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Both mechanical loading and autophagy play important roles in regulating bone growth and remodeling, but the relationship between the two remains unclear. In this study, we examined bone structure with micro-CT imaging and measured bone mechanical properties with three-point bending experiments using bones from wild-type (WT) mice and conditional knockout (cKO) mice with Atg7 deletion in their osteoblasts. We found that the knockout mice had significantly less bone volume, bone thickness, bone ultimate breaking force, and bone stiffness compared to wild-type mice. Additionally, bone marrow cells from knockout mice had reduced differentiation and mineralization capacities in terms of alkaline phosphatase and calcium secretion, as well as Runx2 and osteopontin expression. Knockout mice also had significantly less relative bone formation rate due to mechanical loading. Furthermore, we found that the osteoblasts from wild-type mice had stronger responses to mechanical stimulation compared to autophagy-deficient osteoblasts from knockout mice. When inhibiting autophagy with 3 MA in wild-type osteoblasts, we found similar results as we did in autophagy-deficient osteoblasts. We also found that mechanical loading-induced ATP release is able to regulate ERK1/2, Runx2, alkaline phosphatase, and osteopontin activities. These results suggest that the ATP pathway may play an important role in the possible involvement of autophagy in osteoblast mechanobiology.

Contains fulltext : 291236.pdf (Publisher’s version ) (Open Access) OBJECTIVES: Cleft lip and/or palate (CLP) is a common craniofacial birth defect caused by genetic as well as environmental factors. The phenotypic spectrum of CLP also includes submucous clefts with a defect in the palatal bone. To elucidate the contribution of vitamin A, we evaluated the effects of the vitamin A metabolite all-trans retinoic acid (ATRA) on the osteogenic differentiation and mineralization of mouse embryonic palatal mesenchymal cells (MEPM). SETTING AND SAMPLE POPULATION: MEPM cells were isolated from the prefusion palates of E13 mouse embryos from three different litters. MATERIALS AND METHODS: MEPM cells were cultured with and without 0.5 μM ATRA in osteogenic medium. Differentiation was analysed by the expression of osteogenic marker genes and alkaline phosphatase (ALP) activity after 1, 2, and 7 days. The expression of Wnt marker genes was also analysed. Mineralization was assessed by alizarin red staining after 7, 14, 21, and 28 days. RESULTS: The bone marker genes Sp7, Runx2, Alpl, and Col1a1 were inhibited 10% ± 2%, 59% ± 7%, 79% ± 12% and 57% ± 20% (P

Background Several recent studies have focused on the utility of drug repurposing to expand clinical application of approved therapeutics. Here, we investigate the efficacy of midazolam (MDZ) and cytokines for regenerating calcified tissue, using immortalized porcine dental pulp (PPU7) and mouse skeletal muscle derived myoblast (C2C12) cells, with the goal of repurposing MDZ as a new treatment to facilitate calcified tissue regeneration. Highlights We noted that PPU7 and C2C12 cells cultured with various MDZ regimens displayed increased bone morphogenic protein (BMP-2), transforming growth factor beta (TGF-β), and alkaline phosphatase activity. These increases were highest in PPU7 cells cultured with MDZ alone, and in C2C12 cells cultured with MDZ and BMP-2. PPU7 cells cultured under these conditions demonstrated markedly elevated expression of odontoblastic gene markers, indicating their likely differentiation into odontoblasts. Expression levels of osteoblastic gene markers also increased in C2C12 cells, suggesting that MDZ potentiates the effect of BMP-2, inducing osteoblast differentiation in these cells. Newly formed calcified deposits in both PPU7 and C2C12 cells were identified as hydroxyapatite via crystallographic and crystal engineering analyses. Conclusion MDZ increases ALP activity, inducing expression of specific marker genes for both odontoblasts and osteoblasts while promoting hydroxyapatite production in both PPU7 and C2C12 cells. These responses were cell type specific. MDZ treatment alone could induce these changes in PPU7 cells, but C2C12 cell differentiation required BMP-2 addition.

