High calcium, phosphate and calcitriol supplementation leads to an osteocyte-like phenotype in calcified vessels and bone mineralisation defect in uremic rats.

A link between vascular calcification and bone anomalies has been suggested in chronic kidney disease (CKD) patients with low bone turnover disease. We investigated the vascular expression of osteocyte markers in relation to bone microarchitecture and mineralization defects in a model of low bone turnover CKD rats with vascular calcification. CKD with vascular calcification was induced by 5/6 nephrectomy followed by high calcium and phosphate diet, and vitamin D supplementation (Ca/P/VitD). CKD + Ca/P/VitD group (n = 12) was compared to CKD + normal diet (n = 12), control + normal diet (n = 8) and control + Ca/P/VitD supplementation (n = 8). At week 6, tibia, femurs and the thoracic aorta were analysed by Micro-Ct, histomorphometry and for expression of osteocyte markers. High Ca/P/VitD treatment induced vascular calcification only in CKD rats, suppressed serum parathyroid hormone levels and led to higher sclerostin, DKK1 and FGF23 serum levels. Expression of sclerostin, DKK1 and DMP1 but not FGF23 were increased in calcified vessels from CKD + Ca/P/VitD rats. Despite low parathyroid hormone levels, tibia bone cortical thickness was significantly lower in CKD + Ca/P/VitD rats as compared to control rats fed a normal diet, which is likely the result of radial growth impairment. Finally, Ca/P/VitD treatment in CKD rats induced a bone mineralization defect, which is likely explained by the high calcitriol dose. In conclusion, Ca/P/VitD supplementation in CKD rats induces expression of osteocyte markers in vessels and bone mineralisation anomalies. Further studies should evaluate the mechanisms of high dose calcitriol-induced bone mineralisation defects in CKD.

Exosomes secreted by mice adipose-derived stem cells after low-level laser irradiation treatment reduce apoptosis of osteocyte induced by hypoxia.

OBJECTIVE: Kienböck's disease is a commonly seen posttraumatic avascular necrosis characterized by avascular necrosis of the lunate bone of the wrist which involves the dominant hand. In our study, we aimed to present midterm outcomes of 12 cases treated with radial metaphyseal core decompression. PATIENTS AND METHODS: In our clinic, 12 patients who applied to our outpatient clinic with intractable pain despite at least six weeks of conservative treatment were previously diagnosed and evaluated as Kienböck's disease between the years 2006 and 2014. Patients at early stage received radial metaphyseal core decompression. RESULTS: The patients were evaluated as postoperative grip strength, flexion-extension gap, ulnar-radial deviation gap, VAS, Quick DASH and MAYO wrist scoring and patient satisfaction. CONCLUSIONS: We determined that interventions performed for Kienböck's disease cannot halt radiological progression. We are of the opinion that radial metaphyseal core decompression, aiming at increasing blood perfusion, improve early diagnosis and treatment of Kienböck's disease, increasing the patient satisfaction.

Micrometer-Sized Magnesium Whitlockite Crystals in Micropetrosis of Bisphosphonate-Exposed Human Alveolar Bone.

Osteocytes are contained within spaces called lacunae and play a central role in bone remodelling. Administered frequently to prevent osteoporotic fractures, antiresorptive agents such as bisphosphonates suppress osteocyte apoptosis and may be localized within osteocyte lacunae. Bisphosphonates also reduce osteoclast viability and thereby hinder the repair of damaged tissue. Osteocyte lacunae contribute to toughening mechanisms. Following osteocyte apoptosis, the lacunar space undergoes mineralization, termed "micropetrosis". Hypermineralized lacunae are believed to increase bone fragility. Using nanoanalytical electron microscopy with complementary spectroscopic and crystallographic experiments, postapoptotic mineralization of osteocyte lacunae in bisphosphonate-exposed human bone was investigated. We report an unprecedented presence of ∼80 nm to ∼3 µm wide, distinctly faceted, magnesium whitlockite [Ca18Mg2(HPO4)2(PO4)12] crystals and consequently altered local nanomechanical properties. These findings have broad implications on the role of therapeutic agents in driving biomineralization and shed new insights into a possible relationship between bisphosphonate exposure, availability of intracellular magnesium, and pathological calcification inside lacunae.

