Osteoid Volume Research Articles

In-depth clinical characterization of intravenous iron infusion-induced hypophosphatemic osteomalacia and its resolution

Abstract Iron deficiency anemia is treated by iron supplementation. Increasing evidence has shown that the carbohydrate components in iron infusions can cause hypophosphatemia and subsequent osteomalacia due to excess iFGF23 (intact Fibroblast Growth Factor 23). We here undertook an in-depth characterization of 13 patients with iron infusion-induced osteomalacia (IIIO). Patients were characterized (monocentric institutional practice) by means of laboratory, bone density, HR-pQCT and virtual osteoid volume estimation. We additionally report a patient who was treated with burosumab because iron infusions had to be continued despite osteomalacia. All 13 patients received ferric carboxymaltose (FCM) infusions and presented with low phosphate levels in the laboratory. Stopping the FCM infusions and supportive treatment by substitution of phosphate, calcium, native and/or active vitamin D was the chosen therapeutic approach. Pain, mobility, biochemical data, such as serum phosphate levels, bone mineral density, bone microstructure and virtual osteoid volume were the main outcome measures. Our results indicate biochemical normalization (e. g. phosphate levels pre: 0.50 mmol/l ± 0.23 mmol/l, post: 0.93 mmol/l ± 0.32 mmol/l, P<.001) after stopping the FCM infusion and establishing supportive treatment. Additionally, pain levels on the visual analogue scale (VAS) decreased (VASpre 7.31 ± 1.22, VASpost 2.73 ± 1.27, P<.0001) and areal bone mineral density (expressed as T-score) improved significantly (T-scorepre: -1.85 ± 1.84, T-scorepost: -0.91 ± 2.13, P<.05). One patient requiring ongoing FCM infusions experienced significant additional benefits from burosumab treatment. In conclusion, our results highlight the importance of monitoring phosphate in patients treated with FCM infusions. Stopping FCM infusions is effective in addressing the excess of iFGF23 and thereby phosphate wasting. Supportive therapy enables quick recovery of the musculoskeletal system and improves pain levels in these patients.

Evaluation of Potential Roles of Zinc Finger Homeobox 3 (Zfhx3) Expressed in Chondrocytes and Osteoblasts on Skeletal Growth in Mice.

Bone formation is tightly modulated by genetically encoded molecular proteins that interact to regulate cellular differentiation and secretion of bony matrix. Many transcription factors are known to coordinate these events by controlling gene transcription within networks. However, not all factors involved are known. Here, we identified a novel function forZinc Finger Homeobox 3(Zfhx3), a gene encoding a transcription factor, as a regulator of bone metabolism. We knocked outZfhx3conditionally in mice in either chondrocytes or osteoblasts and characterized their bones by micro-CT in 12-week-old mice. We observed a negative effect in linear bone growth in both knockout mice but reduced bone mass only in mice withZfhx3deleted in osteoblasts. Loss of Zfhx3 expression in osteoblasts affected trabecular bone mass in femurs and vertebrae in both sexes but influenced cortical bone volume fraction only in females. Moreover, transcriptional analysis of femoral bones in osteoblastZfhx3conditional knockout mice revealed a reduced expression of osteoblast genes, and histological evaluation of trabecular bones suggests that Zfhx3 causes changes in bone formation and not resorption. The loss of Zfhx3 causes reductions in trabecular bone area and osteoid volume, but no changes in the expression of osteoclast differentiation markers or number of TRAP stained osteoclasts. These studies introduce Zfhx3 as a relevant factor toward understanding gene regulatory networks that control bone formation and development of peak bone mass.

Biopsies from patients with sacral insufficiency fracture are characterized by low bone matrix mineralization and high turnover.

Sacral insufficiency fractures are known to occur primarily in older women without adequate trauma. While an association with low bone mineral density (ie, osteoporosis) has been reported, more detailed information on local bone quality properties in affected patients is not available. In the present study, core biopsies were obtained from the S1 sacral ala in patients with a bilateral sacral insufficiency fracture (type IV according to the fragility fractures of the pelvis classification) who required surgical stabilization. Dual energy X-ray absorptiometry (DXA) and laboratory bone metabolism analyses were performed. For comparison, control biopsies were acquired from skeletally intact age- and sex-matched donors during autopsy. A total of 31 biopsies (fracture: n = 19; control: n = 12) were evaluated by micro-computed tomography, histomorphometry on undecalcified sections, and quantitative backscattered electron imaging (qBEI). DXA measurements showed mean T-scores in the range of osteoporosis in the fracture cohort (T-scoremin -2.6 ± 0.8). Biochemical analysis of bone metabolism parameters revealed high serum alkaline phosphatase and urinary deoxypyridinoline/creatinine levels. In the biopsies, a loss of trabecular microstructure along with increased osteoid values were detected in the fracture patients compared with controls (osteoid volume per bone volume 5.9 ± 3.5 vs. 0.9 ± 0.5%, p <.001). We also found evidence of microfractures with chronic healing processes (ie, microcallus) as well as pronounced hypomineralization in the biopsies of the fracture cohort compared with the controls as evidenced by lower CaMean measured by qBEI (22.5 ± 1.6 vs. 24.2 ± 0.5wt%, p =.003). In conclusion, this high-resolution biopsy study provides evidence of local hypomineralization in patients with sacral insufficiency fractures, pointing to reduced fracture resistance but also a distinct phenotype other than the predominant loss of trabeculae as in postmenopausal osteoporosis. Our data highlight the importance of therapies that promote bone mineralization to optimally treat and prevent sacral insufficiency fractures.

