iMACs, prehypertrophic and hypertrophic chondrocytes each displayed a specific phenotype. IL-1ß induced a stronger catabolic effect in prehypertrophic chondrocytes than in iMACs. Hypertrophic differentiation of prehypertrophic chondrocytes increased Bmp-2 (95%CI [0.78; 1.98]), Bmp-4 (95%CI [0.89; 1.59]), Cxcl12 (95%CI [2.19; 5.41]), CCL2 (95%CI [3.59; 11.86]), Mmp 3 (95%CI [10.29; 32.14]) and Vegf mRNA expression (95%CI [0.20; 1.74]). Microarray analysis identified IL-34, PTPRZ1 and CSFR1 as being strongly overexpressed in hypertrophic chondrocytes. IL-34 was released by human osteoarthritis cartilage; its receptors were expressed in human osteoarthritis tissues. IL-34 stimulated CCL2 and MMP13 in osteoblasts and hypertrophic chondrocytes but not in iMACs or prehypertrophic chondrocytes. CONCLUSION: Our results identify prehypertrophic chondrocytes as being potentially pivotal in the control of cartilage and subchondral bone integrity. Their differentiation into hypertrophic chondrocytes initiates a remodeling program in which IL-34 may be involved.

The coupling of bone and cartilage turnover in osteoarthritis: opportunities for bone antiresorptives and anabolics as potential treatments?

Osteoarthritis (OA) is the most common form of arthritic disease, and a major cause of disability and impaired quality of life in the elderly. OA is a complex disease of the entire joint, affecting bone, cartilage and synovium that thereby presents multiple targets for treatment. This manuscript will summarise emerging observations from cell biology, preclinical and preliminary clinical trials that elucidate interactions between the bone and cartilage components in particular. Bone and cartilage health are tightly associated. Ample evidence has been found for bone changes during progression of OA including, but not limited to, increased turnover in the subchondral bone, undermineralisation of the trabecular structure, osteophyte formation, bone marrow lesions and sclerosis of the subchondral plate. Meanwhile, a range of investigations has shown positive effects on cartilage health when bone resorption is suppressed, or deterioration of the cartilage when resorption is increased. Known bone therapies, namely oestrogens, selective oestrogen receptor modifiers (SERMs), bisphosphonates, strontium ranelate, calcitonin and parathyroid hormone, might prove useful for treating two critical tissue components of the OA joint, the bone and the cartilage. An optimal treatment for OA likely targets at least these two tissue components. The patient subgroups for whom these therapies are most appropriate have yet to be fully defined but would likely include, at a minimum, those with high bone turnover.

Cartilage, bone and synovial histomorphometry in animal models of osteoarthritis.

OBJECTIVE: This review focuses on histomorphometry for assessing the pathological changes in various compartments of the joint including cartilage, bone and synovium in animal models of osteoarthritis (OA). METHODS: Different methodological approaches are presented concerning sampling, embedding, sectioning, staining, mounting of stained sections and measurement of histomorphometric parameters using automated and semi-automated methods. Notes are provided describing some methods in greater detail. RESULTS: Histomorphometry allows a significant gain of objectivity, accuracy and reproducibility in the quantification of the main histological parameters which best characterize OA in the affected joint (cartilage thickness (CT), chondrocyte size and density, cartilage fissure, proteoglycan (PG) content, subchondral bone plate thickness (SBPT), thickness of synovial living cell layer) in animal models. CONCLUSION: Use of histomorphometry could contribute to a better quantification of histological differences between control and OA animals. Contributing also to the introduction of normative data, it is a major advantage for therapeutic assessments in experimental OA and particularly for the analytical comparison of the efficacy of disease modifying OA drugs (DMOAD).

Cyclodextrin polysulphate protects articular cartilage in experimental lapine knee osteoarthritis.