Alveolar bone defects caused by tooth loss often lead to challenges in implant dentistry, with a need for development of optimal bone biomaterials to predictably rebuild these tissues. To address this problem, we fabricated a novel bone block using platelet-rich fibrin (PRF) and Deproteinized Bovine Bone Mineral (DBBM), and characterized their mechanical and biological properties. The bone block was prepared by mixing DBBM, Liquid-PRF, and Solid-PRF fragments in various combinations as follows: (1) BLOCK-1 made with Solid-PRF fragments + DBBM, (2) BLOCK-2 made with Liquid-PRF + DBBM, (3) BLOCK-3 made with Solid-PRF fragments + Liquid-PRF + DBBM. The time for solidification and the degradation properties were subsequently recorded. Scanning electron microscopy (SEM) and tensile tests were carried out to investigate the microstructure and mechanical properties of each block. The bioactivity of the three groups towards osteoblast differentiation was also evaluated by culturing cells with the conditioned medium from each of the three groups including cell proliferation assay, cell migration assay, alkaline phosphatase (ALP) staining, and alizarin red staining (ARS), as well as by real-time PCR for genes encoding runt-related transcription factor 2 (RUNX2), ALP, collagen type I alpha1(COL1A) and osteocalcin (OCN). BLOCK-3 made with Solid-PRF fragments + Liquid-PRF + DBBM had by far the fastest solidification period (over a 10-fold increase) as well as the most resistance to degradation. SEM and tensile tests also revealed that the mechanical properties of BLOCK-3 were superior in strength when compared to all other groups and further induced the highest osteoblast migration and osteogenic differentiation confirmed by ALP, ARS and real-time PCR. PRF bone blocks made through the combination of Solid-PRF fragments + Liquid-PRF + DBBM had the greatest mechanical and biological properties when compared to either used alone. Future clinical studies are warranted to further support the clinical application of PRF bone blocks in bone regeneration procedures.

Objective : Osteoporosis result from age-related decline in the number of osteoblast progenitors in the bone marrow. Probiotics have beneficial effects on the host, when administered in appropriate amounts. This study investigated the effects of probiotics expressing specific genes, especially the effects of genetically modified bone morphogenetic protein (BMP)-2-expressing Lactobacillus plantarum CJNU 3003 (LP) on ovariectomized rats.Methods : Twenty-eight female Wistar rats (250–300 g, 12 weeks old) were divided into four groups : the sham (control), the ovariectomy (OVX)-induced osteoporosis group (OVX), the OVX and LP (OVX/LP), OVX and genetically modified BMP-2-expressing LP (OVX/LP with BMP) groups. The three groups underwent bilateral OVX and two of these groups were administered two different types of LP via oral gavage daily. At 16 weeks post-OVX, blood was collected from the heart and the bilateral tibiae were extracted and were scanned by ex-vivo micro-computed tomography and stained with hematoxylin-and-eosin (H&E) and Masson’s trichrome stain for pathological assessment. The serum levels of osteocalcin (OC), rat C-telopeptide of type I collagen (CTX-I), BMP-2, and receptor activator of nuclear factor-ĸB ligand (RANKL) were measured.Results : The 3D-micro-computed tomography images showed that the trabecular structure in the OVX/LP with BMP group was maintained compared with OVX and OVX/LP groups. No significant differences were detected in trabecular thickness (Tb.Th) between control and OVX/LP with BMP groups (p>0.05). Furthermore, a tendency toward increased BMD, trabecular bone volume, Tb.Th, and trabecular number and decreased trabecular separation was found in rats in the OVX/LP with BMP groups when compared with the OVX and OVX/LP groups (p>0.05). The H&E and Masson’s trichrome stained sections showed a thicker trabecular bone in the OVX/LP with BMP group compared with the OVX and OVX/LP groups. There was no difference in serum levels of OC, CTX and RANKL control and OVX/LP with BMP groups (p>0.05). In contrast, significant differences were found in OC and CTX-1 levels between the OVX and OVX/LP with BMP groups (p

