Unmineralized Bone Research Articles

Morphological and Immunohistochemical Characterization of Bone Structure and Cell–Cell Communication in a Rat Osteoporosis Model

Bone remodeling is essential for maintaining bone health. The imbalance between bone formation and bone resorption leads to bone diseases such as osteoporosis. Connexin43 (Cx43) is a gap junction molecule that plays an important role in bone homeostasis. The present study investigates the morphological characteristics of bone trabeculae and the distribution of Cx43 in bone cells using osteoporotic rat models to explore the relationship between osteoporosis and bone remodeling. Female Sprague–Dawley rats were divided into three groups: sham, ovarectomy with food deprivation (OVX+diet), and ovarectomy with steroid administration (OVX+steroid) for 3 and 12 months to induce osteoporosis. The lumbar vertebrae were processed for histomorphometric and immunohistochemical evaluation of the trabeculae and the distribution of Cx43 in bone cells. The data showed a significant reduction in trabecular bone in both osteoporotic groups. After 12 months, the OVX+diet treatment resulted in reduced mineralization and an increase in unmineralized bone. The percentage of alkaline phosphatase-positive areas in the OVX+diet vertebrae was lower at 12 months compared to the sham group. A significant increase in tartrate-resistant acid phosphatase (TRAP) positive osteoclasts was observed in the OVX+diet group. Both osteoporotic groups showed a decrease in Cx43-positive osteoblasts areas. An increase in the number of osteoclasts positive for Cx43 was detected in the OVX+diet group. The changes in Cx43 distribution in bone cells, together with trabecular mineralization, suggest that Cx43 may play a role in the progression of osteoporosis and could be a valuable target to improve bone remodeling.

A radiographic and histological study to compare red (650 nm) versus near infrared (810 nm) diode lasers photobiomodulation for alveolar socket preservation

Previous findings indicated that the laser photobiomodulation is more effective than the control or placebo in preserving the alveolar socket. This study aimed to compare two different lasers regarding their effectiveness in aiding alveolar socket preservation. Twenty extraction sockets were selected then divided into two equal groups. Group A was exposed to 650 nm Diode laser, and Group B to 810 nm Diode laser following the same protocol and parameters after a standard alveolar socket preservation procedure with collagen plug. Radiographic analysis with cone beam computed tomography was done to compare the alveolar bone surface area immediately after extraction and three months post-operatively, while bone samples collected before implant drilling were histologically examined for newly formed bone evaluation and histomorphometric analysis in terms of percentage of new bone surface area, percentage of unmineralized bone and finally, immunohistochemical analysis of Osteocalcin reaction surface area as well as optical density. Radiographically, infrared (810 nm) Diode effect on alveolar bone surface area has significantly exceeded the red laser, while histologically, red (650 nm) Diode has demonstrated statistical significance regarding all parameters; newly formed bone surface area percentage, unmineralized bone area percentage and finally Osteocalcin bone marker reaction surface area percentage and optical density. Under the specified conditions and laser parameters, photobiomodulation using the 810 nm Diode got the upper hand radiographically, yet histologically, the red 650 nm Diode managed to dominate all histological parameters when both employed as an adjunct to alveolar socket preservation procedures.

Mesenchymal stem cells enhance targeted bone growth from injectable hydrogels with BMP-2 peptides.