#1552 The role of osteocytes in bone remodeling: comparison between biopsies from patients with osteitis fibrosa and mixed uremic osteodystrophy disease

Abstract Background and Aims Osteocytes represent about 95% of bone cells. They are located in mineralized bone tissue and connect with each other, with osteoblasts, osteoclasts, blood vessels and bone marrow cells. These cells express several proteins that regulate phosphorus-calcium metabolism and bone remodeling. Disorders of mineral metabolism resulting from chronic kidney disease – mineral bone disorder (CKD-MBD) alter tissue function leading to histological changes known as renal osteodystrophy (ROD). ROD is classified: in high turnover – osteitis fibrosa (OF) and mixed uremic osteodystrophy (MUO) and low turnover – adynamic bone disease and osteomalacia. The participation of osteocytes proteins in ROD pathogenesis is not fully explained. The aim of this study was to evaluate the expression of osteocytic proteins in bone biopsies from patients with OF and MUO and analyze clinical, biochemical and bone histomorphometric results. Method We evaluated patients with CKD on hemodialysis, who underwent bone biopsy to elucidate the diagnosis of ROD. Static and dynamic parameters by histomorphometry, including quantification of osteocytes, and the expression of proteins by immunohistochemistry (Sclerostin; Nuclear factor Kappa B ligand – RANKL; Osteoprotegerin – OPG; Dickkopf-related protein 1 - DKK1; Fibroblast growth factor 23 - FGF23; Dentin matrix protein 1 - DMP1; Matrix extracellular phosphoglycoprotein – MEPE; Phosphate regulatory gene with homologies to endopeptidases on the X chromosome – PHEX; podoplanin - E11; and CD44). Results We included 47 patients (24 with OF and 23 with MUO). Table 1 describes the results from the comparison between both groups. Patients with MUO were older and had lower serum P levels compared to patients with OF. Histomorphometric results showed that the structural parameter trabecular number was greater in patients with OF. The formation parameters, as osteoid volume, thickness and surface were greater in patients with MUO. Regarding mineralization, these patients also have lower bone formation rate, and mineralizing surface and a longer mineralization lag time. The expression of osteocytic proteins in both groups showed that DMP-1 was approximately 50% lower, near to statistical significance (p = 0.07) and RANKL was 23% lower in patients with MUO. In Table 2 are described the association found between proteins and histomorphometric parameters. The majority of the proteins involved in bone remodeling are negatively associated with histomorphometric parameters. Conclusion The clinical symptoms of CKD-BMD in patients with OF and MD are similar, and, except for serum P levels, other biochemical parameters did not contribute to differentiate Of and MUO. Bone biopsy, despite being an invasive procedure, revealed differences between the two pathologies, which could better guide clinical treatment, such as, vitamin D supplementation and VD analogs in patients with MUO who present an increase in osteoid parameters, which could improve bone mineralization. Regarding the expression of proteins in osteocytes, we found few differences between the two groups. However, several associations were observed between the expression of these proteins and structural, formation, reabsorption and mineralization parameters in high turnover bone disease.

Histomorphometric analysis of patients with femoral neck fracture and 25-hydroxyvitamin D deficiency: a cross-sectional study.

Vitamin D deficiency causes osteoporosis, bone mineralization disorders, and osteomalacia. Osteomalacia is diagnosed using blood biochemical tests, clinical symptoms, and imaging; however, accurate detection of mineralization disorders requires tissue observation. We investigated the prevalence of bone mineralization disorders and their relationship with serum 25-hydroxyvitamin D (25OHD) levels in patients with untreated osteoporosis with femoral neck fractures. A non-demineralized specimen was prepared from the femoral head removed during surgery in 65 patients. Bone histomorphometry of cancerous bone in the femoral head center was conducted. Osteoid volume per bone volume (OV/BV) and osteoid thickness (O.Th) were measured as indicators of mineralization disorder. The mean serum 25OHD level (11.9 ± 5.7ng/mL) was in the deficiency range (< 12ng/mL). There were no clinically diagnosed cases of osteomalacia (OV/BV > 10% and O.Th > 12.5µm); however, one case of mineralization disorder, considered histologically pre-osteomalacia (OV/BV > 5% and O.Th < 12.5µm), was observed (OB/BV, 17.6%; O.Th, 12.3µm). Excluding this case, those with severe (25OHD < 12ng/mL, at risk of osteomalacia; n = 39) and non-severe deficiency (25OHD ≥ 12ng/mL; n = 25) did not significantly differ in OV/BV (%; 0.77 ± 0.54 vs. 0.69 ± 0.38, p = 0.484) or O.Th (µm; 5.32 ± 1.04 vs. 5.13 ± 0.78, p = 0.410). Further, 25OHD and OV/BV were not significantly correlated (R = -0.124, p = 0.327). This is the first study in the twenty-first century to examine serum 25OHD concentrations and bone mineralization disorders in Japanese patients with osteoporosis. The results indicate that vitamin D deficiency does not necessarily cause bone mineralization disorders and rarely leads to osteomalacia.