OBJECTIVE: To evaluate the in vivo chondroprotective effect of cyclodextrin polysulphate (CDPS) in a rabbit model of experimental osteoarthritis (OA). DESIGN: Experimental OA was induced in rabbits by anterior cruciate ligament transection (ACLT). Forty-eight hours post-surgery, the rabbits were randomised into three treatment groups (n=15 in each group) and a sham-operated control group. The rabbits were either injected subcutaneously with saline, 0.25 mg/kg CDPS or 1 mg/kg CDPS once a week for a period of 12 weeks, and their weight was monitored as a parameter for their general status. The animals were then sacrificed for macroscopic and histological assessment of the knee joints. RESULTS: At the lowest dose, CDPS treatment was unable to induce a significant improvement of cartilage degradation vs the saline control in the experimentally induced knee OA. However, subcutaneous injections of 1 mg/kg CDPS induced a marked inhibition (P<0.05) of osteophyte formation. Additionally, a significant reduction of cartilage degradation revealed an overall chondroprotective effect of CDPS at a concentration of 1 mg/kg. No significant effects on weight gain were noted. CONCLUSIONS: Systemic administration of CDPS is able to protect cartilage in vivo and can therefore be considered as a chondroprotective agent with structure modifying capacities.

Osteopenia and bone-remodeling abnormalities in warfarin-treated lambs.

The physiologic role of osteocalcin (OC), a vitamin K-dependent protein specific to bone, remains elusive. It has been shown that rats maintained on chronic treatment with vitamin K1 and its antagonist warfarin exhibit a marked decrease in bone osteocalcin because noncarboxylated osteocalcin does not bind to bone hydroxyapatite. To assess the role of OC in bone remodeling, we applied the warfarin model to growing lambs. We analyzed the bone changes after 3 months of concurrent warfarin and vitamin K1 treatment. Four groups of four lambs were constituted at birth and received daily a saline solution (control group, CT), 4 mg/kd/day of vitamin K1 (vitamin K group), 4 mg/kg/day of vitamin K1 + 75 or 150 mg/kg/day of warfarin (W75 and W150 group, respectively). In warfarin-treated animals, bone osteocalcin levels were decreased, both in the metaphysis (9% compared to controls) and the diaphysis (30% compared to controls) of the metacarpals. The fraction of noncarboxylated osteocalcin measured every month in the serum was significantly higher in warfarin-treated lambs than in controls at each timing point (37.6 +/- 2.6% in W75 and 48.7 +/- 5.2% in W150 versus 14.4 +/- 3.8% in controls at 3 months). Compared to non-warfarin-treated animals (NW), the main histomorphometric parameters measured on the iliac crest after tetracycline double labeling were significantly reduced in the warfarin-treated lambs: 12.2 +/- 5.2 versus 18.6 +/- 4.7% in NW (p < 0.03) for the cancellous bone area, which reflects the trabecular bone density; 14.7 +/- 6.1 versus 21.0 +/- 3.6% in NW (p < 0.03) for the eroded perimeter, and 0.315 +/- 0.064 versus 0.561 +/- 0.23 microns 3/microns 2/day in NW (p < 0.02) for the tetracycline-based bone formation rate. In conclusion, the depletion of osteocalcin in the bone of lambs induced within 3 months a marked osteopenia that resulted from a decrease in resorption and a more pronounced decrease in bone formation. Our data suggest that the presence of osteocalcin, the major gla-containing protein of bone, may be important for the maintenance of a normal bone mass and remodeling of trabecular bone.

Insulin-like growth factor-I increases trabecular bone formation and osteoblastic cell proliferation in unloaded rats.

We previously found that the inhibition of bone formation and trabecular osteopenia induced by skeletal unloading in rats are associated with reduced proliferation of osteoblastic cells lining the bone surface. In this study, we examined the effects of insulin-like growth factor-I (IGF-I) on trabecular bone formation, bone mineral density, and proliferation of marrow-derived osteoblastic cells in unloaded rats. Skeletal unloading of hind limbs was induced by tail suspension, and recombinant human IGF-I was administered at two different doses (1.3 or 2.0 mg/kg.day) in control and unloaded rats by continuous infusion for 14 days. Treatment with IGF-I had no effect on plasma glucose levels, body weight, or longitudinal bone growth. The double calcein-labeled surface, bone formation rate, and trabecular number measured at the tibial metaphysis were lower in unloaded rats compared to controls and were increased after IGF-I treatment. The increased number of bone-forming sites induced by IGF-I was associated with partial prevention of trabecular bone loss in unloaded rats. In contrast to the beneficial effects of IGF-I on bone formation and bone mineral content in unloaded rats, IGF-I had no effect in control rats. To evaluate the cellular mechanisms of action of IGF-I, marrow stromal cells were derived from the tibia of unloaded and control rats and studied in vitro. Unloading was associated with a decreased proliferation of alkaline phosphatase-positive (ALP+) marrow stromal cells. Treatment with IGF-I increased the number of ALP+ cells in unloaded rats, but not in control rats. IGF-I treatment increased ALP activity and osteocalcin production by marrow-derived cells in suspended and control rats, suggesting that IGF-I stimulated the proliferation and differentiation of osteoblast precursor cells. These results indicate that IGF-I infusion enhanced the recruitment of osteoblastic cells, increased trabecular bone formation, and partially prevented trabecular bone loss in unloaded rats, which supports the hypothesis that IGF-I may mediate in part the effects of loading on bone formation.