Native bone is piezoelectric in nature and generates abundant surface charges under mechanical compression, which regulate osteoblast proliferation, differentiation, adhesion, and so on. Poly (vinylidene fluoride) (PVDF) is becoming one of the most popular piezoelectric polymers because of its easy processability and good biocompatibility. Unfortunately, because only the β and γ crystal phases of PVDF have piezoelectricity, post-treatments, for example, polarizing at high temperature, are required to enhance the piezoelectricity of PVDF scaffolds after fabrication. In this study, we reported a phase-separation-hydration method to fabricate a calcium phosphate silicate (CPS)-doped PVDF scaffold. Our method fabricated a better piezoelectric scaffold than native bone without further processing (∼ 3 pC/N vs. 0.7 pC/N). In addition, the scaffold was mechanically compatible (∼ 7 MPa) with the cancellous bone with sufficient porosity (∼ 45%) to facilitate osteoblast infiltration and bone ingrowth. The in vitro biocompatibility analyses proved that the prepared CPS-PVDF scaffold was biocompatible with osteoblast cells and encouraged osteoblast redifferentiation. In conclusion, our results suggest that this CPS-PVDF scaffold is a promising candidate for bone tissue engineering applications.

Postmenopausal osteoporosis is a systemic disease characterized by the loss of bone mass and increased bone fracture risk largely resulting from significantly reduced levels of the hormone estrogen after menopause. Besides the direct negative effects of estrogen-deficiency on bone, indirect effects of altered immune status in postmenopausal women might contribute to ongoing bone destruction, as postmenopausal women often display a chronic low-grade inflammatory phenotype with altered cytokine expression and immune cell profile. In this context, it was previously shown that various immune cells interact with osteoblasts and osteoclasts either via direct cell-cell contact, or more likely via paracrine mechanisms. For example, specific subtypes of T lymphocytes express TNFα, which was shown to increase osteoblast apoptosis and to indirectly stimulate osteoclastogenesis via B cell-produced receptor-activator of NF-κB ligand (RANKL), thereby triggering bone loss during postmenopausal osteoporosis. Th17 cells release interleukin-17 (IL-17), which directs mesenchymal stem cell differentiation towards the osteogenic lineage, but also indirectly increases osteoclast differentiation. B lymphocytes are a major regulator of osteoclast formation via granulocyte colony-stimulating factor secretion and the RANKL/osteoprotegerin system under estrogen-deficient conditions. Macrophages might act differently on bone cells dependent on their polarization profile and their secreted paracrine factors, which might have implications for the development of postmenopausal osteoporosis, because macrophage polarization is altered during disease progression. Likewise, neutrophils play an important role during bone homeostasis, but their over-activation under estrogen-deficient conditions contributes to osteoblast apoptosis via the release of reactive oxygen species and increased osteoclastogenesis via RANKL signaling. Furthermore, mast cells might be involved in the development of postmenopausal osteoporosis, because they store high levels of osteoclastic mediators, including IL-6 and RANKL, in their granules and their numbers are greatly increased in osteoporotic bone. Additionally, bone fracture healing is altered under estrogen-deficient conditions with the increased presence of pro-inflammatory cytokines, including IL-6 and Midkine, which might contribute to healing disturbances. Consequently, in addition to the direct negative influence of estrogen-deficiency on bone, immune cell alterations contribute to the pathogenesis of postmenopausal osteoporosis., publishedVersion

Aging induces alterations in bone structure and strength through a multitude of processes, exacerbating common aging- related diseases like osteoporosis and osteoarthritis. Cellular hallmarks of aging are examined, as related to bone and the marrow microenvironment, and ways in which these might contribute to a variety of age-related perturbations in osteoblasts, osteocytes, marrow adipocytes, chondrocytes, osteoclasts, and their respective progenitors. Cellular senescence, stem cell exhaustion, mitochondrial dysfunction, epigenetic and intracellular communication changes are central pathways and recognized as associated and potentially causal in aging. We focus on these in musculoskeletal system and highlight knowledge gaps in the literature regarding cellular and tissue crosstalk in bone, cartilage, and the bone marrow niche. While senolytics have been utilized to target aging pathways, here we propose non-pharmacologic, exercise-based interventions as prospective "senolytics" against aging effects on the skeleton. Increased bone mass and delayed onset or progression of osteoporosis and osteoarthritis are some of the recognized benefits of regular exercise across the lifespan. Further investigation is needed to delineate how cellular indicators of aging manifest in bone and the marrow niche and how altered cellular and tissue crosstalk impact disease progression, as well as consideration of exercise as a therapeutic modality, as a means to enhance discovery of bone-targeted therapies.