Microfragmented Fat and Biphasic Calcium Phosphates for Alveolar Cleft Repair: Protocol for a Prospective, Nonblinded, First-in-Human Clinical Study.

Biphasic calcium phosphates (BCP) may serve as off-the-shelf alternatives for iliac crest-derived autologous bone in alveolar cleft reconstructions. To add osteoinductivity to the osteoconductive BCPs to achieve similar regenerative capacity as autologous bone, a locally harvested buccal fat pad will be mechanically fractionated to generate microfragmented fat (MFAT), which has been shown to have high regenerative capacity due to high pericyte and mesenchymal stem cell content and a preserved perivascular niche. Our primary objectives will be to assess the feasibility and safety of the BCP-MFAT combination. The secondary objective will be efficacy, which will be evaluated using radiographic imaging and histological and histomorphometric evaluation of biopsies taken 6 months postoperatively, concomitant with dental implant placement. Eight patients with alveolar cleft (≥15 years) will be included in this prospective, nonblinded, first-in-human clinical study. MFAT will be prepared intraoperatively from the patient's own buccal fat pad. Regular blood tests and physical examinations will be conducted, and any adverse events (AEs) or serious EAs (SAEs) will be meticulously recorded. Radiographic imaging will be performed prior to surgery and at regular intervals after reconstruction of the alveolar cleft with the BCP-MFAT combination. Biopsies obtained after 6 months with a trephine drill used to prepare the implantation site will be assessed with histological and histomorphometric analyses after methylmethacrylate embedding and sectioning. The primary outcome parameter will be safety after 6 months' follow-up, as monitored closely using possible occurrences of SAEs based on radiographic imaging, blood tests, and physical examinations. For efficacy, radiographic imaging will be used for clinical grading of the bone construct using the Bergland scale. In addition, bone parameters such as bone volume, osteoid volume, graft volume, and number of osteoclasts will be histomorphometrically quantified. Recruitment started in November 2019, and the trial is currently in the follow-up stage. This protocol's current version is 1.0, dated September 15, 2019. In this first-in-human study, not only safety but also the histologically and radiographically assessed regenerative potential of the BCP-MFAT combination will be evaluated in an alveolar cleft model. When an SAE occurs, it will be concluded that the BCP-MFAT combination is not yet safe in the current setting. Regarding AEs, if they do not occur at a higher frequency than that in patients treated with standard care (autologous bone) or can be resolved by noninvasive conventional methods (eg, with analgesics or antibiotics), the BCP-MFAT combination will be considered safe. In all other cases, the BCP-MFAT combination will not yet be considered safe. Indonesia Clinical Trial Registry INA-EW74C1N; https://tinyurl.com/28tnrr64. DERR1-10.2196/42371.

Approaching virtual osteoid volume estimation and in-depth tissue characterization in patients with tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) poses a significant diagnostic challenge, leading to increased disease duration and patient burden also by missing clinical suspicion. Today, diagnosis of osteomalacia relies on invasive iliac crest biopsy, if needed. Therefore, a noninvasive method would be beneficial for patients with severe osteomalacia, such as TIO, to inform their clinical management and address specific needs, like estimating the regeneration capacity at high osteoid volumes (OVs) or the potential of a hungry bone syndrome after tumor removal. Furthermore, given the lack of comprehensive histological characterization of TIO, there is a need for additional tissue characterization. Therefore, our assessment encompassed iliac crest biopsies that were examined using quantitative electron backscattered microscopy, Raman spectroscopy, micro-computed tomography, and histology to analyze the biopsy tissue. Our clinical assessment encompassed DXA and high-resolution peripheral quantitative computed tomography (HR-pQCT) alongside with biochemical analyses and clinical evaluations. Combining imaging and clinical data, we established a model to predict the OV. We compared 9 TIO patients with 10 osteoporosis (OPO) patients and 10 healthy controls. Histological analyses confirmed a pronounced OV in TIO patients (OPO: 1.20% ± 1.23% vs TIO: 23.55% ± 12.23%, P < .0005), and spectroscopy revealed lower phosphate levels in TIO biopsies. By combining HR-pQCT and laboratory diagnostics, we developed a linear regression model to noninvasively predict the OV revealing significantly higher modeled OV/BVmodel values of 24.46% ± 14.22% for TIO compared to the control group (5.952% ± 3.44%, P ≤ .001). By combining laboratory diagnostics, namely, ALP and Tt.BMDRadius measured by HR-pQCT, we achieved the calculation of the virtual osteoid volume to bone volume ratio (OV/BVmodel) with a significant correlation to histology as well as reliable identification of TIO patients compared to OPO and control. This novel approach is potentially helpful for predicting OV by noninvasive techniques in diagnostic procedures and improving the clinical management of TIO.