Osteoporosis is the most common chronic metabolic bone disease, and the prevalence of osteoporotic fractures is rapidly increasing with the aging population. While bisphosphonates can reduce bone loss and risk of fracture, these drugs are systemic, rely on long-term use, and patient compliance is low. Recombinant human bone morphogenetic protein-2 (BMP-2) is an FDA-approved protein that can offer a more targeted therapeutic than systemic treatments. DWIVA is a peptide sequence corresponding to the wrist epitope of BMP-2, and DWIVA-functionalized hydrogels feature osteoinductive propertiesin vitro and in vivo. This study reports that self-forming DWIVA-functionalized hydrogels injected into the intramedullary canal of rat femurs induce a local increase in trabecular bone in as little as 2 weeks. Increases in bone volume, trabecular thickness, and trabeculae count from DWIVA-laden hydrogels persist for at least 4 weeks, and the inclusion of mesenchymal stem cells (MSCs) significantly enhances the development of mineralized bone. Histological analysis of decalcified femurs also shows that hydrogel injections containing DWIVA peptide and MSCs stimulate unmineralized bone tissue formation and induce an increased count of osteoblasts and osteoclasts at the injection site after 4 weeks. Overall, the MSC-laden DWIVApeptide-functionalized hydrogels presented rapidly induce targeted bone formation and have the potential to form nascent bone within bones in jeopardy of an osteoporotic fracture such as the femur.

Radiographic and histological evaluation of bone formation induced by rhBMP-2-incorporated biomimetic calcium phosphate material in clinical alveolar sockets preservation

PurposeWe assessed the efficiency of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) incorporated biomimetic calcium phosphate on β-tricalcium phosphate (β-TCP) (rhBMP-2/BioCaP/β-TCP) on bone formation in a model of socket preservation using cone beam computed tomography (CBCT) scanning and histological examination.MethodsForty patients undergoing minimally invasive single-root tooth extraction for dental implantation were randomized to three groups according to the material used for socket preservation: filling with rhBMP-2/BioCaP/β-TCP, β-TCP, or natural healing (kept unfilled) (controls). The alveolar sockets (including the control group) were covered by two-layer collagen membranes and sutured. Two CBCT scans were taken, one immediately after socket preservation procedure (baseline) and another 6 weeks later. Gray values (GVs) obtained from CBCT were recorded. During insertion of the dental implant, biopsies were taken and analyzed histologically for new bone formation, residual material, and unmineralized bone tissue at the core of the biopsy.ResultsThe mean (± standard deviation) changes of GVs of the CBCT scans at the central area of filled materials were as follows: 373.19 ± 157.16 in the rhBMP-2/BioCaP/β-TCP group, 112.26 ± 197.25 in the β-TCP group, and -257 ± 273.51 in the control group. The decrease of GVs in the rhBMP-2/BioCaP/β-TCP group as compared with the β-TCP group was statistically significant (P < 0.001). Differences in new bone formation (P = 0.006) were also found: 21,18% ± 7.62% in the rhBMP-2/BioCaP/β-TCP group, 13.44% ± 6.03% in the β-TCP group, and 9.49% ± 0.08% in controls. The residual material was10.04% ± 4.57% in the rhBMP-2/BioCaP/β-TCP group vs. 20.60% ± 9.54%) in the β-TCP group (P < 0.001). Differences in unmineralized bone tissue (P < 0.001) were also found (68.78% ± 7.67%, 65.96% ± 12.64%, and 90.38% ± 7.5% in the rhBMP-2/BioCaP/β-TC, β-TCP, and control groups, respectively).ConclusionsThis study shows that rhBMP-2/BioCaP/β-TCP is a promising bone substitute with fast degradation and potent pro-osteogenic capacity that can be useful for socket preservation in implant dentistry.Trial registration: ChiCTR, ChiCTR2000035263. Registered 5 August 2020, https://www.chictr.org.cn/ChiCTR2000035263.Graphical

The LRP5 high-bone-mass mutation causes alveolar bone accrual with minor craniofacial alteration.