Serum osteocalcin (bone Gla-protein), an index of bone growth in lambs. Comparison with age-related histomorphometric changes.

In 96 normal male sheep, we studied the variations with age of serum osteocalcin (bone Gla-protein), measured with an assay specific for ovine osteocalcin. We compared serum osteocalcin with the main histomorphometric parameters of bone growth measured on the metacarpus of 20 normal lambs from birth to 90 days of age. Serum osteocalcin significantly decreased with age (r = -0.70, p less than 0.001), particularly during the first 90 days of life (r = -0.85, p less than 0.001). During this growth period, serum osteocalcin was significantly correlated with the appositional rate (r = +0.73, p less than 0.001), the rate of longitudinal bone growth (r = +0.68, p less than 0.002), the rate of production of chondrocytes in the growth plate (r = +0.60, p less than 0.007), and the thickness of the growth plate (r = +0.79, p less than 0.001). In low birth weight male lambs (growth-retarded animals), serum osteocalcin was significantly lower at birth when compared to normal lambs (271 +/- 156 vs. 535 +/- 169 micrograms/l, p less than 0.001), and was also significantly correlated with histomorphometric parameters. We conclude that serum osteocalcin, which is already known as a sensitive and specific marker of bone formation, is also a sensitive biochemical marker of skeletal growth in normal and growth-retarded lambs. In addition, sheep appears as a valid animal for experimental studies on bone growth.

Dose effects on ewe bone remodeling of short-term sodium fluoride administration--a histomorphometric and biochemical study.

The early effects of two doses of sodium fluoride (NaF) on bone remodeling was studied in 14 ewes divided into two groups. Group I received orally 1 mg NaF/kg/day and group II received a five-fold greater dose. No calcium supplement was given. Transiliac bone biopsies and blood samples were taken before treatment (T0) and after 45 (T45) days of treatment. Bone fluoride content significantly increased in group II. In both groups, a significant decrease of serum calcium and phosphorus, and a slight but nonsignificant augmentation in serum parathyroid hormone were noted. Osteoid perimeter and area were significantly increased. The osteoid width significantly increased in both groups, but was twice higher in group II than I. At T45, the osteoblast perimeter increased in both groups. Osteoid perimeter was significantly correlated with serum osteocalcin values (r = 0.74; p less than 0.001) and bone fluoride content (r = 0.64; p less than 0.01). The bone formation rate at tissue level tended to increase in both groups. Concerning the apposition rate, a decrease was noted which was 1.5-fold higher in group II than in I. The increased formation period resulted from a prolonged inactive period in group II. These results point out a stimulatory effect of fluoride on the birth rate of osteoblasts. However, fluoride prolonged the lifespan of osteoblasts that had reduced activity.

The anabolic effect of human PTH (1-34) on bone formation is blunted when bone resorption is inhibited by the bisphosphonate tiludronate--is activated resorption a prerequisite for the in vivo effect of PTH on formation in a remodeling system?