Angiogenesis, hematopoiesis and osteogenesis are fundamental processes mediating complex and essential biological functions. In the bone marrow, endothelial cells (ECs) are a principal mediator of regulatory signals that govern hematopoietic and mesenchymal stem cells. EC and osteoblast interactions and niche functions of ECs are fundamental in maintaining bone health and coordinating repair and regeneration following injury. These cellular interactions are subject to dysregulation and deterioration under stress, aging, chronic disease states and malignancy. Thus, the prospect of manipulating the bone vasculature has tremendous potential to advance therapeutic interventions for the management of bone diseases. This review discusses the current state of vascular-skeletal tissue interactions focusing on osteoblast and hematopoietic stem cells interaction with ECs.

Octacalcium phosphate and collagen composite (OCPcol) promotes osteogenic differentiation and angiogenesis, thereby enhancing bone regeneration. Although a newly developed freeze-dried composite of OCPcol and teriparatide (OCPcolTPTD) reinforced bone regeneration more than OCPcol, the mechanism of bone regeneration remains unresolved. In this study, disks containing OCPcolTPTD, OCPcol, or β-tricalcium phosphate (β-TCP) col were inserted into rodents with calvarial bone defects, before euthanasia 4 weeks later. Immunohistochemical and histochemical analyses were performed on bone samples to evaluate bone matrix development, angiogenesis, and osteoclast and osteoblast localization. In the OCPcolTPTD and OCPcol groups, bone regeneration was observed at the surface of calvarial dura mater and around acidophilic granular cells with abundant collagenous fiber-containing cells. Furthermore, the newly formed bone in the OCPcolTPTD group showed a larger total area and individual separated area than the other groups. Various osteogenic proteins were detected in the regenerated bone and peri-bone tissues by histochemistry and immunohistochemistry. Although the expression of several osteogenic biomarkers in the OCPcolTPTD group after 4 weeks of implantation was significantly lower than that in the OCPcol group, new bone formation by OCPcolTPTD in the center of the defect, where bone regeneration is difficult, tended to be superior to that by OCPcol. These results suggest that OCPcolTPTD enhanced bone regeneration more evenly and homogenously than OCPcol. Impact statement Our study suggests that octacalcium phosphate and collagen (OCPcol) together with a TPTD enhances bone regeneration in rodents with calvarial bone defects. Furthermore, we believe that composite of OCPcol and teriparatide (OCPcolTPTD) could be developed into novel clinical technique for the regeneration or repair of bone.

Background Identifying the factors affecting osteoblast differentiation ofperiodontal ligamentcells (PDLCs) can help enhance the regeneration of periodontal tissue.LncRNAplasmacytoma variant translocation 1 (lncPVT1) is an important regulatory factor involved in many biological processes, but its role in osteogenesisremains unclear. Methods Expressionsof osteogenic markers were detected by quantitative reverse transcription polymerase chain reaction and Western blot analysis. Alkaline phosphatase staining was conducted for early osteoblast differentiation and alizarin red S staining was used for mineral deposition. RNA sequencing was used to identify the miRNAs regulated by lncPVT1 during osteogenesis. Cell transfection was used to overexpress or knockdown lncPVT1 and miR-10a-5p. Dual luciferase reporter assayswere conducted to analyze the binding of miR-10a-5p to brain-derived neurotrophic factor (BDNF). Results LncPVT1 was significantly increased during osteogenic induction of PDLCs. Overexpression of lncPVT1 promoted osteogenesis, whereas lncPVT1 knockdown inhibited this process. RNA sequencing showed that miR-10a-5p expression was significantly increased after lncPVT1 knockdown.RNA immunoprecipitation assay further demonstrated the binding potential of lncPVT1 and miR-10a-5p. MiR-10a-5p inhibited the osteogenesis of PDLCs, and partially reversed the stimulatory effects of lncPVT1.Subsequently, we identified a predicted binding site for miR-10a-5p on BDNF and confirmed it using dual luciferase reporter assays. Moreover, lncPVT1 upregulated the expression of BDNF, while miR-10a-5p downregulated BDNF expression. BDNF promoted osteogenesis and partially rescued the si-lncPVT1-mediated inhibition of PDLCs osteogenic differentiation. Conclusion LncPVT1 positively regulated the osteogenic differentiation of PDLCs via miR-10a-5p and BDNF.Our resultsprovide a promising target for enhancing the osteogenic potential of PDLCs. This article is protected by copyright. All rights reserved.

