Osteoblast Population Research Articles

Suppressive Effects of Geoje Raspberry (Rubus tozawae Nakai ex J.Y. Yang) on Post-Menopausal Osteoporosis via Its Osteogenic Activity on Osteoblast Differentiation

Background: Osteoporosis is a metabolic bone disease with a high mortality rate due to non-traumatic fractures. The risk of osteoporosis is increasing globally due to an increasing aging population. Current therapies are limited to delaying disease progression. Recently, the need to discover foods with osteogenic activity for the prevention and treatment of osteoporosis has been emphasized. We focused on bone formation via osteoblast differentiation, considering bone formation and resorption during bone homeostasis. Rubus tozawae Nakai ex J. Y. Yang (RL, Geoje raspberry) is a deciduous subshrub that has been traditionally eaten for its fruit. Methods and Results: We identified the third subfraction of n-hexane fraction (RL-Hex-NF3) of RL, an endemic Korean plant with osteogenic activity, which increased bone density in ovariectomized mice, a representative animal model of osteoporosis, via the depletion of female hormones, which resulted from the increase in the osteoblast population. RL-Hex-NF3 induced osteoblast differentiation and the expression of osteogenic markers in MC3T3-E1 pre-osteoblasts. Seven compounds were identified from RL-Hex-NF3 using NMR spectroscopy. Of these, three compounds, namely, 3β-hydroxy-18α,19α-urs-20-en-28-oic acid, betulinic acid, and (1S,6R,7S)-muurola-4,10(14)-diene-15-ol, showed strong osteogenic activity. Conclusions: RL-Hex-NF3 and its compounds suppress bone loss via their osteogenic properties, suggesting that they could be a potent candidate to treat osteoporosis.

A detailed methodology for a three-dimensional, self-structuring bone model that supports the differentiation of osteoblasts towards osteocytes and the production of a complex collagen-rich mineralised matrix.

There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Effects of Hydrogen-Rich Water on Interleukin-1β, Number of Osteoclasts and Osteoblasts in Streptozotocin-induced Diabetic Rats with Orthodontic Tooth Movement

BACKGROUND: Orthodontic tooth movement (OTM) may increase the risk of treatment-related complications for some diabetes mellitus (DM) patients. Hydrogen-rich water (HRW) has been demonstrated in many studies to reduce oxidative stress and cell damage. This study aimed to examine the levels of interleukin (IL)-1β, blood glucose level, body weight, tooth displacement, and population of osteoclasts and osteoblasts in diabetic rats with OTM.METHODS: Thirty rats (Rattus novergicus) were divided into 6 groups: OTM, HRW, DM, DM+OTM, DM+HRW, and DM+OTM+HRW. DM, DM+OTM, DM+HRW, and DM+OTM+HRW groups were induced with streptozotocin (STZ) after 8 weeks of high-fat diet (HFD) and continued being fed HFD for 4 weeks. OTM, DM+OTM, and DM+OTM+HRW groups were placed on an orthodontic device for the orthodontic treatment. HRW, DM+HRW, and DM+OTM+HRW groups were administered HRW via oral gavage 3 times a day for 4 weeks. At the end of the study, all rats were euthanized and blood samples were collected for IL-1β measurement using enzyme-linked immunosorbent assay (ELISA) kits. Meanwhile, the rats’ maxilla was taken to measure tooth movement, and the number of osteoclast and osteoblast were counted.RESULTS: The highest increase of IL-1β was in the DM+OTM group (140.07±5.14 pg/mL) and the lowest was in the HRW group (92.80±2.89 pg/mL). The average number of osteoblasts were higher in tension sites, while osteoclasts were higher in pressure sites.CONCLUSION: Consumption of HRW in STZ-induced diabetic rats with OTM can reduce IL-1β levels, reduce tooth mobility, and promote bone remodeling.KEYWORDS: diabetes mellitus, hydrogen rich water, interleukin-1β, orthodontic tooth movement, osteoblast, osteoclast

Osteoprogenitor recruitment and differentiation during intracortical bone remodeling of adolescent humans.