Mutations in low-density lipoprotein receptor-related protein 5 (LRP5) cause various bone diseases. Several mouse models were generated to study the role of LRP5 in bone development. But most of the studies were confined to the appendicular skeleton. The role of LRP5 in the axial skeleton, especially in the craniofacial skeleton, is largely unknown. The aim of this study was to investigate the craniofacial phenotype with the LRP5G171V mutation. To understand how LRP5 affects craniofacial bone properties, we analyzed LRP5 high-bone-mass mutant mice carrying the G171V missense mutation (LRP5HBM ). Quantitative microcomputed tomographic imaging and histomorphometric analyses were used to study craniofacial phenotypes and bone density. Histology, immunohistochemistry, and in vivo fluorochrome labeling were used to study molecular mechanisms. LRP5HBM mice showed overall minor changes in the craniofacial bone development but with increased bone mass in the interradicular alveolar bone, edentulous ridge, palatine bone, and premaxillary suture. Elevated osteocyte density was observed in LRP5HBM mice, along with increased Runx2 expression and unmineralized bone surrounding osteocytes. Meanwhile, LRP5HBM mice exhibited increased osteoprogenitors, but no significant changes were observed in osteoclasts. This led to a high-bone-mass phenotype, and an increased osteocyte density in the alveolar bone and edentulous ridge. LRP5HBM mice display increased bone mass in the alveolar bone with minor changes in the craniofacial morphology. Collectively, these data elucidated the important role of LRP5 in axial bone development and homeostasis and provided clues into the therapeutical potential of LRP5 signaling in treating alveolar bone loss.

Continuous Two-Zone In Vitro Co-culture Model of the Enthesis

The enthesis is the interfacial tissue between ligament or tendon, and bone, which connects tissues of distinctly different mechanical properties. Although ligament and enthesis injury is commonplace, the development and healing mechanisms of these tissues are yet unclear. Current models for investigating these mechanisms are primarily in vivo animal models as in vitro models have been limited in their structural and mechanical biomimicry of the native enthesis. In this study, an in vitro enthesis model was developed using a modified gel aspiration-ejection method. Continuous two-zone aligned dense collagen (ADC) hydrogels with an overlapping interface were fabricated within 2 h. The mechanical properties of acellular two-zone ADC confirmed the continuous nature of this model, as the mechanical properties showed no significant difference compared to single-zone ADC and maintained comparable structural and mechanical characteristics of immature ligaments and unmineralized bone. Human anterior cruciate ligament fibroblasts and human spine vertebral osteoblasts were isolated from donor tissues and were seeded to form the enthesis model. These were cultured for 14 days, at which the viability and proliferation was observed to be 85 ± 7.5% and 230 ± 52%, respectively. Histological and immunofluorescence analyses at day 14 revealed extensive cell-driven matrix remodelling, and the seeded fibroblasts and osteoblasts maintained their phenotype within their compartments of the layered co-culture model. These results demonstrate the rapid fabrication of a two-zone co-culture system that can be utilized as an in vitro model to further understand the degenerative and regenerative mechanisms within the enthesis.

A new model of bone remodeling and turnover set up in the framework of generalized continuum mechanics

In the past few decades, numerous studies have attempted to address the various phenomena that take place simultaneously during bone remodeling. Drawing from Frost’s “mechanostat theory,” multiple phenomenological models of varying complexity have been developed to describe bone remodeling in terms of evolution of bone porosity, tissue properties, and mineralization. The main goal of this paper is to present the general theory of a novel macroscopic and comprehensive model of bone remodeling accounting for the interactions of mechanics and biochemistry at the microscale. Two independent remodeling mechanisms are considered: the rotation of the material axes and the turnover of bone material. The former mechanism is related to the change of orientation of bone microstructure. Bone turnover refers to the dynamic process of bone resorption by osteoclasts, formation of unmineralized bone by osteoblasts, and mineralization. The model is set up in the framework of generalized continuum mechanics. The evolution of bone tissue is thus described through its macroscopic deformation as well as macroscopic variables related to the orientation of bone microstructure and bone tissue composition (porosity, unmineralized and mineralized bone matrix). Thermodynamically consistent evolution laws of bone material are obtained by enforcing suitable statements of the virtual power principle and of the dissipation principle. Moreover, additional constitutive hypotheses are formulated to develop a phenomenological law of bone turnover. The turnover model is discussed on the basis of a number of numerical simulations. Although the model can capture the main features of bone turnover, it cannot describe satisfactorily the complexity of the underlying biological crosstalk, highlighting the need of a more refined mechanobiological constitutive theory of bone turnover.