Recent studies have demonstrated the importance of bone as an endocrine organ that produces biologically active substances, which regulate both local bone metabolism and metabolic functions throughout the body. In the process of bone remodeling (formation/destruction), the active cells secrete specific biomarkers that help detect osteometabolic dysfunction. Among bone hormones, osteocalcin plays an important role as a coordinator of bone modeling processes, energy homeostasis, metabolism of glucose, lipids and minerals. Osteocalcin is a structural protein of the bone matrix, which is synthesized by osteoblasts and enters the bloodstream in the process of bone resorption. The level of osteocalcin in the serum is used as a specific marker of bone formation. Osteocalcin promotes pancreatic β-cell proliferation and insulin secretion, and also affects the insulin sensitivity of peripheral tissues. The inverse association of glycemia with the level of osteocalcin was revealed. Patients with type 2 diabetes mellitus usually have normal or even slightly elevated bone mineral density compared to age-appropriate controls. Decreased bone quality and increased risk of fractures are associated with changes in bone microarchitecture and local humoral environment. An imbalance in osteoblast/osteoclast activity may be due to oxidative stress and the accumulation of glycosylation end products, which contributes to chronic inflammation and bone resorbtion in patients with diabetes. It is shown that the level of osteocalcin in the blood serum is significantly reduced compared to healthy controls, both in patients with type 1 diabetes mellitus and, especially, in type 2 diabetes mellitus. Given the importance of developing new approaches to the diagnosis and correction of metabolic disorders in diabetic patients, the study of the influence of bone hormones on hormonal and metabolic parameters and bone status, including the risk of fractures, remains relevant in modern diabetology., Дослідження останнього часу продемонстрували значення кістки як ендокринного органа, що продукує біологічно активні речовини, які регулюють як місцевий кістковий метаболізм, так і метаболічні функції у всьому організмі. Під час кісткового ремоделювання (формування/руйнування) активні клітини виділяють специфічні біомаркери, які допомагають виявляти остеометаболічну дисфункцію. Серед гормонів кісткової тканини важлива роль належить остеокальцину як координатору процесів кісткового моделювання, енергетичного гомеостазу, метаболізму глюкози, ліпідів та мінеральних речовин. Остеокальцин — структурний білок кісткового матриксу, що синтезується остеобластами і надходить до кровотоку під час резорб­ції кісткової тканини. Рівень остеокальцину в сироватці крові використовується як специфічний маркер формування кісткової тканини. Остеокальцин сприяє проліферації β-клітин підшлункової залози та секреції інсуліну, а також впливає на інсуліночутливість периферичних тканин. Виявлено обернену асоціацію глікемії з рівнем остеокальцину. У хворих на цукровий діабет 2-го типу (ЦД2) зазвичай спостерігається нормальна або навіть трохи підвищена мінеральна щільність кісткової тканини порівняно з контролем відповідного віку. Зниження якості кісток та підвищений ризик переломів пов’язують зі змінами кісткової мікроархітектури та місцевого гуморального середовища. Дисбаланс активності остеобластів/остеокластів може бути зумовлений окиснювальним стресом та накопиченням кінцевих продуктів глікозилювання, що сприяє хронічному запаленню та резорбції кісток у пацієнтів із діабетом. Показано, що рівень остеокальцину в сироватці крові значно знижується порівняно зі здоровим контролем як у хворих на ЦД1, так і, особливо сильно, при ЦД2. З урахуванням важливості розробки нових підходів до діагностики та корекції метаболічних порушень у хворих на ЦД вивчення впливу кісткових гормонів на гормонально-метаболічні показники та стан кісткової тканини, включаючи ризик переломів, зберігає актуальність у сучасній діабетології.