Recruitment and proliferation of osteoprogenitors during the reversal-resorption phase, and their differentiation into mature bone-forming osteoblasts is crucial for initiation of bone formation during bone remodeling. This study investigates the osteoprogenitors' gradual recruitment, proliferation, and differentiation into bone-forming osteoblasts within intracortical remodeling events of healthy adolescent humans. The study was conducted on cortical bone specimens from 11 adolescent human controls - patients undergoing surgery due to coxa valga. The osteoprogenitor recruitment route and differentiation into osteoblasts were backtracked using immunostainings and in situ hybridizations with osteoblastic markers (CD271/NGFR, osterix/SP7, COL3A1 and COL1A1). The osteoblastic cell populations were defined based on the pore surfaces, and their proliferation index (Ki67), density and number/circumference were estimated in multiplex-immunofluorescence (Ki67, TRAcP, CD34) stained sections. During the reversal-resorption phase, osteoclasts are intermixed with (COL3A1+NFGR+) osteoblastic reversal cells, which are considered to be osteoprogenitors of (COL1A1+SP7+) bone-forming osteoblasts. Initiation of bone formation requires a critical density of these osteoprogenitors (43±9 cells/mm), which is reached though proliferation (4.4±0.5% proliferative) and even more so through recruitment of osteoprogenitors, but challenged by the ongoing expansion of the canal circumference. These osteoprogenitors most likely originate from osteoblastic bone lining cells and mainly lumen osteoprogenitors, which expand their population though proliferation (4.6±0.3%) and vascular recruitment. These lumen osteoprogenitors resemble canopy cells above trabecular remodeling sites, and like canopy cells they extend above bone-forming osteoblasts where they may rejuvenate the osteoblast population during bone formation. Initiation of bone formation during intracortical remodeling requires a critical density of osteoprogenitors on eroded surfaces, which is reached though proliferation and recruitment of local osteoprogenitors: bone lining cells and lumen osteoprogenitors.

The synergistic effects of polyphenols and intestinal microbiota on osteoporosis.

Osteoporosis is a common metabolic disease in middle-aged and elderly people. It is characterized by a reduction in bone mass, compromised bone microstructure, heightened bone fragility, and an increased susceptibility to fractures. The dynamic imbalance between osteoblast and osteoclast populations is a decisive factor in the occurrence of osteoporosis. With the increase in the elderly population in society, the incidence of osteoporosis, disability, and mortality have gradually increased. Polyphenols are a fascinating class of compounds that are found in both food and medicine and exhibit a variety of biological activities with significant health benefits. As a component of food, polyphenols not only provide color, flavor, and aroma but also act as potent antioxidants, protecting our cells from oxidative stress and reducing the risk of chronic disease. Moreover, these natural compounds exhibit anti-inflammatory properties, which aid in immune response regulation and potentially alleviate symptoms of diverse ailments. The gut microbiota can degrade polyphenols into more absorbable metabolites, thereby increasing their bioavailability. Polyphenols can also shape the gut microbiota and increase its abundance. Therefore, studying the synergistic effect between gut microbiota and polyphenols may help in the treatment and prevention of osteoporosis. By delving into how gut microbiota can enhance the bioavailability of polyphenols and how polyphenols can shape the gut microbiota and increase its abundance, this review offers valuable information and references for the treatment and prevention of osteoporosis.

The beneficial effects of simultaneous supplementation of Lactobacillus reuteri and calcium fluoride nanoparticles on ovariectomy-induced osteoporosis

BackgroundThe development of new strategies to inhibit and/or treat osteoporosis as a chronic systemic disease is one of the most crucial topics. The present study aimed to investigate the simultaneous effects of calcium fluoride nanoparticles (CaF2 NPs) and lactobacillus reuteri ATCC PTA 6475 (L. reuteri) against osteoporosis in an ovariectomized rat model (OVX).MethodsIn this study, 18 matured Wistar female rats were randomly assigned into 6 groups, including control, OVX, sham, OVX + L. reuteri, OVX + CaF2 NPs, and OVX + L. reuteri + CaF2 NPs. We used OVX rats to simulate post-menopausal osteoporosis, and the treatments were begun two weeks before OVX and continued for four weeks. All groups’ blood samples were collected, and serum biomarkers (estrogen, calcium, vitamin D3, and alkaline phosphatase (ALP)) were measured. The tibia and Femur lengths of all groups were measured. Histopathological slides of tibia, kidney, and liver tissues were analyzed using the Hematoxylin and Eosin staining method.ResultsOur results revealed that dietary supplementation of L. reuteri and CaF2 NPs in low doses for 6 weeks did not show adverse effects in kidney and liver tissues. The tibial and femoral lengths of OVX rats as well as the population of osteoblasts and osteocytes and newly generated osteoid in the tibia remarkably increased in the combination therapy group. Moreover, there was a significant increase in serum estrogen levels and a significant decrease in serum calcium and alkaline phosphatase levels in combination treatment groups compared to the OVX groups not receiving the diet.ConclusionsOur results suggest the favorable effects of the simultaneous supplementation of L. reuteri and CaF2 NP to reduce post-menopausal bone loss.Graphical abstract