Evaluation of the effects of photobiomodulation on vertebras in two rat models of experimental osteoporosis.

The aim of this study was to evaluate the effects of photobiomodulation (PBM) on cancellous bone in rat models of ovariectomized induced osteoporosis (OVX-D) and glucocorticoid-induced osteoporosis (GIOP). The experiment comprised of nine groups. A group of healthy rats was used for baseline evaluations. The OVX-D rats were further divided into groups as follows: control rats with osteoporosis, OVX-D rats that received alendronate (1 mg/kg 60 days), OVX-D rats treated with pulsed wave laser (890 nm, 80 Hz, 900 s, 0.0061 W/cm2, 5.5 J/cm2, three times a week, 60 days), and OVX-D rats treated with alendronate + pulsed laser. Dexamethasone was administered to the remaining rats that were split into four groups: control, alendronate-treated rats, laser-treated rats, and GIOP rats treated with alendronate + laser. T12, L1, L2, and L3 vertebrae were subjected to laser. Results of the current study demonstrated that OVX-D and GIOP significantly decreased some stereological parameters, and type 1 collagen gene expression compared to the healthy group. There was a significant increase in osteoclast number in both OVX-D and glucocorticoid administration compared to the healthy group. However, the detrimental effect of the OVX-D procedure on bone was more serious than glucocorticoid administration. Results showed that laser alone had a detrimental effect on trabecular bone volume, and cortical bone volume in groups GIOP and OVX-D compared to those in the healthy group. Alendronate significantly improved total vertebral bone volume, trabecular bone volume, and cortical bone volume, in GIOP and OVX-D groups compared to the laser-treated groups. Furthermore, the alendronate + laser in OVX-D rats and GIOP rats produced significantly increased osteoblast number and type 1 collagen gene expression and caused a significant decrease in osteoclast number compared to the controls.

Bone natural autofluorescence and confocal laser scanning microscopy: Preliminary results of a novel useful tool to distinguish between forensic and ancient human skeletal remains.

The fast, high-throughput distinction between palaeoanthropological/archaeological remains and recent forensic/clinical bone samples is of vital importance in the field of medico-legal science. In this paper, a novel dating method was developed using the autofluorescence of human bones and the confocal laser scanning microscope as the means to distinguish between archaeological and forensic anthropological skeletal findings. Human bones exhibit fluorescence, typically induced by natural antibiotics that are absorbed by collagen, and provide secondary, exogenous fluorophores. However, primary natural fluorescence (or autofluorescence) caused by enigmatic endogenous fluorophores is also present as a micro-phenomenon, whose nature is still obscure. Here, we show that the endogenous fluorophores are mucopolysaccharides of the Rouget-Neumann sheath and, more relevant, that the intensity of the natural fluorescence in human bone decreases in a relationship to the antiquity of the samples. These results suggest that the autofluorescence of bone is a promising technique for the assessment of skeletal remains that may be potentially of medico-legal interest. A larger study is proposed to confirm these findings and to create a predictive model between the autofluorescence intensity and the time since death.

Inflammatory Bowel Disease in a Rodent Model Alters Osteocyte Protein Levels Controlling Bone Turnover.

Bone loss is a common comorbidity of inflammatory bowel disease (IBD), leading to elevated fracture risk in these patients. Inflammatory factors associated with IBD cause increased bone resorption and decreased bone formation with multiple factors implicated as instigators of these alterations. In this project, we examined the influence of IBD on osteocyte proteins in male rats (2 months old) divided into two groups: induced gut inflammation via 2,4,6-trinitrobenzenesulfonic acid (TNBS) enema, and vehicle control. We examined the prevalence of two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), an anti-inflammatory cytokine, interleukin-10 (IL-10), the anabolic factor insulin-like growth factor-I (IGF-I), osteoclastogenesis regulators RANKL and OPG, and the bone formation inhibitor sclerostin in osteocytes in three bone compartments 4 weeks after initiation of gut inflammation. Histomorphometry of the proximal tibia and fourth lumbar vertebra revealed lower bone volume, lower bone formation rate (BFR), lower osteoid surface (OS), and higher osteoclast surface (Oc.S) with TNBS. Tibial mid-shaft periosteal BFR was also lower with TNBS. Immunohistochemical staining of the distal femur demonstrated that %TNF-α+ , %IL-6+ , %RANKL+ , and %OPG+ osteocytes were elevated in cancellous bone in TNBS animals compared to vehicle. These changes were coincident with increased bone resorption. With regression analysis, %RANKL+ osteocytes statistically predicted the increase in cancellous Oc.S (R2 = 0.565). Increased %sclerostin+ osteocytes observed in the TNBS treatment predicted declines in cancellous OS (R2 = 0.581) as well as BFR in cancellous and cortical bone (R2 = 0.674, R2 = 0.908, respectively). Contrary to our hypothesis, %IGF-I+ osteocytes increased in TNBS animals. In conclusion, the IBD model produced a systemic inflammation that altered the regulatory protein profile in osteocytes that control bone resorption and bone formation, likely contributing to IBD-induced bone loss. These data highlight a potential mechanistic role of osteocytes in inflammatory bone loss associated with IBD and systemic inflammation. © 2017 American Society for Bone and Mineral Research.