Aim Biomimetic implant surface coatings can improve osteoblast response and enhance osseointegration. This study aimed to assess the response of osteoblast-like cell line (MG-63) coating of injectable platelet-rich fibrin on titanium discs. Materials and methods Injectable-PRF (i-PRF) was prepared by centrifugation of blood at 700 rpm for 3 minutes without any anti-coagulant in i-PRF tubes. Ten commercially pure titanium discs were divided into control groups comprising plain discs, and test group, titanium discs were coated with i-PRF. These were then added to the cultured MG-63 cells. Cell proliferation, alkaline phosphatase production, and mineralization were assessed in both groups at day 1, 7, 14, and 21. Results The cell proliferation, alkaline phosphatase production, and mineralization increased significantly from day 1 to day 21 in both test and control groups and was significantly higher in the test group than in control group at day 1, 7, 14, and 21 (p Conclusions Coating of titanium discs with i-PRF causes increased proliferation, alkaline phosphatase production, and increased mineralization at day 1, 7, 14, and 21 in MG-63 osteoblast-like cells. Clinical significance Improved osteoblast proliferation and mineralization demonstrate enhanced activity on the surface of an implant, which in turn may lead to increased bone to implant contact and faster/ and/or enhanced osseointegration.

MicroRNAs (miRNAs) regulate osteogenic differentiation and influence osteoporosis (OP). The aim of this study was to determine the potential role of miR-874-3p in OP. The expression levels of miR-874-3p and leptin (LEP) in the femoral neck trabeculae of 35 patients with or without OP were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The effects of miR-874-3p or LEP on the cell proliferation and alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) levels were observed by upregulating miR-874-3p in human bone marrow mesenchymal stem cells (hBMSCs). Additionally, calcium deposition levels were evaluated using alizarin red staining (ARS). Molecular mechanisms of miR-874-3p and LEP underlying the osteogenic differentiation of hBMSCs were also evaluated using bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays. The miR-874-3p levels were significantly lower in the femoral neck trabeculae of patients with OP than those of the control group, while the opposite was observed regarding the levels of LEP. Expression levels of miR-874-3p in hBMSCs were upregulated during osteogenic differentiation, while those of LEP were downregulated. Moreover, miR-874-3p upregulation promoted ALP, RUNX2, OCN, and OSX mRNA expression, cell proliferation, and calcium deposition in hBMSCs. LEP was found to be a target gene of miR-874-3p. Overexpression of LEP inhibited the expression of osteoblast markers and reversed the effect of osteogenic differentiation induced by the upregulation of miR-874-3p. In conclusion, miR-874-3p promoted the proliferation and differentiation of hBMSCs by downregulating the expression of LEP, thus inhibiting OP. Abbreviations : miRNAs: microRNAs; OP: osteoporosis; hBMSCs: human Bone Marrow Mesenchymal stem cells; LEP: leptin; DEGs: differentially expressed genes

Objectives Periodontal ligament-derived stem cells (PDLSCs) are regarded as a viable option for periodontal regeneration using cell sheet technology. The objective of the present in vitro study was to characterize human PDLSCs based on their phenotypic and biological properties and to evaluate the ascorbic acid (AA or vitamin C)-induced cell sheet by analyzing the molecular markers. Methods PDLSCs were established from premolars, and their morphology, viability, proliferation, phenotypic marker expression, and ability to differentiate into osteocytes and adipocytes were analyzed. PDLSCs were then induced to form cell sheets using 100 μM AA, and gene expression was examined by real-time polymerase chain reaction. Results PDLSCs showed fibroblastic morphology with >95% viability. The cells were highly proliferative and positive for surface antigens CD29, CD73, and CD90 but negative for CD34 and CD45. They were capable of differentiating into osteocytes and adipocytes. Induction with 100 μM AA transformed PDLSCs into two-to three-layered cell sheets. There was no significant upregulation in ALP and RUNX2 expression in the AA-induced cell sheet. However, the expression levels of late osteoblast differentiation marker (bone gamma-carboxy glutamate protein); cementogenic markers (cementum attachment protein and CP23), and genes encoding extracellular matrix (ECM) proteins [collagen type 1 alpha 1 and integrin beta 1) were higher in AA-induced cell sheets by PDLSCs. Conclusions The stimulating effect of AA on cell sheet formation by PDLSCs was confirmed by the expression of typical markers involved in osteogenesis/cementogenesis and ECM secretion, which makes this procedure a prospective option for periodontal tissue regeneration applications.