Alendronate induces skeletal alterations in the chicken embryonic development model

Alendronate, a nitrogen-containing bisphosphonate, has reported long-term clinical success in the management of distinct bone-related conditions, particularly in the modulation of post-menopausal osteoporosis. Nonetheless, whether the inhibitory activity over osteoclastic cells' functionality is widely acknowledged, contradictory evidence arises from the assessment of alendronate activity over osteoblastic populations. This may be of particular relevance in situations in which bone formation exceeds bone resorption, with further emphasis on embryonic development, since alendronate can cross the placental barrier and alendronate-based therapies are being extended into women of reproductive age. Accordingly, the present study aims to assess the effects of alendronate, at distinct concentrations (1.5E−10M to 1.5E−7M) on bone tissue development, within a translational animal model – the embryonic chicken development model. Embryos, at the beginning of osteogenesis (day 7) were exposed to different alendronate concentrations for 4 days. Embryos were following characterized for skeletal development by histomorphometric analysis upon histochemical staining, microtomographic analysis, and gene expression assessment of genes related to osteoclastogenic/osteoclastic and osteoblastogenic/osteogenic differentiation, as well as to the immuno-inflammatory activation. The findings revealed that exposure to alendronate had a dose-dependent impact on skeletal growth and mineralization. This effect was evidenced by diminished bone volume and reduced bone surface parameters, with the 1.5E−7M concentration leading to a remarkable reduction of over 50%. Additionally, a decreased osteoclastogenic/osteoclastic gene expression was verified, associated with a diminished osteoblastogenic/osteogenic program – within the 30–50% range for 1.5E-7 M, supporting the diminished bone formation process. An increased inflammatory activation may contribute, at least in part, to the attained outcomes. Overall present findings suggest a negative influence of alendronate on the embryonic bone development process in a dose-dependent manner, highlighting the potential risk of alendronate use during embryonic development.

A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations

Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models. Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease.

Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.

In situ fabrication of cerium-incorporated hydroxyapatite/magnetite nanocomposite coatings with bone regeneration and osteosarcoma potential.

With the ultimate goal of providing a novel platform able to inhibit bacterial adhesion, biofilm formation, and anticancer properties, cerium-doped hydroxyapatite films enhanced with magnetite were developed via spin-coating. The unique aspect of the current study is the potential for creating cerium-doped hydroxyapatite/Fe3O4 coatings on a titanium support to enhance the functionality of bone implants. To assure an increase in the bioactivity of the titanium surface, alkali pretreatment was done before deposition of the apatite layer. Scanning electron microscopy (SEM) in conjunction with energy-dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD) analysis, and Fourier transform-infrared (FTIR) spectroscopy were used to evaluate coatings. Coatings demonstrated good efficacy against Staphylococcus aureus and Escherichia coli, with the latter showing the highest efficacy. In vitro bioactivity in simulated body fluid solution showed this material to be proficient for bone-like apatite formation on the implant surface. Electrochemical impedance spectroscopy was undertaken on intact coatings to examine the barrier properties of composites. We found that spin-coating at 4000 rpm could greatly increase the total resistance. After seeding with osteoblastic populations, Ce-HAP/Fe3O4 materials the adhesion and proliferation of cells. The heating capacity of the Ce-HAP/Fe3O4 film was optimal at 45 °C at 15 s at a frequency of 318 kHz. Osseointegration depends on many more parameters than hydroxyapatite production, so these coatings have significant potential for use in bone healing and bone-cancer therapy.

Angiotensin II Modulates Calcium/Phosphate Excretion in Experimental Model of Hypertension: Focus on Bone.