Parathyroid hormone (PTH) and its (1-34) fragment are stimulators of bone turnover that have an anabolic effect increasing trabecular bone mass when administered intermittently by daily subcutaneous injections. Its clinical use in osteoporosis, however, has been limited by the concomitant increased bone resorption and deleterious effect on cortical bone. To evaluate if a treatment combining PTH and a potent inhibitor of bone resorption would retain the anabolic effect of PTH without increasing bone resorption, we analyzed the effects of PTH (1-34) (500 IU/d) with or without the bisphosphonate tiludronate (1 mg/kg per day) for 3 months on biochemical and histological indices of bone turnover in old female sheep, an animal model which has a slow bone remodeling activity that resembles the one of elderly women. As expected, PTH (1-34) induced a significant increase of urinary pyridinoline and hydroxyproline (reflecting bone resorption), and of serum osteocalcin and alkaline phosphatase (reflecting bone formation), that were consistent with an increase of resorption and tetracycline-based formation of bone measured on iliac crest biopsy. In contrast, all biochemical and histological indices of bone turnover were decreased in sheep receiving tiludronate, a potent inhibitor of bone resorption. Surprisingly, in the combined therapy group, biochemical and histological indices of both resorption and formation did not differ from the control groups. Thus, the model of old sheep, which closely resembles the situation in old human, shows that the anabolic effect of PTH on bone is not maintained when PTH is coadministered with a bisphosphonate, in marked contrast to results noted in the growing rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of parathyroid hormone and agonists of the adenylyl cyclase and protein kinase C pathways on bone cell proliferation.

The anabolic effect of parathyroid hormone (PTH) on bone is partly due to a stimulation of osteoblast proliferation. The PTH signal is transduced by the pathways of adenylyl cyclase (AC)/protein kinase (PK) A and phospholipase C/PKC/Ca++. There is still uncertainty about the relative contribution of the two pathways to the proliferative effects of the hormone. In our study, PTH(1-34), AC/PKA agonists, and phorbol 12-myristate-13-acetate (PMA, a PKC activator) stimulated cell proliferation in cultured mouse calvariae. In isolated osteoblasts, only PMA stimulated proliferation, whereas AC/PKA agonists and PTH(1-34) inhibited it. As already known, PTH in the presence of supramaximal concentrations of transforming growth factor-beta (TGF-beta) stimulated osteoblast growth; under these same conditions, AC/PKA agonists reproduced the stimulatory effect of PTH(1-34), whereas PMA became inhibitory. PTH(1-31), which stimulates AC without affecting PKC, acted similarly to the fully active PTH(1-34) in both calvaria and isolated osteoblasts. On the contrary, midregion fragments that activate only PKC stimulated calvaria cell proliferation faintly in comparison with PTH(1-34); no effect was seen in osteoblasts, either with or without TGF-beta. Our study shows that the effects of PTH on proliferation can be mimicked by agonists of the AC/cAMP pathway. Although PMA is indeed able to stimulate cell growth in tissue explants, its effects on isolated osteoblasts markedly diverge from those of PTH. We conclude that activation of the AC/PKA pathway is the main component of the proliferative effects of PTH.

Development of an enzyme-linked immunoassay for the quantification of YKL-40 (cartilage gp-39) in guinea pig serum using hen egg yolk antibodies.

An indirect competition immunoassay for the quantification of YKL-40 (cartilage gp-39, Chondrex) in guinea pig serum has been developed using egg yolk antibodies (IgY). The immune response of hens to YKL-40 was verified by immunoblot analyses. Highly specific antibodies were obtained 30 days after the first injection. The ELISA was developed in 96-well microtiter plates with quadruplicate determinations for each point. The assay was based on the ability of YKL-40 present in serum to displace the binding of antibodies to the coated antigen. An inhibition mixture containing standard YKL-40 or guinea pig serum, diluted 1/5, and primary antibodies, diluted 1/5000, was allowed to equilibrate for 2 h at room temperature and dispensed for 16 h at 4 degrees C in wells coated with 1 microg/ml of YKL-40. Detection was achieved by the addition of rabbit anti-chicken antibodies conjugated to peroxidase followed by tetramethylbenzidine. Specificity was assessed by parallelism between a dilution curve of serum and standard YKL-40. The sensitivity of detection was 10 ng/ml. Intra- and interassay coefficients of variation were both 8.7%. The analytical recovery was 101.5+/-5.4% (mean+/-standard deviation (SD), n=9). The YKL-40 concentration in serum from 12 adult guinea pigs was 330+/-216 ng/ml (mean+/-SD) with a lower value of 164 ng/ml and an upper value of 982 ng/ml. In contrast to the rat, a dilution curve of rabbit serum gave parallelism with the guinea pig standard, suggesting recognition of a similar epitope. Possible applications of the assay in the guinea pig include disease models where YKL-40 is overexpressed and could be used as a marker, i.e. osteoarthritis, rheumatoid arthritis, cancer, liver fibrosis, atherosclerosis and more generally, pathologies with increased tissue remodeling.