Effects of Potentilla fulgens as a Prophylactic Agent in Tibial Defects in Rats.

OBJECTIVE: To investigate the effects of Potentilla fulgens as a prophylactic agent on tibial defects in the rat. STUDY DESIGN: Twenty-eight male Wistar albino rats weighing 200-215 g each were divided into 3 experimental groups. The tibial bone defect group served as the control group. The experimental groups were Potentilla fulgens with tibial defect (14 days) and Potentilla fulgens with tibial defect (28 days). Extract of Potentilla fulgens was mixed with water (400 mg/kg/day) and given to groups 14 and 28 as drinking water. The histopathological and immunohistochemical characteristics of each tibial bone cavity within each group were observed. The trabecular new bone formation was evaluated by expression rate of osteonectin and osteopontin. RESULTS: In the Potentilla fulgens + tibial defect group (14 days), trabecular bone had started combining extensive new bone formation, osteocyte cells were evident, and lamellar bone was formed. Osteoblasts showed a positive reaction with osteonectin. Osteopontin expression was positively observed between fibrous structures and in the osteoblast and osteocyte cells. This can be considered indicative of new bone formation. In the Potentilla fulgens + tibial defect group (28 days), an increase in expansion in trabecular bone and myeloid tissue was observed. Osteoblastic activity and osteocyte cells began to be observed in new bone fragments. CONCLUSION: In our study we show that Potentilla fulgens extract provided a protective effect on new bone formation and aided in the development of osteocytes and secretion of matrix in osteoblasts. Additionally, we show the inductive effect of the extract on new bone formation. In particular, the expression of osteopontin and osteonectin was also supported with the Western blot technique on the development of osteoblasts and osteocytes, showing a similar trend with our results.

Immobilization and long-term recovery results in large changes in bone structure and strength but no corresponding alterations of osteocyte lacunar properties.

The ability of osteocytes to demineralize the perilacunar matrix, osteocytic osteolysis, and thereby participate directly in bone metabolism, is an aspect of osteocyte biology that has received increasing attention during the last couple of years. The aim of the present work was to investigate whether osteocyte lacunar properties change during immobilization and subsequent recovery. A rat cortical bone model with negligible Haversian remodeling effects was used, with temporary immobilization of one hindlimb induced by botulinum toxin. Several complementary techniques covering multiple length scales enabled correlation of osteocyte lacunar properties to changes observed on the organ and tissue level of femoral bone. Bone structural parameters measured by µCT and mechanical properties were compared to sub-micrometer resolution SR µCT data mapping an unprecedented number (1.85 million) of osteocyte lacunae. Immobilization induced a significant reduction in aBMD, bone volume, tissue volume, and load to fracture, as well as the muscle mass of rectus femoris. During the subsequent recovery period, the bone structural and mechanical properties were only partly regained in spite of a long-term (28weeks) study period. No significant changes in osteocyte lacunar volume, density, oblateness, stretch, or orientation were detected upon immobilization or subsequent recovery. In conclusion, the bone architecture and not osteocyte lacunar properties or bone material characteristics dominate the immobilization response as well as the subsequent recovery.

Repair of Critical Calvarias Defects With Systemic Epimedium sagittatum Extract.