A link between hypertension and long-term bone health has been suggested. The aim of this study was to investigate the effects of chronic angiotensin II administration on urinary calcium/phosphate excretion, bone mineral density, bone remodeling and osteoblast population in a well-established experimental model of hypertension, in the absence of possible confounding factors that could affect bone metabolism. Male Sprague-Dawley rats, divided in the following groups: (a) Angiotensin II (Ang II, 200 ng/kg/min, osmotic minipumps, sub cutis, n = 8); (b) Ang II+losartan (Los, 50 mg/kg/day, per os, n = 6); (c) control group (physiological saline, sub cutis, n = 9); and (d) control+losartan (n = 6) were treated for four weeks. During the experimental period, 24-hour diuresis, urinary calcium, phosphate and sodium excretion were measured prior to the treatment, at two weeks of treatment, and at the end of the treatment. Systolic blood pressure was measured by plethysmography technique (tail cuff method). At the end of the experimental protocol, the rats were euthanized and peripheral quantitative computed tomography at the proximal metaphysis and at the diaphysis of the tibiae and quantitative bone histomorphometry on distal femora were performed. Angiotensin II-dependent hypertension is associated with increased calcium and phosphate excretion. AT1 receptor blockade prevented the increase of blood pressure and phosphate excretion but did not affect the increase of calcium excretion. These changes took place without significantly affecting bone density, bone histology or osteoblast population. In conclusion, in our experimental conditions, angiotensin II-dependent hypertension gave rise to an increased urinary excretion of calcium and phosphate without affecting bone density.

Mechanistic PK-PD model of alendronate treatment of postmenopausal osteoporosis predicts bone site-specific response.

Alendronate is the most widely used drug for postmenopausal osteoporosis (PMO). It inhibits bone resorption, affecting osteoclasts. Pharmacokinetics (PK) and pharmacodynamics (PD) of alendronate have been widely studied, but few mathematical models exist to simulate its effect. In this work, we have developed a PK model for alendronate, valid for short- and long-term treatments, and a mechanistic PK-PD model for the treatment of PMO to predict bone density gain (BDG) at the hip and lumbar spine. According to our results, at least three compartments are required in the PK model to predict the effect of alendronate in both the short and long terms. Clinical data of a 2-year treatment of alendronate, reproduced by our PK-PD model, demonstrate that bone response is site specific (hip: 7% BDG, lumbar spine: 4% BDG). We identified that this BDG is mainly due to an increase in tissue mineralization and a decrease in porosity. The difference in BDG between sites is linked to the different loading and dependence of the released alendronate on the bone-specific surface and porosity. Osteoclast population diminishes quickly within the first month of alendronate treatment. Osteoblast population lags behind but also falls due to coupling of resorption and formation. Two dosing regimens were studied (70 mg weekly and 10 mg daily), and both showed very similar BDG evolution, indicating that alendronate accumulates quickly in bone and saturates. The proposed PK-PD model could provide a valuable tool to analyze the effect of alendronate and to design patient-specific treatments, including drug combinations.

The Effect of Osteoblast Isolation Methods from Adult Rats on Osteoclastogenesis in Co-Cultures.

Co-cultures of osteoblasts and osteoclasts are on the rise because they enable a more complex study. Diseases such as osteoporosis are related to a higher age. Thus, cell isolation from adult individuals is necessary. Osteoblasts can be isolated from the rat femur by three methods: explant culture, explant culture with enzymatic pre-treatment, or enzymatic treatment. The isolation methods yield different populations of osteoblasts which, in a co-culture with peripheral blood mononuclear cells, might result in differences in osteoclastogenesis. Therefore, we examined the differences in osteogenic markers, cell proliferation, and the metabolic activity of isolated osteoblast-like cells in a growth and differentiation medium. We then evaluated the effect of the isolated populations of osteoblast-like cells on osteoclastogenesis in a subsequent co-culture by evaluating osteoclast markers, counting formed osteoclast-like cells, and analyzing their area and number of nuclei. Co-cultures were performed in the presence or absence of osteoclastogenic growth factors, M-CSF and RANKL. It was discovered that enzymatic isolation is not feasible in adult rats, but explant culture and explant culture with enzymatic pre-treatment were both successful. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation than explant culture in a growth medium. The differentiation medium reduced differences in proliferation during the culture. Some differences in metabolic activity and ALP activity were also found between the osteoblast-like cells isolated by explant culture or by explant culture with enzymatic pre-treatment, but only on some days of cultivation. According to microscopy, the presence of exogenous growth factors supporting osteoclastogenesis in co-cultures was necessary for the formation of osteoclast-like cells. In this case, the formation of a higher number of osteoclast-like cells with a larger area was observed in the co-culture with osteoblast-like cells isolated by explant culture compared to the explant culture with enzymatic pre-treatment. Apart from this observation, no differences in osteoclast markers were noted between the co-cultures with osteoblast-like cells isolated by explant culture and the explant culture with enzymatic pre-treatment. The TRAP and CA II activity was higher in the co-cultures with exogenous growth than that in the co-cultures without exogenous growth factors on day 7, but the opposite was true on day 14. To conclude, explant culture and explant culture with enzymatic pre-treatment are both suitable methods to yield osteoblast-like cells from adult rats capable of promoting osteoclastogenesis in a direct co-culture with peripheral blood mononuclear cells. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation. The explant culture yielded osteoblast-like cells which induced the formation of a higher number of osteoclast-like cells with a larger area compared to the explant culture with enzymatic pre-treatment when cultured with exogenous M-CSF and RANKL.