1,3-Diaryl-4,5,6,7-tetrahydro-2H-isoindole derivatives: a new series of potent and selective COX-2 inhibitors in which a sulfonyl group is not a structural requisite.

Novel tetrahydro-2H-isoindoles have been prepared and evaluated as inhibitors of the COX-2 isoenzyme. A 1,3-diaryl substitution on the central polycyclic ring system and absence of a sulfonyl moiety are the two structural features of this chemical series. A short and easy synthetic pathway produced several derivatives which were shown to be potent and selective COX-2 vs COX-1 inhibitors (IC(50) = 0. 6-100 nM for COX-2, 100->1000 nM for COX-1). Structural modifications established that a bicyclic ring appended to the pyrrole nucleus and 4,4'-difluoro substitution on the phenyl rings were optimal for high inhibitory potency. Activity was confirmed in the human whole blood assay and subsequently in the murine air-pouch model in which in vivo PGE2 inhibitory activity was evaluated with respect to gastric tolerance (ED(50) for inhibition of exudate PGE2 of 3 mg/kg and gastric PGE2 of 20 mg/kg). Gastric tolerance was further assessed after administration to mice of high doses (up to 400 mg/kg) of the inhibitors by measurement of gastric damage. This panel of studies allowed selection of a number of tetrahydro-2H-isoindoles which were compared in the adjuvant-induced arthritis model. Compounds 32 and 37 showed the most potent activity with ED(50) values for edema inhibition in the noninjected paw of 0. 35 and 0.15 mg/kg/day, respectively, after oral administration. In addition, this interesting antiinflammatory profile was accompanied by a protective effect against arthritis-induced osteopenia, the decrease being 50% with a dose of 0.25 mg/kg/day.

Influence of treadmill running on femoral bone in young orchidectomized rats.

Forty 6-wk-old male Wistar rats weighing 308 +/- 24 g were divided into two groups. On day 0, the 20 animals in one group were surgically castrated and the other group was sham operated. Within each group, 10 rats were selected for treadmill running (60% maximal O2 consumption, 1 h/day, 6 days/wk for 15 wk). The 20 sedentary rats were used as controls. At the time the rats were killed (day 105), running had no significant effect on femoral mechanical properties either in castrated or in sham-operated rats. Femoral bone density was lower in orchidectomized than in sham-operated rats. Nevertheless, it was higher in exercised than in sedentary rats. Femoral Ca content paralleled changes in bone density. Treadmill running had no significant effect on plasma osteocalcin concentration but inhibited the increase in urinary deoxypyridinoline excretion observed in castrated rats. Image analysis (measured at the distal femoral diaphysis) revealed that these effects mainly resulted from decreased trabecular bone resorption in castrated exercised rats.

Effects of ossein-hydroxyapatite compound on ewe bone remodeling: biochemical and histomorphometric study.

Ossein-hydroxyapatite compound (OHC) is a protein-mineral complex derived from bovine bone. Its effects on bone remodeling were studied in old ewes which have seasonal variations in bone remodeling. Seven animals received 200 mg OHC/kg b.w./day for 90 days from July to September. The control group consisted of 7 untreated animals followed for the same period of time. OHC was administered through a fistula into the fourth stomach. A significant decrease of bone histomorphometric parameter values was noted in controls at the end of the experiment, due to seasonal variations: the cancellous eroded perimeter decreased by 45%, the osteoblastic perimeter by 60% and the bone formation rate at the cell level by 20%. In contrast, in the treated-group, these parameters tended to increase or did not change. In conclusion, counteracting the significant seasonal reduction of bone remodeling in ewes, OHC seems able to stimulate directly or indirectly bone metabolism, especially when osteoblast activity is reduced and may partly prevent the seasonal reduction of bone turnover.

Effects of a chronic GRF treatment on lambs having low or normal birth weight.