It is well established in reconstructive surgery the repair of great bone defects is a difficult goal to be achieved. The aim of this study was to evaluate the influence of an extract rich in icariin on bone neoformation in critically sized defects in rat calvaria. Under continual saline irrigation, a circular bone defect was created in 40 rat calvarias with an 8-mm diameter trephine drill. Animals were randomly divided into a test group that received an Epimedium sagittatum extract (containing 5.8 mg/mL of icariin) and a control group that received an equal volume of saline solution. Substances were administered daily through a feeding tube until euthanasia. After 7, 14, 21, and 42 days, 5 animals from each group were euthanized. Calvaria defect samples were fixed in 10% formalin for 48 hours, X-rayed, and histologically processed. In the test group, there was a significant reduction in the bone defect area on X-ray images and an increase in new bone area in all of the experimental periods in the test group. At 42 days, the bone in the test group also exhibited a significant reduction in osteocyte (P = 0.002) and osteoclast density (P = 0.041). The authors conclude that administration of systemic Epimedium extracts containing high concentrations of icariin can induce bone neoformation and reduce osteocyte and osteoclast densities, thereby altering the normal deposition and remodeling patterns that are present in critically sized bone defects.

Rapamycin reduces severity of senile osteoporosis by activating osteocyte autophagy.

SUMMARY: Osteocyte is the orchestrator of bone remolding and decline in osteocyte autophagy is involved in senile osteoporosis. Our results suggested that rapamycin, at least in part by activating osteocyte autophagy, reduced the severity of age-related bone changes in trabecular bone of old male rats. INTRODUCTION: Previous literatures have showed that osteocyte is the orchestrator of bone remolding and age-related decline in osteocyte number is associated with senile osteoporosis. Autophagy is an important cellular protective mechanism which can preserve osteocyte viability and failure of autophagy in osteocyte with age has been linked to senile osteoporosis. The purpose of this study was to explore whether rapamycin, one activator of autophagy, has protective effects on senile osteoporosis through inducing osteocyte autophagy. METHODS: Fifty-two 24-month-old male Sprague-Dawley (SD) rats were randomly divided into two groups. Rapamycin (1 mg/kg weight/day) or DMSO vehicle control was administered intraperitoneally for 12 weeks. BMD and bone microstructure were determined by Micro-CT. Fluorochrome labeling of the bones was performed to measure the mineral apposition rate (MAR). TRAP staining was performed to evaluate osteoclast number. The plasma levels of bone turnover markers were also analyzed. The effects of rapamycin on osteocyte autophagy were determined by immunohistochemistry, Western blot, and q-PCR. TUNEL was used to determine the prevalence of osteocyte apoptosis. RESULTS: Micro-CT evaluation demonstrated that rapamycin had a protective effect on age-related bone loss in trabecular bone. Besides, rapamycin resulted in an obvious increase of MAR and a decrease of osteoclast number in contrast to the control group. Furthermore, rapamycin also induced autophagy in osteocyte demonstrated by increased LC3-positive osteocyte and increased LC3 turnover. In addition, rats treated with rapamycin exhibited decreased apoptosis of osteocyte determined by TUNEL. CONCLUSIONS: These results suggested that rapamycin, at least in part by activating osteocyte autophagy, reduced the severity of age-related bone changes in trabecular bone of old male rats. Therefore, rapamycin might be a feasible therapeutic approach for senile osteoporosis.

Stereological study of the effect of black olive hydroalcoholic extract on osteoporosis in vertebra and tibia in ovariectomized rats.