Osteomalacia and Vitamin D Status: A Clinical Update 2020.

ABSTRACTHistorically, rickets and osteomalacia have been synonymous with vitamin D deficiency dating back to the 17th century. The term osteomalacia, which literally means soft bone, was traditionally applied to characteristic radiologically or histologically documented skeletal disease and not just to clinical or biochemical abnormalities. Osteomalacia results from impaired mineralization of bone that can manifest in several types, which differ from one another by the relationships of osteoid (ie, unmineralized bone matrix) thickness both with osteoid surface and mineral apposition rate. Osteomalacia related to vitamin D deficiency evolves in three stages. The initial stage is characterized by normal serum levels of calcium and phosphate and elevated alkaline phosphatase, PTH, and 1,25‐dihydroxyvitamin D [1,25(OH)2D]—the latter a consequence of increased PTH. In the second stage, serum calcium and often phosphate levels usually decline, and both serum PTH and alkaline phosphatase values increase further. However, serum 1,25(OH)2D returns to normal or low values depending on the concentration of its substrate, 25‐hydroxyvitamin D (25OHD; the best available index of vitamin D nutrition) and the degree of PTH elevation. In the final stage, hypocalcemia and hypophosphatemia are invariably low with further exacerbation of secondary hyperparathyroidism. The exact,or even an approximate, prevalence of osteomalacia caused by vitamin D deficiency is difficult to estimate, most likely it is underrecognized or misdiagnosed as osteoporosis. Signs and symptoms include diffuse bone, muscle weakness, and characteristic fracture pattern, often referred to as pseudofractures, involving ribs, scapulae, pubic rami, proximal femurs, and codfish‐type vertebrae. The goal of therapy of vitamin D‐deficiency osteomalacia is to alleviate symptoms, promote fracture healing, restore bone strength, and improve quality of life while correcting biochemical abnormalities. There is a need for better understanding of the epidemiology of osteomalacia. Simplified tools validated by concurrent bone histology should be developed to help clinicians promptly diagnose osteomalacia. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Conductive Hearing Loss in the Hyp Mouse Model of X-Linked Hypophosphatemia Is Accompanied by Hypomineralization of the Auditory Ossicles.

X-linked hypophosphatemia (XLH) is a hereditary musculoskeletal disorder caused by loss-of-function mutations in the PHEX gene. In XLH, increased circulating fibroblast growth factor 23 (FGF23) levels cause renal phosphate wasting and low concentrations of 1,25-dihydroxyvitamin D, leading to an early clinical manifestation of rickets. Importantly, hearing loss is commonly observed in XLH patients. We present here data from two XLH patients with marked conductive hearing loss. To decipher the underlying pathophysiology of hearing loss in XLH, we utilized the Hyp mouse model of XLH and measured auditory brain stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) to functionally assess hearing. As evidenced by the increased ABR/DPOAE threshold shifts in the mid-frequency range, these measurements indicated a predominantly conductive hearing loss in Hyp mice compared to wild-type (WT) mice. Therefore, we carried out an in-depth histomorphometric and scanning electron microscopic analysis of the auditory ossicles. Quantitative backscattered electron imaging (qBEI) indicated a severe hypomineralization of the ossicles in Hyp mice, evidenced by lower calcium content (CaMean) and higher void volume (ie, porosity) compared to WT mice. Histologically, voids correlated with unmineralized bone (ie, osteoid), and the osteoid volume per bone volume (OV/BV) was markedly higher in Hyp mice than WT mice. The density of osteocyte lacunae was lower in Hyp mice than in WT mice, whereas osteocyte lacunae were enlarged. Taken together, our findings highlight the importance of ossicular mineralization for hearing conduction and point toward the potential benefit of improving mineralization to prevent hearing loss in XLH. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Utilizing Osteocyte Derived Factors to Enhance Cell Viability and Osteogenic Matrix Deposition within IPN Hydrogels.