Nisin probiotic prevents inflammatory bone loss while promoting reparative proliferation and a healthy microbiome

Dysbiosis of the oral microbiome mediates chronic periodontal disease. Realignment of microbial dysbiosis towards health may prevent disease. Treatment with antibiotics and probiotics can modulate the microbial, immunological, and clinical landscape of periodontal disease with some success. Antibacterial peptides or bacteriocins, such as nisin, and a nisin-producing probiotic, Lactococcus lactis, have not been examined in this context, yet warrant examination because of their biomedical benefits in eradicating biofilms and pathogenic bacteria, modulating immune mechanisms, and their safety profile in humans. This study’s goal was to examine the potential for nisin and a nisin-producing probiotic to abrogate periodontal bone loss, the host inflammatory response, and changes in oral microbiome composition in a polymicrobial mouse model of periodontal disease. Nisin and a nisin-producing Lactococcus lactis probiotic significantly decreased the levels of several periodontal pathogens, alveolar bone loss, and the oral and systemic inflammatory host response. Surprisingly, nisin and/or the nisin-producing L. lactis probiotic enhanced the population of fibroblasts and osteoblasts despite the polymicrobial infection. Nisin mediated human periodontal ligament cell proliferation dose-dependently by increasing the proliferation marker, Ki-67. Nisin and probiotic treatment significantly shifted the oral microbiome towards the healthy control state; health was associated with Proteobacteria, whereas 3 retroviruses were associated with disease. Disease-associated microbial species were correlated with IL-6 levels. Nisin or nisin-producing probiotic’s ability to shift the oral microbiome towards health, mitigate periodontal destruction and the host immune response, and promote a novel proliferative phenotype in reparative connective tissue cells, addresses key aspects of the pathogenesis of periodontal disease and reveals a new biomedical application for nisin in treatment of periodontitis and reparative medicine.

Impaired vertebral development following hyperglycemic induction and recovery in Danio rerio

Diabetes mellitus is a chronic metabolic condition that can result in numerous comorbidities, including diabetes‐induced osteoporosis. Regardless of age, diabetics are at an increased risk of bone fracture as a result of decreased bone quality and mass. In the Clark lab we have previously established a glucose immersion model of hyperglycemic induction in larval zebrafish (Danio rerio). Zebrafish are a viable model to study diabetic complications as they glucoregulate similar to mammals and have robust regenerative abilities previously shown to regenerate pancreatic b‐cells, peripheral nerves, cardiac muscle, and fins. Given the negative impact diabetes has on bones, we hypothesized that hyperglycemia would negatively impact vertebral development; but given zebrafish’s ability to regenerate we also hypothesized that after a recovery from hyperglycemia, the vertebrae would have a similar phenotype to controls. Larval zebrafish were incubated in 120 mM glucose solution (treatment), or egg water (control), for seven days beginning five days post fertilization (dpf). To assess vertebral development following a recovery period, zebrafish were placed in normal egg water for an additional ten days, and control fish were kept in egg water for an equal amount of time. Using alizarin red to visualize mineralized vertebrae after 7‐day incubation in glucose solution (12dpf) showed a reduced percentage of zebrafish with any mineralized vertebrae compared to controls (****p<0.0001). The vertebrae that were present following treatment had significantly less area of mineralized centra compared to controls (****p<0.0001). This suggests that hyperglycemia negatively impacted initial vertebral development. Following recovery (22 dpf), the treatment fish had significantly reduced total body length than controls (****p<0.0001). Treatment fish continued to have significantly less mineralized centra area (****p<0.0001) and significantly increased area of intervertebral domain (****p<0.0001) when compared to size matched controls. This suggests that the vertebrae of treatment fish are mineralizing and expanding into the intervertebral domain at a slower rate than controls. Additionally, glucose‐treated fish had significantly reduced percentage of fish with mineralized neural (****p<0.0001) and hemal (****p<0.0001) arches present on their vertebra compared to controls. These results suggest a dramatic impairment of vertebral development that extends beyond both treatment and recovery periods, leading us to believe the reduced mineralization may be responsible for the behavioral deficits through reductions in their ability to swim. Furthermore, our results may indicate profound effects of gestation and childhood diabetes on the skeletal development of children. Future experiments will investigate osteoblast and chondrocyte populations following hyperglycemic induction and recovery to elucidate the mechanisms that result in impaired vertebral mineralization. This discovery would guide further research into potential therapeutics for diabetes‐induced osteoporosis.