UNLABELLED: A cocktail of many different antioxidants might be more effective than supplementation with a single molecule, and it closely resembles the natural environment in which active compounds were found. This is the first study well-grounded in stereological examination that showed that black olive extract effectively can ameliorate the quantitative changes of the bone structure and prevented bone loss in this osteoporosis animal model. INTRODUCTION: The aim of this study was to quantitatively evaluate the effects of black olive extract consumption on treatment of ovariectomized (OVX) induced osteoporosis in rats. This is the first study well-grounded in stereological examination. METHODS: Ninety adult rats were allocated to control, sham-operated, OVX, and olive-supplemented OVX groups (received 250-, 500-, and 750-mg/kg body weight black olive hydroalcoholic extract orally) for 16 weeks. At the end of the experiment, blood samples were collected, and plasma levels of calcium, phosphorus, and alkaline phosphatase were assayed. Then, the specimens from both the tibia and fifth lumbar vertebra (L5) bones were processed, and stereological analysis was performed. RESULTS: Administration of extract resulted in decrease of alkaline phosphatase level during the treatment. After treatment of OVX rats with three doses of extract, the total number of the osteocytes revealed an increment in 500- and 750-mg/kg treated groups in comparison to the OVX group. This increment was significant only in L5. Compared to the OVX group, a significant increase was observed in the number of osteoblastsin L5 vertebra in three doses of extract-treated groups. However, this increment in tibia was statistically significant only in 750-mg/kg black olive hydroalcoholic extract-treated group. Moreover, the number of osteoclast cells were significantly decreased in vertebra and tibia in the treated groups compared to the OVX group (P < 0.05). CONCLUSION: Black olive hydroalcoholic extract effectively can ameliorate the quantitative changes of the bone structure and prevented bone loss in this osteoporosis animal model. Thus, it can be a promising candidate for treatment of accelerated bone loss especially in postmenopausal osteoporosis.

Experimental evaluation of the effects of Ankaferd Blood Stopper and collagenated heterologous bone graft on bone healing in sinus floor augmentation.

PURPOSE: The aim of this study was to evaluate the effect of collagenated heterologous bone graft (CHBG) and Ankaferd Blood Stopper (ABS), a plant extract, on bone healing after sinus floor augmentation. MATERIALS AND METHODS: Thirty-six New Zealand rabbits were used. Bilateral sinus augmentation was performed, and 72 bone defects were created. The maxillary sinuses were grafted with four different biomaterials: blood clot (control group), CHBG (Apatos Mix, OsteoBiol, Tecnoss) (graft group), ABS (ABS group), and ABS + CHBG (ABS+graft group). The rabbits were sacrificed at 1, 4, and 8 weeks after surgery. Histochemical and immunohistochemical examinations were performed on all samples. Staining with hematoxylin-eosin and Masson trichrome was performed, and bone marker activity was evaluated. RESULTS: Lymphocyte infiltration was high at the first week in all groups and decreased from 1 to 8 weeks. All materials were biocompatible. Osteoclast numbers increased in the control group from 1 to 8 weeks and decreased in the other groups. There was no new bone formation in week 1 in all groups. New bone formation increased in all groups from 1 to 8 weeks, and at the fourth week, new bone formation was greater in the ABS and ABS+graft groups than in the other groups. There were osteoclasts around the bone graft materials, but degeneration of the graft was seen only in the ABS+graft group at week 8. CONCLUSION: ABS accelerates bone healing in sinus augmentation procedures and can be used alone or with CHBG. CHBG has osteoconductive properties, and ABS can accelerate bone graft degeneration.

Effects of local application of simvastatin on bone regeneration in femoral bone defects in rabbit.

Simvastatin (SIM), which is widely used in hyperlipidemia treatment, has also attracted attention due to its anabolic effects on bones. This study is designed to investigate the effectiveness of 2 mg SIM combined with 3 different carriers as delivery systems. Bone defects were surgically created in the femoral bones of 14 New Zealand white rabbits. The carriers used were the inorganic bovine bone graft (BOS), the hydroxyapatite combined with calcium sulfate (HACS), and the collagen sponge (COS). The bone defects were divided for each time period into 7 groups, as follows: passive control group (CONT), active control groups (BOS), (HACS) and (COS) (no simvastatin), and groups (BOS + SIM), (HACS + SIM) (carrier and simvastatin combination). Animal were sacrificed at 4 and 8 weeks postoperatively, and bone defects areas were prepared for histological examination and histomorphometric evaluation. Analysis of variance demonstrated statistically significant differences between groups depending on the carrier used. At 4 weeks, the BOS + SIM group presented higher rates of new bone formation, whereas at 8 weeks more new bone formation was noted for the HACS + SIM group. This study suggests that local application of simvastatin, combined with an appropriate carrier, can promote new bone formation.

Hyperbaric oxygen therapy as a prevention modality for radiation damage in the mandibles of mice.