Purpose To investigate the feasibility and the optimum condition of human adipose-derived stem cells cultured on the mineralized collagen material; and to further explore the mechanism of osteogenic differentiation of the human Adipose-derived stem cells stimulated by the mineralized collagen material. Methods Primary human adipose-derived stem cells (HADSCs) were isolated from human adipose tissue using centrifugal stratification, which had been passed repeatedly to later generations and purified. Human adipose-derived stem cells were cultured on the bone graft material and the optimum concentration was explored by Alamar blue colorimetric method. The rest experiment was conducted according to the result. The experimental groups are shown below: group A (HADSCs + bone graft material); group B (HADSCs). Morphological observation was taken by scanning electronic microscope (SEM). Alkaline phosphatase activities were tested by histochemical method. Calcium deposition was investigated by alizarin red staining. The quantity access of osteogenic-related mRNA: ALP (alkaline phosphatase), BMP2 (bone morphogenetic protein 2) and RUNX2 (runt-related transcription factor 2) were detected using RT-PCR. Results The cultured cells grew stably and proliferated rapidly. The optimum condition was 0.5 mg/cm2 bone graft material coated on the bottom of medium. After culturing on the material 14 days, the alizarin red staining showed that more calcium deposition was detected in group A and alkaline phosphatase activities of group A was higher than group B (p ˃ 0.05). Similarly, after culturing for 14 days, the ALP, BMP2 and RUNX2 transcription activity of group A was higher than group B (p ˃ 0.05). Conclusion Human adipose-derived stem cells cultured on bone graft material were dominantly differentiated into osteoblast in vitro. Thus it provided a new choice for bone tissue engineering.

Background and Objective: Osteoporosis is a worldwide healthcare challenge. Conventional medications for osteoporosis prevention are not clinically effective or associated with gastrointestinal tract adverse effects. The present study aimed to comparatively investigate the effects of technetium-99 conjugated with methylene diphosphonate (99Tc-MDP) and calcium carbonate and alendronate on the prevention and treatment of osteoporosis in glucocorticoid-induced osteoporosis rabbit model through evaluating bone alkaline phosphatase (B-ALP), TRAP-5b levels and histopathological parameters. Methods: Forty healthy female New Zealand rabbits were randomly divided into five groups (each n=8), including control group (Control Group), osteoporosis model group (GIO Group), osteoporosis model + 99Tc-MDP group (99Tc-MDP Group), osteoporosis model + alendronate group (Alendronate Group), and osteoporosis model + calcium carbonate group (calcium carbonate Group). Animals in each group were treated with corresponding interventions for 14 weeks. The blood samples were collected at the first and 14th week, and B-ALP and TRAP-5b levels were detected by enzyme- linked immunosorbent assay (ELISA). The rabbits were anesthetized at the 14th week, and pathological cytological observation was performed on both femurs. Results: The age and weights of rabbits in different groups had no statistically significant differences (P>0.05). B-ALP levels in serum of all groups except for the Control Group decreased after treatment, but the differences were not statistically significant (P>0.05). TRAP-5b levels in serum of all groups increased after treatment. Specifically, differences in the GIO Group and calcium carbonate group were statistically significant (P99Tc-MDP Group and alendronate Group were not statistically significant (P99Tc- MDP group and alendronate group was similar to control group and GIO group. Moreover, the calcium carbonate group and GIO group exhibited similar bone tissue morphology. Conclusions: 99Tc-MDP has a preventive effect on the glucocorticoid-induced osteoporotic rabbit model. This osteoporosis preventive effect might be attributed to the capacities of 99Tc-MDP in promoting the osteoblasts generation and inhibiting the generation and reducing the activity of osteoclasts.