Many bone defects arising due to traumatic injury, disease, or surgery are unable to regenerate, requiring intervention. More than four million graft procedures are performed each year to treat these defects making bone the second most commonly transplanted tissue worldwide. However, these types of graft suffer from a limited supply, a second surgical site, donor site morbidity, and pain. Due to the unmet clinical need for new materials to promote skeletal repair, this study aimed to produce novel biomimetic materials to enhance stem/stromal cell osteogenesis and bone repair by recapitulating aspects of the biophysical and biochemical cues found within the bone microenvironment. Utilizing a collagen type I–alginate interpenetrating polymer network we fabricated a material which mirrors the mechanical and structural properties of unmineralized bone, consisting of a porous fibrous matrix with a young’s modulus of 64 kPa, both of which have been shown to enhance mesenchymal stromal/stem cell (MSC) osteogenesis. Moreover, by combining this material with biochemical paracrine factors released by statically cultured and mechanically stimulated osteocytes, we further mirrored the biochemical environment of the bone niche, enhancing stromal/stem cell viability, differentiation, and matrix deposition. Therefore, this biomimetic material represents a novel approach to promote skeletal repair.

In Situ Endoscopic Analysis of Vascular Supply and Regenerated Alveolar Bone in β-TCP Grafted and Ungrafted Postextraction Sites before Implant Placement: A Prospective Case Control Study.

Background Endoscopy has seen a significant development over recent years in various medical fields with its application expanding from the support of minimal invasive surgery to in situ imaging. In this context, the application of endoscopic techniques to assess the quality of the regenerated bone in situ in the drill hole before implant placement is an appealing approach. Aim The aim of this study was to use short distance support immersion endoscopy (SD-SIE) to compare the quality of regenerated bone in healed postextraction sites, which are grafted with an in situ hardening β-TCP, against ungrafted sites, before implant placement. This assessment was based on microscopic bone analysis in combination with the blood vessel count. Method 13 spontaneously healed and 13 grafted postextraction sites in 3 men and 6 women, aged 26–83 years, were evaluated using SD-SIE after 4–6 months. SD-SIE was applied in drill holes before implant placement, and videos were taken from representative central buccal areas. The video recordings were analyzed using Image J software for (1) number of blood vessels per area (NBV), (2) relative area of vessels (VA), (3) relative area of mineralized bone (MBA), (4) relative area of unmineralized bone (UMBA), and (5) relative area of bone substitute (BSA). Results The grafted sites showed more (1) NBV as well as (2) VA (8.6 ± 1.1; 2.03 ± 0.28%) than the ungrafted sites (2.5 ± 0.6; 1.18 ± 0.36%) (independent t-test; p < 0.05); (3) MBA and (4) UMBA were similar to those in the grafted sites (86.3 ± 2.2 %; 13.7 ± 2.2 %) and to the ungrafted sites (89.5 ± 3.7%; 10.5 ± 3.6%) (independent t-test; p > 0.05); and (5) BSA in the grafted sites was 18.2 ± 5.4%. Conclusion SD-SIE is an interesting new approach for in situ assessment of bone quality and blood supply before implant placement. The regenerated bone in β-TCP grafted extraction sockets showed an increased vascularization compared to ungrafted sites providing a vital support for subsequent implant placement.