Genetic Analysis, Phenotypic Spectrum and Functional Study of Rare Osteogenesis Imperfecta Caused by CRTAP Variants.

Deficiency of cartilage-associated protein (CRTAP) can cause extremely rare autosomal recessive osteogenesis imperfecta (OI) type VII. We investigated the pathogenic mechanisms of CRTAP variants through functional studies on bones of patients with OI. Two nonconsanguineous families with CRTAP mutations were included and their phenotypes and genotypes were evaluated. Bone specimens were obtained from 1 patient with OI and a normal control during orthopedic surgery. The impacts of the novel variant on the CRTAP transcript were confirmed. The expression levels of CRTAP mRNA and CRTAP protein were analyzed. The quantification of prolyl 3-hydroxylation in the α1 chain of type I collagen was evaluated. Patients with OI type VII had early-onset recurrent fractures, severe osteoporosis, and bone deformities. The c.621 + 1G > A and c.1153-3C > G mutations were identified in CRTAP in the patients with OI. The c.621 + 1G > A variant was a novel mutation that could impair mRNA transcription, leading to a truncated CRTAP protein. In a patient with c.621 + 1G > A and c.1153-3C > G mutations in CRTAP, the mRNA and protein levels of CRTAP in osteoblasts were significantly decreased and the osteoid volume and osteoblast numbers were markedly reduced compared with those in the normal control individual. This was simultaneously accompanied by significantly reduced prolyl 3-hydroxylation at Pro986 in the α1 chain of type I collagen and invisible active bone formation in bone. The novel c.621 + 1G > A mutation in CRTAP expands the genotypic spectrum of type VII OI. Biallelic mutations of c.621 + 1G > A and c.1153-3C > G in CRTAP can lead to reduced CRTAP mRNA and deficient CRTAP protein in osteoblasts, which reduces 3-hydroxylation in Pro986 of the α1 chain of type I collagen and impairs bone formation, thus contributing to severe OI type VII.

Bone Structure and Turnover in Postmenopausal Women With Long-Standing Type 1 Diabetes.

Compromised bone structural and mechanical properties are implicated in the increased fracture risk in type 1 diabetes (T1D). We investigated bone structure and turnover by histomorphometry in postmenopausal women with T1D and controls without diabetes using tetracycline double-labeled transiliac bone biopsy. After in vivo tetracycline double labeling, postmenopausal women with T1D of at least 10 years and without diabetes underwent transiliac bone biopsy. An expert blinded to the study group performed histomorphometry. Static and dynamic histomorphometry measurements were performed and compared between the two groups. The analysis included 9 postmenopausal women with T1D (mean age 58.4 ± 7.1 years with 37.9 ± 10.9 years of diabetes and HbA1c 7.1% ± 0.4%) and 7 postmenopausal women without diabetes (mean age 60.9 ± 3.3 years and HbA1c 5.4% ± 0.2%). There were no significant differences in serum PTH (38.6 ± 8.1 versus 51.9 ± 23.9 pg/mL), CTX (0.4 ± 0.2 versus 0.51 ± 0.34 ng/mL), or P1NP (64.5 ± 26.2 versus 87.3 ± 45.3 ng/mL). Serum 25-hydroxyvitamin D levels were higher in T1D than in controls (53.1 ± 20.8 versus 30.9 ± 8.2 ng/mL, p < 0.05). Bone structure metrics (bone volume, trabecular thickness, trabecular number, and cortical thickness) were similar between the groups. Indices of bone formation (osteoid volume, osteoid surface, and bone formation rate) were 40% lower in T1D and associated with lower activation frequency. However, the differences in bone formation were not statistically significant. Long-standing T1D may affect bone turnover, mainly bone formation, without significantly affecting bone structure. Further research is needed to understand bone turnover and factors affecting bone turnover in people with T1D. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Osteomalacia Prevalence, Biochemical Profile, and Histology in Patients with Low-Energy Hip Fractures Over the Age of 45.

Our objective was to determine the prevalence of osteomalacia in low-energy hip fracture patients over the age of 45, based on biochemical and histological measures. This cross-sectional study included 72 patients over 45 with low-energy mechanism hip fractures. Samples of fasting venous blood were taken for hemograms and serum biochemistry analyses. Bicortical biopsies of the iliac crest were obtained, processed, and evaluated by an expert pathologist for osteomalacia. Biochemical osteomalacia (b-OM) is defined according to a distinct criterion. A low level of serum calcium, phosphorus, albumin, and 25OHD was found in 43.1, 16.7, 73.6, and 59.7% of patients, respectively. 50.0% of patients had high serum alkaline phosphatase (ALP) levels. b-OM was found in 30 (41.7%), and no significant association was found with PTH, Cr, Alb, age, sex, fracture type, side of the trauma, and season were not associated with osteomalacia. Osteomalacia was diagnosed on histopathological analysis in 19/72 (26.7%), and 54/72 (75.0%) of all cases fulfilled b-OM criteria. In the histologic evaluation, osteoid seam width, osteoid surface, and osteoid volume were 28.5µm, 25.6, and 12.1%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the biochemical test for detecting osteomalacia were 73.6, 64.2, 42.4, 87.2, and 66.7%, respectively. Up to 30% of elderly patients with low-energy hip fractures are affected by osteomalacia. A biochemical screening along with a bone biopsy and histopathologic evaluation may be logical in a high-risk population for osteomalacia diagnosis.