BACKGROUND: Radiation therapy (RT) as part head and neck cancer treatment often leads to irradiation of surrounding normal tissue, such as mandibular bone. A reduced reparative capacity of the bone can lead to osteoradionecrosis (ORN). Hyperbaric oxygen therapy (HBOT) is used to treat ORN, based on its potential to raise the oxygen tension in tissues. However, prevention of radiation-induced damage is of great interest. Our purpose was to investigate whether HBOT could prevent radiation-induced damage to murine mandibles. METHODS: Twenty-eight mice were irradiated in the head and neck region with a single dose (15 Gy) and half of them were subsequently subjected to HBOT. Another 14 mice did not receive any treatment and served as controls. Ten and 24 weeks after RT, mandibles were harvested and analysed histologically and by microcomputed tomography (micro-CT). RESULTS: Micro-CT analysis showed a reduction in relative bone volume by RT, which was partly recovered by HBOT. Trabecular thickness and separation were also positively influenced by HBOT. Morphologically, HBOT suppressed the osteoclast number, indicating decreased resorption, and decreased the amount of lacunae devoid of osteocytes, indicating increased bone viability. CONCLUSIONS: HBOT was able to partly reduce radiation-induced effects on microarchitectural parameters, resorption, and bone viability in mouse mandibles. HBOT could therefore potentially play a role in the prevention of radiation-induced damage to human mandibular bone.

Pulsed electromagnetic fields protect the balance between adipogenesis and osteogenesis on steroid-induced osteonecrosis of femoral head at the pre-collapse stage in rats.

This study was designed to investigate the effects of pulsed electromagnetic fields (PEMF) on the balance of adipogenesis and osteogenesis on steroid-induced osteonecrosis of the femoral head (OFH) in rats. Forty-two rats were divided into three groups: Steroid group (S, n = 16); Steroid + PEMF group (S + P, n = 16); and Control group (C, n = 10). For groups S and S + P, all rats were first intravenously given 10 µg/kg lipopolysaccharide on day 1, and then intramuscularly injected with 20 mg/kg methylprednisolone acetate on days 2, 3, and 4, with an interval of 24 h. After 4 weeks, the S + P group was treated with PEMF (4.5-ms square pulse, repeated at 15 Hz, with a peak of 1.2 mT) for 4 h a day for the next 8 weeks. Group S was not exposed to PEMF. Group C was chosen as the control group, without steroid use and exposure to PEMF. After 8 weeks of treatment, the histological changes, and mRNA and protein expressions of PPAR-γ2 and Runx2 were measured and analyzed. Compared with the S group, lower incidence of osteonecrosis (31% vs. 69%, P < 0.05) and empty osteocyte lacuna rate (36.16 ± 15.34 vs. 59.55 ± 21.70, P < 0.01) was observed in the S + P group. Furthermore, PEMF suppressed the expressions of PPAR-γ2 and improved the expressions of Runx2 in the femoral head (P < 0.05). All data suggest that PEMF is an effective physiotherapy in the treatment of steroid-induced ONFH, and the possible underlying mechanisms include protecting the balance between adipogenesis and osteogenesis.

Osteoblast ontogeny and implications for bone pathology: an overview.

Osteoblasts are specialized mesenchyme-derived cells accountable for bone synthesis, remodelling and healing. Differentiation of osteoblasts from mesenchymal stem cells (MSC) towards osteocytes is a multi-step process strictly controlled by various genes, transcription factors and signalling proteins. The aim of this review is to provide an update on the nature of bone-forming osteoblastic cells, highlighting recent data on MSC-osteoblast-osteocyte transformation from a molecular perspective and to discuss osteoblast malfunctions in various bone diseases. We present here the consecutive stages occurring in the differentiation of osteoblasts from MSC, the transcription factors involved and the role of miRNAs in the process. Recent data concerning the pathogenic mechanisms underlying the loss of bone mass and architecture caused by malfunctions in the synthetic activity and metabolism of osteoblasts in osteoporosis, osteogenesis imperfecta, osteoarthritis and rheumatoid arthritis are discussed. The newly acquired knowledge of the ontogeny of osteoblasts will assist in unravelling the abnormalities taking place during their differentiation and will facilitate the prevention and/or treatment of bone diseases by therapy directed against altered molecules and mechanisms.

The effects of minimally invasive laser needle system on suppression of trabecular bone loss induced by skeletal unloading.