Abstract LB-260: Development and validation of propeptide therapeutics for treating bone metastatic prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in men in the United States. Most prostate cancer related fatalities are not due to tumors at origination sites, but rather due to prostate cancer metastasis throughout the body. Although the relative 5-year survival rate for localized prostate cancer nears 100%, unfortunately, upon metastasis the 5-year survival rate drops to 29.8%. Bone metastases disrupt tissue homeostasis and weaken the skeleton, resulting in an increased risk of severe bone pain and fracture. This is due to increased formation of immature unmineralized bone by osteoblasts, and increased osteolysis by osteoclasts, which induce high levels of bone resorption. Thus, a primary therapeutic objective for malignant bone disease is to eliminate tumors while promoting bone homeostasis. Current therapies are very limited and include palliative radiotherapy, and biphosphate or anti-RANK treatments, which target osteoclasts and decrease the rate of bone resorption. However, these treatments only delay, but do not prevent, skeletal related injuries. Our laboratory has recently developed a strategy aimed at disrupting malignant cytokines signals using inhibitory peptides targeting IL-6 receptors. I here present a novel strategy to express these peptides in vivo following gene delivery as targeted and multifunctional “propeptides.” These propeptides target bone metastases via EGFR antagonist (tumor-homing peptide) and contain dual therapeutic domains consistent of IL-6Ra antagonistic peptides and a pro-osteogenesis peptide, osteostatin. The propeptides are activated by metalloproteases (MMP2/9), which are overexpressed in metastatic prostate tumors and associated bone lesions. We can detect expression of the propeptides following C2C12 muscle cell transfection via real time quantitative PCR and are currently optimizing biochemical strategies for detecting propeptide products via western blot. We hypothesize that optimized secretion and targeting of the therapeutic propeptides will be effective in eliminating prostate tumor metastases, and restoring bone homeostasis. Future studies will examine the expression of propeptide in vivo following gene delivery to muscle and efficacy of propeptides in treating bone metastatic prostate tumors in a TRAMP-C2Ras tibial implantation model. Citation Format: Janelle W. Salameh, Charles S. Umbaugh, Marxa L. Figueiredo. Development and validation of propeptide therapeutics for treating bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-260.

Static osteogenesis does not precede dynamic osteogenesis in periosteal ossification of Pleurodeles (Caudata, Amphibia) and Pogona (Squamata, Lepidosauria).

Two successive mechanisms have been described in perichondral ossification: (1) in static osteogenesis, mesenchymal cells differentiate into stationary osteoblasts oriented randomly, which differentiate into osteocytes in the same site; (2) in dynamic osteogenesis, mesenchymal cells differentiate into osteoblasts that are all oriented in the same direction and move back as they secrete collagen fibers. This study is aimed at testing the hypothesis that the ontogenetic sequence static then dynamic osteogenesis observed in the chicken and in the rabbit is homologous and was acquired by the last common ancestor of amniotes or at a more inclusive node. For this we analyze the developmental patterns of Pleurodeles (Caudata, Amphibia) and those of the lizard Pogona (Squamata, Lepidosauria). We processed Pleurodeles larvae and Pogona embryos, prepared thin and ultrathin sections of appendicular bones, and analyzed them using light and transmission electron microscopy. We show that static osteogenesis does not precede dynamic osteogenesis in periosteal ossification of Pleurodeles and Pogona. Therefore, the null hypothesis is rejected and according to the parsimony method the ontogenetic sequence observed in the chicken and in the rabbit are convergent. In Pleurodeles and Pogona dynamic osteogenesis occur without a previous rigid mineralized framework, whereas in the chicken and in the rabbit dynamic osteogenesis seems to take place over a mineralized support whether bone (in perichondral ossification) or calcified cartilage (in endochondral ossification). Interestingly, in typical dynamic osteogenesis, osteoblasts show an axis (basal nucleus-distal endoplasmic reticulum) perpendicular to the front of secreted unmineralized bone matrix, whereas in Pleurodeles and Pogona this axis is parallel to the bone matrix.