Blocking CCN2 Reduces Established Bone Loss Induced by Prolonged Intense Loading by Increasing Osteoblast Activity in Rats.

We have an operant model of reaching and grasping in which detrimental bone remodeling is observed rather than beneficial adaptation when rats perform a high-repetition, high-force (HRHF) task long term. Here, adult female Sprague-Dawley rats performed an intense HRHF task for 18 weeks, which we have shown induces radial trabecular bone osteopenia. One cohort was euthanized at this point (to assay the bone changes post task; HRHF-Untreated). Two other cohorts were placed on 6 weeks of rest while being simultaneously treated with either an anti-CCN2 (FG-3019, 40 mg/kg body weight, ip; twice per week; HRHF-Rest/anti-CCN2), or a control IgG (HRHF-Rest/IgG), with the purpose of determining which might improve the trabecular bone decline. Results were compared with food-restricted control rats (FRC). MicroCT analysis of distal metaphysis of radii showed decreased trabecular bone volume fraction (BV/TV) and thickness in HRHF-Untreated rats compared with FRCs; responses improved with HRHF-Rest/anti-CCN2. Rest/IgG also improved trabecular thickness but not BV/TV. Histomorphometry showed that rest with either treatment improved osteoid volume and task-induced increases in osteoclasts. Only the HRHF-Rest/anti-CCN2 treatment improved osteoblast numbers, osteoid width, mineralization, and bone formation rate compared with HRHF-Untreated rats (as well as the latter three attributes compared with HRHF-Rest/IgG rats). Serum ELISA results were in support, showing increased osteocalcin and decreased CTX-1 in HRHF-Rest/anti-CCN2 rats compared with both HRHF-Untreated and HRHF-Rest/IgG rats. These results are highly encouraging for use of anti-CCN2 for therapeutic treatment of bone loss, such as that induced by chronic overuse. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

HIGH - GRADE OSTEOBLASTIC OSTEOSARCOMA OF THE MAXILLA: DIAGNOSIS, TREATMENT, AND BUCCOMAXILLOFACIAL REHABILITATION

Osteosarcoma is one of the most common malignant bone lesions; however, it comprises only 6%-8% of all osteosarcomas. It is a lesion commonly found between the third and fourth decade of life, in addition to having a similar proportion of appearing between the mandible and maxilla. This study aims to report the case of a 64-year-old Caucasian female patient, affected by a high-grade osteoblastic osteosarcoma. The patient presented an edema in the posterior region of the maxilla, paresthesia, and nasal obstruction. Panoramic radiography and CT scans, along with anatomopathological examination, were performed. Histopathologically, an intense volume of osteoid was found. Left radial maxillectomy was the chosen treatment. The patient was first rehabilitated with a provisional palatal obturator prosthesis in order to improve her nutritional status. Her rehabilitation was planned and accomplished with a mandibular removable partial denture and a maxillary removable partial obturator denture. Osteosarcoma is one of the most common malignant bone lesions; however, it comprises only 6%-8% of all osteosarcomas. It is a lesion commonly found between the third and fourth decade of life, in addition to having a similar proportion of appearing between the mandible and maxilla. This study aims to report the case of a 64-year-old Caucasian female patient, affected by a high-grade osteoblastic osteosarcoma. The patient presented an edema in the posterior region of the maxilla, paresthesia, and nasal obstruction. Panoramic radiography and CT scans, along with anatomopathological examination, were performed. Histopathologically, an intense volume of osteoid was found. Left radial maxillectomy was the chosen treatment. The patient was first rehabilitated with a provisional palatal obturator prosthesis in order to improve her nutritional status. Her rehabilitation was planned and accomplished with a mandibular removable partial denture and a maxillary removable partial obturator denture.

Bone vitality and vascularization of mandibular and maxillary bone grafts in maxillary sinus floor elevation: A retrospective cohort study.