The effects of a long term treatment with human GRF(1-29)NH2 on plasma growth hormone (GH), somatomedin C (Sm-C), histomorphometric parameters of bone growth and body composition were investigated in normal and low birthweight male lambs. The animals were divided into two groups according to their birthweight: 24 normal birthweight (NBW) lambs weighing more than 4 kg and 22 low birthweight (LBW) lambs weighing less than 2.5 kg at birth. Half of the animals in each group received two daily subcutaneous injections (8 micrograms/kg body weight) of hGRF(1-29) NH2 (GRF) from birth to slaughter at 45 or 90 days of age. The other animals received the solvent only. At the beginning and at the end of the treatment, plasma GH and serum Sm-C concentrations were measured in all groups. After slaughter, a histomorphometric study was performed on undecalcified sections of metacarpal growth plates, and the remaining of the carcass was pulverized to study the chemical body composition. GRF induced GH release in both GRF-treated groups. However, plasma GH reached higher (P less than .001) concentrations and the GRF-induced GH peak lasted longer in LBW than in NBW lambs. At day 45, the GRF treatment increased (P less than .05) serum Sm-C concentrations in LBW. Most of histomorphometric parameters reflecting the metacarpal growth in length, were not statistically modified under GRF treatment. However, the size of degenerative cells was smaller (P less than .05) in LBW treated lambs as compared to controls. Consequently, the cell production in the growth plate was increased (P less than .05) under GRF treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Should subchondral bone turnover be targeted when treating osteoarthritis?

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritic disease, and it is a major cause of disability and impaired quality of life in the elderly. OA is a complex disease of the entire joint, including bone and cartilage, thereby presenting alternative approaches for treatment. This review summarizes emerging observations from cell biology to preliminary clinical trials, describing interactions between the bone and cartilage components. We speculate whether a treatment for OA would be possible without targeting the bone compartment? METHODS: Peer-reviewed articles found using pre-defined search criteria and published in the PubMed database until June 2007 are summarized. In addition, abstracts from the OsteoArthritis Research Society International (OARSI) conferences in the time period 2000-2007 were included. RESULTS: Bone and cartilage health seem to be tightly associated. Ample evidence is found for bone changes during progression of OA, including, but not limited to, increased turnover in the subchondral bone, thinning of the trabecular structure, osteophytes, bone marrow lesions and sclerosis of the subchondral plate. In addition, a range of investigations has described secondary positive effects on cartilage health when bone resorption was suppressed, or deterioration of the cartilage when resorption is increased. CONCLUSION: An optimal treatment for OA might include targeting both the bone and cartilage compartments. Hence, as several cell systems are to be targeted in a safe manner, limited options seem possible.

Measurement of knee cartilage thickness using MRI: a reproducibility study in a meniscectomized guinea pig model of osteoarthritis.

The in vivo precision (reproducibility) of quantitative MRI is of particular importance in osteoarthritis (OA) progression of small magnitude and response to therapy. In this study, three-dimensional high-resolution MRI performed at 7 T was used to assess the short-term reproducibility of measurements of mean tibial cartilage thickness in a meniscectomized guinea pig model of OA. MR image acquisition was repeated five times in nine controls (SHAM) and 10 osteoarthritic animals 3 months after meniscectomy (MNX), in vivo. The animals were then killed for histomorphometric assessment and correlation with the MRI-based measurements. Medial tibial cartilage thickness was measured on MR images using semi-automatic dedicated 3D software developed in-house. The reproducibility of measurements of cartilage thickness was assessed by five repeated MRI examinations with a short recovery delay between examinations (48 h). The computed coefficients of variation were 8.9% for the SHAM group and 8.2% for the MNX group. The coefficients of variation were compatible with expected thickness variations between normal and pathological animals. A positive agreement and significant partial correlation (Spearman r' = 0.74; P < 0.01) between the MRI and histomorphometric data was established. Three-dimensional high-resolution MRI is a promising non-invasive research tool for in vivo follow-up. This modality could be used for staging and monitoring therapy response in small-animal models of OA.

Knee cartilage thickness measurements using MRI: a 4(1/2)-month longitudinal study in the meniscectomized guinea pig model of OA.