This study was aimed to evaluate the effects of low-level laser therapy (LLLT) in the treatment of trabecular bone loss induced by skeletal unloading. Twelve mice have taken denervation operation. At 2 weeks after denervation, LLLT (wavelength, 660 nm; energy, 3 J) was applied to the right tibiae of 6 mice (LASER) for 5 days/week over 2 weeks by using a minimally invasive laser needle system (MILNS) which consists of a 100 µm optical fiber in a fine needle (diameter, 130 µm) [corrected]. Structural parameters and histograms of bone mineralization density distribution (BMDD) were obtained before LLLT and at 2 weeks after LLLT. In addition, osteocyte, osteoblast, and osteoclast populations were counted. Two weeks after LLLT, bone volume fraction, trabeculae number, and trabeculae thickness were significantly increased and trabecular separations, trabecular bone pattern factor, and structure model index were significantly decreased in LASER than SHAM (p < 0.05). BMDD in LASER was maintained while that in SHAM was shifted to lower mineralization. Osteocyte and osteoblast populations were significantly increased but osteoclast population was significantly decreased in LASER when compared with those in SHAM (p < 0.05). The results indicate that LLLT with the MILNS may enhance bone quality and bone homeostasis associated with enhancement of bone formation and suppression of bone resorption.

Parathyroid hormone [1-34] improves articular cartilage surface architecture and integration and subchondral bone reconstitution in osteochondral defects in vivo.

OBJECTIVE: The 1-34 amino acid segment of the parathyroid hormone (PTH [1-34]) mediates anabolic effects in chondrocytes and osteocytes. The aim of this study was to investigate whether systemic application of PTH [1-34] improves the repair of non-osteoarthritic, focal osteochondral defects in vivo. DESIGN: Standardized cylindrical osteochondral defects were bilaterally created in the femoral trochlea of rabbits (n = 8). Daily subcutaneous injections of 10 µg PTH [1-34]/kg were given to the treatment group (n = 4) for 6 weeks, controls (n = 4) received saline. Articular cartilage repair was evaluated by macroscopic, biochemical, histological and immunohistochemical analyses. Reconstitution of the subchondral bone was assessed by micro-computed tomography. Effects of PTH [1-34] on synovial membrane, apoptosis, and expression of the PTH receptor (PTH1R) were determined. RESULTS: Systemic PTH [1-34] increased PTH1R expression on both, chondrocytes and osteocytes within the repair tissue. PTH [1-34] ameliorated the macro- and microscopic aspect of the cartilaginous repair tissue. It also enhanced the thickness of the subchondral bone plate and the microarchitecture of the subarticular spongiosa within the defects. No significant correlations were established between these coexistent processes. Apoptotic levels, synovial membrane, biochemical composition of the repair tissue, and type-I/II collagen immunoreactivity remained unaffected. CONCLUSIONS: PTH [1-34] emerges as a promising agent in the treatment of focal osteochondral defects as its systemic administration simultaneously stimulates articular cartilage and subchondral bone repair. Importantly, both time-dependent mechanisms of repair did not correlate significantly at this early time point and need to be followed over prolonged observation periods.

Oxytocin promotes bone formation during the alveolar healing process in old acyclic female rats.

OBJECTIVE: OT was reported to be a direct regulator of bone mass in young rodents, and this anabolic effect on bone is a peripheral action of OT. The goal of this study was to investigate the peripheral action of oxytocin (OT) in the alveolar healing process in old female rats. MATERIALS AND METHODS: Females Wistar rats (24-month-old) in permanent diestrus phase, received two ip (12h apart) injections of saline (NaCl 0.15M - control group) or OT (45µg/rat - treated group). Seven days later, the right maxillary incisor was extracted and analyses were performed up to 28 days of the alveolar healing process (35 days after saline or OT administration). RESULTS: Calcium and phosphorus plasma concentrations did not differ between the groups. The plasma biochemical bone formations markers, alkaline phosphatase (ALP) and osteocalcin were significantly higher in the treated group. Histomorphometric analyses confirmed bone formation as the treated group presented the highest mean value of post-extraction bone formation. Tartrate-resistant acid phosphatase (TRAP) was significantly reduced in the treated group indicating an anti-resorptive effect of OT. Immunohistochemistry reactions performed in order to identify the presence of osteocalcin and TRAP in the bone cells of the dental socket confirmed these outcomes. CONCLUSIONS: OT was found to promote bone formation and to inhibit bone resorption in old acyclic female rats during the alveolar healing process.

A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells.

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.