Mandibular retromolar (predominantly cortical) and maxillary tuberosity (predominantly cancellous) bone grafts are used in patients undergoing maxillary sinus floor elevation (MSFE) for dental implant placement. The aim of this retrospective cohort study was to investigate whether differences exist in bone formation and vascularization after grafting with either bone source in patients undergoing MSFE. Fifteen patients undergoing MSFE were treated with retromolar (n= 9) or tuberosity (n= 6) bone grafts. Biopsies were taken 4months postoperatively prior to dental implant placement, and histomorphometrically analyzed to quantify bone and osteoid area, number of total, apoptotic, and receptor activator of nuclear factor-κB ligand (RANKL)-positive osteocytes, small and large-sized blood vessels, and osteoclasts. The grafted area was divided in three regions (caudal-cranial): RI, RII, and RIII. Bone volume was 40% (RII, RIII) higher and osteoid volume 10% (RII) lower in retromolar compared to tuberosity-grafted areas. Total osteocyte number and number of RANKL-positive osteocytes were 23% (RII) and 90% (RI, RII) lower, but osteoclast number was higher (retromolar: 12, tuberosity: 0) in retromolar-grafted areas. The total number of blood vessels was 80% (RI) to 60% (RIII) lower, while the percentage of large-sized blood vessels was 86% (RI) to 25% (RIII) higher in retromolar-grafted areas. Number of osteocyte lacunae and apoptotic osteocytes were similar in both bone grafts used. Compared to the retromolar bone, tuberosity bone showed increased bone vitality and vascularization in patients undergoing MSFE, likely due to faster bone remodeling or earlier start of new bone formation. Therefore, tuberosity bone grafts might perform better in enhancing bone regeneration.

MicroRNA-101a enhances trabecular bone accrual in male mice

High-throughput microRNA sequencing was performed during differentiation of MC3T3-E1 osteoblasts to develop working hypotheses for specific microRNAs that control osteogenesis. The expression data show that miR-101a, which targets the mRNAs for the epigenetic enzyme Ezh2 and many other proteins, is highly upregulated during osteoblast differentiation and robustly expressed in mouse calvaria. Transient elevation of miR-101a suppresses Ezh2 levels, reduces tri-methylation of lysine 27 in histone 3 (H3K27me3; a heterochromatic mark catalyzed by Ezh2), and accelerates mineralization of MC3T3-E1 osteoblasts. We also examined skeletal phenotypes of an inducible miR-101a transgene under direct control of doxycycline administration. Experimental controls and mir-101a over-expressing mice were exposed to doxycycline in utero and postnatally (up to 8 weeks of age) to maximize penetrance of skeletal phenotypes. Male mice that over-express miR-101a have increased total body weight and longer femora. MicroCT analysis indicate that these mice have increased trabecular bone volume fraction, trabecular number and trabecular thickness with reduced trabecular spacing as compared to controls. Histomorphometric analysis demonstrates a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. Remarkably, while female mice also exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). Hence, miR-101a upregulation during osteoblast maturation and the concomitant reduction in Ezh2 mediated H3K27me3 levels may contribute to the enhanced trabecular bone parameters in male mice. However, the sex-specific effect of miR-101a indicates that more intricate epigenetic mechanisms mediate physiological control of bone formation and homeostasis.

Prevention of Hypomineralization In Auditory Ossicles of Vitamin D Receptor (Vdr) Deficient Mice.

Intact mineralization of the auditory ossicles - the smallest bones in the body - is essential for sound transmission in the middle ear, while ossicular hypomineralization is associated with conductive hearing loss. Here, we performed a high-resolution analysis of the ossicles in vitamin D receptor deficient mice (Vdr-/- ), which are characterized by hypocalcemia and skeletal mineralization defects, and investigated whether local hypomineralization can be prevented by feeding a calcium-rich rescue diet (Vdr-/- res ). In Vdr-/- mice fed a regular diet (Vdr-/- reg ), quantitative backscattered electron imaging (qBEI) revealed an increased void volume (porosity, p<0.0001) along with lower mean calcium content (CaMean, p=0.0008) and higher heterogeneity of mineralization (CaWidth, p=0.003) compared to WT mice. Furthermore, a higher osteoid volume per bone volume (OV/BV; p=0.0002) and a higher osteocyte lacunar area (Lc.Ar; p=0.01) were found in histomorphometric analysis in Vdr-/- reg mice. In Vdr-/- res mice, full rescue of OV/BV and Lc.Ar (both p>0.05 vs. WT) and partial rescue of porosity and CaWidth (p=0.02 and p=0.04 vs. WT) were observed. Compared with Hyp mice, a model of X-linked hypophosphatemic rickets, Vdr-/- reg mice showed a lower osteoid volume in the ossicles (p=0.0002), but similar values in the lumbar spine. These results are consistent with later postnatal impairment of mineral homeostasis in Vdr-/- mice than in Hyp mice, underscoring the importance of intact mineral homeostasis for ossicle mineralization during development. In conclusion, we revealed a distinct phenotype of hypomineralization in the auditory ossicles of Vdr-/- mice that can be partially prevented by a rescue diet. Since a positive effect of a calcium-rich diet on ossicular mineralization was demonstrated, our results open new treatment strategies for conductive hearing loss. Future studies should investigate the impact of improved ossicular mineralization on hearing function.

Effects of Primary Kidney Disease Etiology on Renal Osteodystrophy in Pediatric Dialysis Patients.