Caspase-8 Deficient Osteoblastic Cells Display Alterations in Non-Apoptotic Pathways.

Caspase-8 is the key component of the receptor-mediated (extrinsic) apoptotic pathway. Immunological localization of active caspase-8 showed its presence in osteoblasts, including non-apoptotic ones. Further in vivo exploration of caspase-8 functions in the bone is hindered by the fact that the caspase-8 knock-out is lethal prenatally. Examinations were thus performed using individual cell populations in vitro. In this study, caspase-8 was eliminated by the CRISPR/cas9 technology in MC3T3-E1 cells, the most common in vitro model of osteoblastic populations. The aim of the work was to specify the consequences of caspase-8 deficiency on non-apoptotic pathways. The impact on the osteogenic gene expression of the osteoblastic cells along with alterations in proliferation, caspase cascades and rapamycin induced autophagy response were evaluated. Osteogenic differentiation of caspase-8 deficient cells was inhibited as these cells displayed a decreased level of mineralization and lower activity of alkaline phosphatase. Among affected osteogenic genes, based on the PCR Array, major changes were observed for Ctsk, as down-regulated, and Gdf10, as up-regulated. Other significantly down-regulated genes included those coding osteocalcin, bone morphogenetic proteins (-3, -4 and -7), collagens (-1a1, -14a1) or Phex. The formation of autophagosomes was not altered in rapamycin-treated caspase-8 deficient cells, but expression of some autophagy-related genes, including Tnfsf10, Cxcr4, Dapk1 and Igf1, was significantly downregulated. These data provide new insight into the effects of caspase-8 on non-apoptotic osteogenic pathways.

Transcriptional Regulation of Jaw Osteoblasts: Development to Pathology

Craniofacial and jaw bones have unique physiological specificities when compared to axial and appendicular bones. However, the molecular profile of the jaw osteoblast (OB) remains incomplete. The present study aimed to decipher the bone site-specific profiles of transcription factors (TFs) expressed in OBs in vivo. Using RNA sequencing analysis, we mapped the transcriptome of confirmed OBs from 2 different skeletal sites: mandible (Md) and tibia (Tb). The OB transcriptome contains 709 TF genes: 608 are similarly expressed in Md-OB and Tb-OB, referred to as “OB-core”; 54 TF genes are upregulated in Md-OB, referred to as “Md-set”; and 18 TF genes are upregulated in Tb-OB, referred to as “Tb-set.” Notably, the expression of 29 additional TF genes depends on their RNA transcript variants. TF genes with no previously known role in OBs and bone were identified. Bioinformatics analysis combined with review of genetic disease databases and a comprehensive literature search showed a significant contribution of anatomical origin to the OB signatures. Md-set and Tb-set are enriched with site-specific TF genes associated with development and morphogenesis (neural crest vs. mesoderm), and this developmental imprint persists during growth and homeostasis. Jaw and tibia site-specific OB signatures are associated with craniofacial and appendicular skeletal disorders as well as neurocristopathies, dental disorders, and digit malformations. The present study demonstrates the feasibility of a new method to isolate pure OB populations and map their gene expression signature in the context of OB physiological environment, avoiding in vitro culture and its associated biases. Our results provide insights into the site-specific developmental pathways governing OBs and identify new major OB regulators of bone physiology. We also established the importance of the OB transcriptome as a prognostic tool for human rare bone diseases to explore the hidden pathophysiology of craniofacial malformations, among the most prevalent congenital defects in humans.