Hypophosphatasia: Rare but there

ABSTRACTHypophosphatasia (HP) is an inherited disorder characterized by defective bone and teeth mineralization and deficiency of serum and bone alkaline phosphatase (AP) activity. For severe forms, disease frequency has been estimated at 1/100,000. Symptoms of the disease are highly variable in their clinical expression ranging from stillbirth with unmineralized bone to early loss of tooth without any bone symptoms. The transmission of severe form is autosomal recessive while milder forms get transmitted as dominant or recessive autosomal traits. The diagnosis can be done by performing serum AP assay and molecular analysis of the tissue-nonspecific alkaline phosphatase (TNSALP) gene.How to cite this articleJhingala VK, Tangade P, Punia H, Gupta V, Singh V, Kalra M, Chaudhary S. Hypophosphatasia: Rare but there. J Contemp Dent 2016;6(3):184-188.

A Mouse Model for Human Osteogenesis Imperfecta Type VI

Osteogenesis imperfecta type VI (OI type VI) has recently be linked to a mutation in the SERPINF1 gene, which encodes pigment epithelium-derived factor (PEDF), a ubiquitously expressed protein originally described for its neurotrophic and antiangiogenic properties. In this study, we characterized the skeletal phenotype of a mouse with targeted disruption of Pedf. In normal mouse bone, Pedf was localized to osteoblasts and osteocytes. Micro-computed tomography (µCT) and quantitative bone histomorphometry in femurs of mature Pedf null mutants revealed reduced trabecular bone volume and the accumulation of unmineralized bone matrix. Fourier transform infrared microscopy (FTIR) indicated an increased mineral:matrix ratio in mutant bones, which were more brittle than controls. In vitro, osteoblasts from Pedf null mice exhibited enhanced mineral deposition as assessed by Alizarin Red staining and an increased mineral:matrix determined by FTIR analysis of calcified nodules. The findings in this mouse model mimic the principal structural and biochemical features of bone observed in humans with OI type VI and consequently provide a useful model with which to further investigate the role of PEDF in this bone disorder.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 ( Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice ( Trpv6 −/− ) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6 −/− mice receiving a normal (∼ 1%) or low (∼ 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6 +/+ and Trpv6 −/− mice (− 35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6 −/− mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

Rapid and consistent porcine model for chronic total occlusions in the peripheral vasculature

Development and testing of treatment options for chronic total occlusions (CTO) still remains one of the last frontiers of vascular interventions. The lack of a consistent CTO animal model is one of the hurdles precluding efficient treatment advances in this field.We describe here a porcine model of a calcified CTO achieved by an intravascular delivery of de‐mineralized bone matrix (DBM) products in the femoral arteries. DBM is essentially de‐mineralized allograft bone particles with osteoinductive properties. In this study, the DBM material was delivered to each vessel via a guide catheter based approach. At 28 days, each treated vessel was evaluated angiographically, the animals were sacrificed and the treated vessels segments were assessed histopathologically. CTO was detected in 73% of the treated vessels. Morphologically, the CTO lesions were characterized by a mixed tissue growth composed of well vascularized collagenous matrix, mineralized and un‐mineralized bone fragments and variable mononuclear inflammation. This tissue reaction appeared well adhered to the vascular tunica media. Vascular channels lined by endothelium were present in vessels with incomplete chronic occlusion.This animal model can provide a platform for 1) development and testing of new CTO crossing devices, 2) evaluation of stents in calcified vessels 3) identification and evaluation of pharmacological entities in context of calcification and 4) development and evaluation of decalcification treatments.

Distinctive anabolic roles of 1,25-dihydroxyvitamin D3 and parathyroid hormone in teeth and mandible versus long bones