ABSTRACTCongenital diseases of the kidney and urinary tract (CAKUT) and glomerulonephritis are the main causes of chronic kidney disease (CKD) in children. Although renal osteodystrophy (ROD) and indices of mineral metabolism have been characterized in dialyzed children, the impact of primary kidney disease on ROD is unknown. We performed a cross‐sectional study of bone biopsies performed in 189 pediatric dialysis patients aged 12.6 ± 5.4 years. Patients were classified into three groups according to primary kidney disease: CAKUT (n = 82), hereditary (n = 22), or glomerular disease (n = 85). Serum concentrations of calcium, phosphate, alkaline phosphatase (ALP), parathyroid hormone (PTH), and 25(OH) vitamin D were measured at the time of biopsy. Fibroblast growth factor 23 (FGF23) levels were measured in a subset of 59 patients. Levels of calcium, phosphate, PTH, and 25(OH) vitamin D were similar across groups. CAKUT patients had higher serum ALP and lower C‐terminal FGF23 levels. Bone turnover and bone volume parameters did not differ across groups. However, osteoid volume (OV/BV), osteoid surface (OS/BS), and osteoid maturation time (OMT) were highest in the CAKUT group and lowest in the hereditary group. Multiple regression analysis revealed that calcium, phosphate, ALP, and PTH were independently associated with OV/BV and osteoid thickness (O.Th). PTH was an independent factor affecting bone formation rate. The relationship between CKD etiology and bone histomorphometric variables was abrogated after adjustment for biochemical parameters in the multivariable models. Overall, bone histology differed according to CKD etiology in the unadjusted analysis; however, this association could not be confirmed independently of biochemical parameters. Although CAKUT patients had a greater mineralization defect with elevated serum ALP levels, longitudinal studies will be needed to elucidate mediation pathways that might be involved in the complex interplay of CKD‐mineral bone disease (MBD). © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Association of marine Collagen/Biosilicate composites and photobiomodulation in the process of bone healing using an experimental model of calvarial defect

The study comparing the bone regenerative capacity in an experimental model of cranial bone defects in rats, into 3 groups: G1: bone defects irradiated with photobiomodulation; G2: Biosilicate + photobiomodulation and G3: Biosilicate and Spongin + photobiomodulation. Histocompatibility and bone responses were performed after 15 and 45 days of implantation. Histological analysis demonstrated that photobiomodulation irradiated animals presented an increased amount of newly formed over time. Histomorphometry showed higher values for bone volume for G3 and G1, higher values for osteoid volume and number of osteoblasts observed for G3 compared to G2. TGF-β immunolabelling was higher for G2. The values found for VEGF were higher for biosilicate (with or without spongin) 15 days of implantation with an increased difference being observed for G1, 45 days after surgery. In conclusion, the stimulus provided by photobiomodulation associated to the biomimetic composite increased bone formation in the cranial bone defect in rats. Consequently, these data highlight the potential of the introduction of spongin into biosilicate and irradiated with photobiomodulation to improve the biological performance for bone regeneration applications.

Effect of serum 25-hydroxyvitamin D concentrations on skeletal mineralization in black and white women

There is controversy over the adverse effect of vitamin D deficiency on bone mineralization. The purpose of this study was to determine the ethnical differences in vitamin D and bone mineralization as well as the association between vitamin D deficiency and bone mineralization defects. We examined serum 25-hydroxyvitamin D (25(OH)D) levels and transiliac bone biopsies in 92 healthy black and white women, aged 20-73 years. The major bone mineralization indices include osteoid volume per bone volume (OV/BV), osteoid surfaces per bone surface (OS/BS), osteoid thickness (O.Th) and mineralization lag time (Mlt). 25(OH)D levels were significantly lower and prevalence of 25(OH)D deficiency was significantly higher in blacks than in whites. However, none of the mineralization indices showed significant difference between the two groups. In addition, there was no significant correlation between 25(OH)D levels and mineralization indices in both black and white cohorts. Only one case had O.Th marginally greater than 12.5 µm, which is the cutoff value for identifying bone mineralization defects. OV/BV and OS/BS, but not O.Th, were significantly positively correlated with activation frequency (Ac.f). Our study indicated: (1) vitamin D deficiency is common, but bone mineralization is not impaired in black women, and (2) there are no significant correlations between serum 25(OH)D levels and bone mineralization indices, suggesting that vitamin D deficiency may not be an independent factor contributing to bone mineralization defects and osteomalacia.