DIVERSE HISTOMORPHOLOGY OF OSTEOSARCOMA; ILLUSTRATION OF TWELVE CASES

<h3>Background</h3> Head and neck osteosarcomas are rare and comprise less than 1% of tumors of the head and neck. Their management is relegated to specialist head and neck centers, which means that only a small team of head and neck pathologists analyzes the resected tumors. Their rarity and the need to identify malignant osteoid in biopsies makes their diagnosis challenging. <h3>Objective</h3> To highlight the varied histomorphology of head and neck osteosarcoma through a pictorial presentation of 12 cases. <h3>Methods</h3> The histology database at University College London Hospital recorded 124 cases of osteosarcoma in 15 years. Histology reports were reviewed to identify cases with varied histomorphology. <h3>Results</h3> Twelve osteosarcomas were selected, 3 of surface origin and 9 of intramedullary origin. Three were chondroblastic, 7 osteoblastic, and 2 fibroblastic. Seven of the medullary tumors and 2 of the surface tumors were high grade, and 2 medullary and 1 surface tumors were low grade. Diverse features seen in these cases were psammomatoid calcifications, ganglion-like cells, osteosclerosis, osteoclast-like giant cells, and areas resembling benign fibro-osseous lesions, chondromyxoid fibroma, osteochondroma, aneurysmal bone cysts, and osteoblastoma. <h3>Conclusions</h3> Diagnosis of intramedullary and surface osteosarcoma relies on identification of malignant osteoid produced by atypical mesenchymal cells. Other features such as myxochondroid stroma, sclerotic bone, pseudocystic spaces, psammomatoid calcifications, fibroblastic proliferation, chondroblastic tissue, epithelioid osteoblastic population, osteoclast-like giant cells, and osteochondroma-like presentation can be misleading, leading to aberrant diagnosis such as benign fibro-osseous lesion, aneurysmal bone cyst, osteoblastoma, and osteochondroma.

Control of osteoblast regeneration by a train of Erk activity waves.

SummaryRegeneration is a complex chain of events that restores a tissue to its original size and shape. The tissue-wide coordination of cellular dynamics needed for proper morphogenesis is challenged by the large dimensions of regenerating body parts. Feedback mechanisms in biochemical pathways can provide effective communication across great distances1-5, but how they might regulate growth during tissue regeneration is unresolved6,7. Here, we report that rhythmic traveling waves of Erk activity control the growth of bone in time and space in regenerating zebrafish scales, millimetre-sized discs of protective body armour. We find that Erk activity waves travel as expanding concentric rings, broadcast from a central source, inducing ring-like patterns of osteoblast tissue growth. Using a combination of theoretical and experimental analyses, we show that Erk activity propagates as excitable trigger waves able to traverse the entire scale in approximately two days, with the frequency of wave generation controlling the rate of scale regeneration. Furthermore, periodic induction of synchronous, tissue-wide Erk activation in place of travelling waves impairs tissue growth, indicating that wave-distributed Erk activation is key to regeneration. Our findings reveal trigger waves as a regulatory strategy to coordinate cell behaviour and instruct tissue form during regeneration.

Osteoblast Lineage Support of Hematopoiesis in Health and Disease.

In mammals, hematopoiesis migrates to the bone marrow during embryogenesis coincident with the appearance of mineralized bone, where hematopoietic stem cells (HSCs) and their progeny are maintained by the surrounding microenvironment or niche, and sustain the entirety of the hematopoietic system. Genetic manipulation of niche factors and advances in cell lineage tracing techniques have implicated cells of both hematopoietic and nonhematopoietic origin as important regulators of hematopoiesis in health and disease. Among them, cells of the osteoblast lineage, from stromal skeletal stem cells to matrix-embedded osteocytes, are vital niche residents with varying capacities for hematopoietic support depending on stage of differentiation. Here, we review populations of osteoblasts at differing stages of differentiation and summarize the current understanding of the role of the osteoblast lineage in supporting hematopoiesis. © 2022 American Society for Bone and Mineral Research (ASBMR).