OBJECTIVE: The aim of this study was to follow, over a 4(1/2)-month period, the medial tibia cartilage thickness on a meniscectomy (MNX) guinea pig osteoarthritis (OA) model and to compare with control animals, using three-dimensional high-resolution magnetic resonance imaging (3D HR-MRI). METHODS: MRI experimentations were performed in vivo at 7 T on guinea pig knee joints. 3D HR-MR images were acquired in 60 controls (SHAM) and 45 osteoarthritic animals (MNX) at four time-points (15, 45, 90 and 135 days) after surgery. Medial tibial cartilage thickness was measured from MRI images using in-house dedicated 3D software followed by a statistical analysis. At each time-point 15 SHAM and 15 MNX animals were sacrificed for histomorphometric assessments. RESULTS: No significant difference of mean cartilage thickness between the groups was found at early stage (D45) using MRI; however, significant differences were found between the groups at D90 (P<0.001) and D135 (P<0.001). Histomorphometry data confirmed the pathological status of the animals and was well correlated with MRI at D15 (r=0.79, P<0.01), D45 (r=0.67, P<0.01), and D135 (r=0.39, P<0.05) for SHAM, and at D45 (r=0.63, P<0.01), and D135 (r=0.81, P<0.01) for MNX. CONCLUSION: Medial tibial cartilage measurement based on HR-MR images enables the monitoring of longitudinal cartilage thickness changes. This technique showed significant differences between SHAM and MNX as from D90 after surgery. It could be used as a noninvasive and reproducible tool to monitor therapeutic response in this OA model.

Measurement of serum bone gla-protein (BGP) in humans with an ovine BGP-based radioimmunoassay.

Most RIAs of serum bone gla-protein (BGP; also called osteocalcin) used for clinical investigation are based on bovine BGP for standard, tracer, and immunogen because of the homology between bovine and human BGP. However, ovine BGP differs from human BGP by only five amino acids, being identical from residues 11 to 49, as compared with homology at residues 20-49 between bovine and human BGP. In screening various anti-ovine BGP polyclonal anti-sera we selected one (R310) that exhibits apparently complete cross-reactivity with human BGP, as assessed by dilutions of 13 human sera from normal subjects and from patients with bone disease. This RIA gave a 42% binding at a 10,000-fold final dilution, with intra- and interassay variations less than 7% and 11%, respectively. Gel-filtration chromatography of human serum showed a single immunoreactive peak. Synthetic fragments of human BGP 1-10, 7-19, 25-37, and 37-49 were not recognized by R310, suggesting that either a mid-molecule region or a conformational epitope was its target. Using this RIA, we determined that serum BGP increased with age in women (P less than 0.02), by a mean of 90% from ages 30 to 70 years. Serum BGP was also increased in patients with primary hyperparathyroidism, renal osteodystrophy, and Paget's disease. In contrast with the "normal" concentrations of BGP detected with an anti-bovine BGP antiserum (R102), serum BGP was increased in patients with postmenopausal osteoporosis as measured with the R310 ovine assay, suggesting a greater sensitivity for the latter assay.

Specific evaluation of localized bone mass and bone loss in the rat using dual-energy X-ray absorptiometry subregional analysis.

Dual-energy X-ray absorptiometry (DXA), together with the use of ultra-high resolution software, recently appeared as an accurate method for determining bone mineral density (BMD) in the rat. In order to assess the ability of this technique to detect changes in bone mass in the rat rapidly and precisely, we measured BMD at various sites of the femur using DXA subregional analysis. In particular, we studied the BMD of the metaphyseal part of the femur (M-BMD) rich in trabecular bone, and compared the values obtained with the cancellous bone volume measured by histomorphometry. In short-term ovariectomized animals (experiment 1), M-BMD was the only parameter to differentiate statistically between 10 ovariectomized (OVX) and 10 SHAM-operated (SHAM) rats (-11.2%, p < 0.01) 9 days after surgery. M-BMD still expressed the greatest variation between OVX and SHAM rats 42 days following ovariectomy (experiment 2) (-16.1%, p < 0.001 v -6.2%, p < 0.01 for the total femur BMD) and confirmed previous data demonstrating a greater loss of cancellous than cortical bone after cessation of ovarian activity. M-BMD was highly correlated with cancellous bone volume (BV) in normal (r = 0.82, p < 0.001, n = 30), OVX (r = 0.77, p < 0.001, n = 22) and SHAM (r = 0.88, p < 0.001, n = 21) rats.(ABSTRACT TRUNCATED AT 250 WORDS)