MO574MINERAL AND BONE DISORDERS CHANGES IN RENAL TRANSPLANTED PATIENTS

Abstract Background and Aims Successful renal transplant restores many physiologic abnormalities, including improvement of chronic kidney disease- mineral and bone disorder (CKD-MBD) syndrome. The primary aims of this study were: analyse the changes and evolution of the 3 components of CKD-MBD pre and 1 year post renal transplantation: the mineral abnormalities, the bone disorders and the vascular calcifications; and to correlate fibroblast grow factor 23 (FGF23), klotho and sclerostin serum levels with bone histomorphometric parameters and CV disease. The secondary aims were to study the evolution of other bone related parameters and correlate those with bone biopsies data, as well as to validate Adragão vascular calcification score in a population of renal transplant patients. Method We performed a prospective cohort study of a consecutive sample of de novo single renal transplanted patients in our unit. At inclusion, demographic, clinical and transplant-related data were collected, X-ray of the pelvis and hands (for Adragão score) and echocardiographic findings were recorded. All patients were submitted to a bone biopsy and laboratorial evaluation at baseline (time 0 – T0). Patients were followed for 12 months (time 1 – T1), after which performed laboratorial evaluation, a 2nd bone biopsy, echocardiogram, X-ray of pelvis and hands, bone densitometry and non-contrast cardiac CT (Agatston score). Continuous variables are presented as medians and categorical variables as frequencies. Differences between T0 and T1 were accessed by Wilcoxon matched-pairs test and paired McNemar test. Correlations between bone histomorphometric findings and severity of vascular calcifications with demographic and laboratorial parameters were obtained with Wilcoxon rank-sum test or Kruskall Wallis test. STATA software was used and p &amp;lt; 0.05 was considered statistically significant. Results We recruited 84 patients in a 28 month-period. At the end of 12 months, 69 patients performed a 2nd evaluation. Median age 53 years, 48 men, 53 caucasian, median dialysis vintage 55 months. We observe a significant reduction on phosphorus, magnesium, PTH, calcitonin, sclerostin, bone alkaline phosphatase and FGF23. Both calcium and alpha-klotho serum levels increase, with no significant changes in vitamin D levels. 68% of the patients presented renal osteodystrophy at the 2nd bone biopsy, and we observed a significant increase in the development of low turnover bone disorder, with no major changes in volume or mineralization. Changes in alpha-klotho, bAP and sclerostin (from T0 to T1) were important determinants of changes in turnover, mineralization and volume, respectively. Despite not being statistically significant, we were able to observe an improvement in the cortical bone porosity. Vascular calcifications and echocardiographic findings weren’t different comparing to the baseline (Median Adragão score was 1 in both evaluations, and valve calcifications were present in 22% and 23% of patients, with no changes in LVMI). The median Agatston score was 48.5, being the median adjusted percentile of 82%. FGF23 and sclerostin were found to be independent risk factors for extra-osseous calcifications, as well as low bone volume, cortical porosity and osteoid volume. Adragão score and valve calcifications correlated well with the increased severity of coronary calcifications determined by Agatston score (absolute and percentile). Conclusion In conclusion, renal transplantation improves two of the three components of CKD-MBD (biochemical and bone disorders), slowing the progression of vascular calcifications. FGF23, sclerostin and bAP seemed to be key parameters in understanding the bone changes observed in post transplant period, and these hormones also interfere with extra osseous calcification severity. Adragão score seems to be a good tool to access vascular calcifications in renal transplanted patients.

Greater heterogeneity of the bone mineralisation density distribution and low bone matrix mineralisation characterise tibial subchondral bone marrow lesions in knee osteoarthritis patients.

Tibial subchondral bone marrow lesions (BMLs) identified by MRI have been recognised as potential disease predictors in knee osteoarthritis (KOA), and may associate with abnormal bone matrix mineralisation and reduced bone quality. However, these tissue-level changes of BMLs have not been extensively investigated. Thus, the aim of this study was to quantify the degree of subchondral bone matrix mineralisation (both plate and trabeculae) in relation to histomorphometric parameters of bone remodelling and osteocyte lacunae (OL) characteristics in the tibial plateau (TP) of KOA patients with and without BMLs (OA-BML and OA No-BML, respectively) in comparison to nonOA cadaveric controls (CTL). Osteochondral (cartilage-bone) tissue was sampled from the BML signal region within the medial compartment for each OA-BML TP, and from a corresponding medial region for OA No-BML and CTL TPs. The tissue samples were embedded in resin, and sections stained with Von-Kossa Haematoxylin and Eosin (H&E) for quantitation of static indices of bone remodelling. Resin blocks were then further polished, and carbon-coated for quantitative backscattered electron imaging (qBEI) to determine the bone mineralisation density distribution (BMDD), as well as OL characteristics. It was found that OA-BML contained higher osteoid volume per tissue volume (OV/TV; %) and per bone volume (OV/BV; %) in both subchondral plate and trabecular bone compared to OA No-BML and CTL. The BMDD of OA-BML in both subchondral plate and trabecular bone was shifted toward a lower degree of mineralisation. Typically, an increase in both the heterogeneity of mineralisation density (Ca Width; wt%Ca) and the percentage of lower calcium (Ca Low; % B.Ar) in trabecular bone with OA-BML versus CTL was observed. Further, unmineralised OL density (#/mm2) in subchondral plate was distinctly higher in OA-BML samples compared to CTL. The KOA patients with and without BMLs had significantly decreased density of mineralised OL (#/mm2) in trabecular bone compared to CTL. Taken together, these findings indicate that tibial BMLs in advanced KOA patients are characterised by significantly hypo-mineralised subchondral bone compared with CTL. These differences associated with evidence of increased bone remodelling in OA-BML, and may influence the mechanical properties of the subchondral bone, with implications for the overlying cartilage.