To assess the roles of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) and parathyroid hormone (PTH) in hard tissue formation in oro-facial tissues, we examined the effect of either 1,25(OH)(2)D or PTH deficiency on dentin and dental alveolar bone formation and mineralization in the mandibles, and osteoblastic bone formation in long bones of 1alpha-hydroxylase knockout (1alpha(OH)ase(-/-)) mice. Compared with wild-type mice, the mineral density was decreased in the teeth and mandibles, and unmineralized dentin (predentin and biglycan immunopositive dentin) and unmineralized bone matrix in the dental alveolar bone were increased in 1alpha(OH)ase(-/-) mice. The dental volume, reparative dentin volume, and dentin sialoprotein immunopositive areas were reduced in 1alpha(OH)ase(-/-) mice. The cortical thickness, dental alveolar bone volume, and osteoblast number were all decreased significantly in the mandibles; in contrast, the osteoblast number and surface were increased in the trabecular bone of the tibiae in 1alpha(OH)ase(-/-) mice consistent with their secondary hyperparathyroidism. The expression of PTH receptor and IGF1 was reduced slightly in mandibles, but enhanced significantly in the long bones in the 1alpha(OH)ase(-/-) mice. To control for the role of secondary hyperparathyroidism, we also examined teeth and mandibles in 6-week-old PTH(-/-) mice. In these animals, dental and bone volumes in mandibles were not altered when compared with their wild-type littermates. These results suggest that 1,25(OH)(2)D(3) plays an anabolic role in both dentin and dental alveolar bone as it does in long bones, whereas PTH acts predominantly in long bones rather than mandibular bone.

Metabolic bone disease in HIV infection

HIV mainly replicates in CD4þ T lymphocytes andmonocyte/macrophages causing severe immunologicalimpairment. In addition to the immune system, HIVinfection affects tissues and organs such as kidney, liver,the central nervous system, heart and bone showing acomplex pathogenesis [1].The advent and widespread use of highly activeantiretroviral therapy (HAART) in the last two decadeshasled toa markedimprovementinthe treatmentofHIVdisease even though viral infection cannot be eradicatedbecause HAART does not completely eliminate the viralreservoirs [2]. HAART has dramatically changed thecourseofHIVinfection froma fatalinfectiontoachronicand relatively manageable disease. The increased lifeexpectancyofHIVpatientsandtheeffectsofHAARThavechanged the management of HIV infection. Nowadaysmedical treatment is no longer focused solely on HIVinfection, opportunistic diseases and monitoring immunederangement, but also includes the control of metabolic,cardiovascular, liver, bone and kidney complications. Inparticular, bone alterations have been observed in thecourse of HIV disease representing a pivotal clinicalprobleminthemanagementofHIVpatientsespeciallyforapossible development of bone fractures [3]. The majorbonelesionsdetectableinHIVpatientsarerelatedtobonedemineralization (osteopenia/osteoporosis and osteoma-lacia) and osteonecrosis ([4] for a review).This report will discuss the pathogenesis, diagnosis andtreatment of major bone complications represented bybone demineralization diseases during HIV infection andHAART treatment.

Mineralization and Bone Resorption Are Regulated by the Androgen Receptor in Male Mice

Androgens play a key role in skeletal growth and bone maintenance; however, their mechanism of action remains unclear. To address this, we selectively deleted the androgen receptor (AR) in terminally differentiated, mineralizing osteoblasts using the Cre/loxP system in mice (osteocalcin-Cre AR knockouts [mOBL-ARKOs]). Male mOBL-ARKOs had decreased femoral trabecular bone volume compared with littermate controls because of a reduction in trabecular number at 6, 12, and 24 wk of age, indicative of increased bone resorption. The effects of AR inactivation in mineralizing osteoblasts was most marked in the young mutant mice at 6 wk of age when rates of bone turnover are high, with a 35% reduction in trabecular bone volume, decreased cortical thickness, and abnormalities in the mineralization of bone matrix, characterized by increased unmineralized bone matrix and a decrease in the amount of mineralizing surface. This impairment in bone architecture in the mOBL-ARKOs persisted throughout adulthood despite an unexpected compensatory increase in osteoblast activity. Our findings show that androgens act through the AR in mineralizing osteoblasts to maintain bone by regulating bone resorption and the coordination of bone matrix synthesis and mineralization, and that this action is most important during times of bone accrual and high rates of bone remodeling.