Pure hydroxyapatite (HA) as bone graft substitute has excellent osteogenic activity, but it has lower fracture toughness and its biological activity is harmed by the addition of toughening phases, which limits its clinical application. To alleviate the contradiction, columnar β-Si3N4 as the toughening phase and La3+/Y3+ sintering aid as active ions are used in this study to prepare β-Si3N4/HA composite biomaterials. According to the results, hardness and toughness of 10 wt% β-Si3N4/HA composite prepared by cold press sintering at 1300 °C were 6.44 GPa and 1.69 MPa m1/2, respectively, being 110% and 140% higher than those of pure HA. Moreover, 10 wt% β-Si3N4/HA composite exhibited better protein adsorption capacity and obviously stimulated adhesion, proliferation, and osteogenic differentiation of osteoblast. In addition, it was found that sintering aid not only facilitated the improvement of mechanical properties, but also promoted the formation of 100–200 nm nanostripe structure, which was beneficial to cell adhesion. Determination of La3+concentration combined with biological experiments also proved that its concentration range from 4×10−8 M to 6 × 10−8M was beneficial to cell proliferation and osteogenic differentiation. In summary, β-Si3N4 and sintering aids were shown to improve mechanical properties of HA at maintaining the biological activity of the latter.

Polymeric coatings incorporated by biocomposites and antimicrobials have become attractive in recent years due to their ability to make the surface of Ti and its alloys antimicrobial and pro-osteogenic. Thus, this review aimed to answer the “Do the polymeric hybrid coatings applied on titanium surfaces and their alloys for dental implant purposes present antibacterial and osteogenic activity?”.

Implant provisional restorations should ideally be nontoxic to the contacting and adjacent tissues, create anatomical and biophysiological stability, and establish a soft tissue seal through interactions between prosthesis, soft tissue, and alveolar bone. However, there is a lack of robust, systematic, and fundamental data to inform clinical decision making. Here we systematically explored the biocompatibility of fibroblasts and osteoblasts in direct contact with, or close proximity to, provisional restoration materials. Human gingival fibroblasts and osteoblasts were cultured on the “contact” effect and around the “proximity” effect with various provisional materials: bis-acrylic, composite, self-curing acrylic, and milled acrylic, with titanium alloy as a bioinert control. The number of fibroblasts and osteoblasts surviving and attaching to and around the materials varied considerably depending on the material, with milled acrylic the most biocompatible and similar to titanium alloy, followed by self-curing acrylic and little to no attachment on or around bis-acrylic and composite materials. Milled and self-curing acrylics similarly favored subsequent cellular proliferation and physiological functions such as collagen production in fibroblasts and alkaline phosphatase activity in osteoblasts. Neither fibroblasts nor osteoblasts showed a functional phenotype when cultured with bis-acrylic or composite. By calculating a biocompatibility index for each material, we established that fibroblasts were more resistant to the cytotoxicity induced by most materials in direct contact, however, the osteoblasts were more resistant when the materials were in close proximity. In conclusion, there was a wide variation in the cytotoxicity of implant provisional restoration materials ranging from lethal and tolerant to near inert, and this cytotoxicity may be received differently between the different cell types and depending on their physical interrelationships.

Background and objectives MiR-148a-3p has been reported to regulate the differentiation of marrow stromal cell osteoblast. In this study, whether miR-148a-3p regulated the odontoblastic differentiation of human dental pulp stem cells (hDPSCs) or not was explored. Methods and results The hDPSCs were isolated and identified via flow cytometry. Targets of miR-148a-3p were identified via bioinformatics and dual-luciferase reporter assay. After the cell was cultured in the odontogenic differentiation medium or infected, cell viability, invasion, and odontoblastic differentiation were detected via MTT, transwell, and Alizarin Red S staining, respectively. The miR-148a-3p, Wnt1, β-catenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 expressions were determined by RT-qPCR and Western blot. The hDPSCs odontoblastic differentiation downregulated the miR-148a-3p expression and upregulated Wnt1 expression. Wnt1 was determined as the target for miR-148a-3p. MiR-148a-3p mimic and siWnt1 suppressed the cell viability, invasion, and odontoblastic differentiation of hDPSCs and inhibited the Wnt1, β-catenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 expressions. In contrast, miR-148a-3p inhibitor and overexpressed Wnt1 promoted the cell viability, invasion, and odontoblastic differentiation of hDPSCs, and upregulated the Wnt1, β-catenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 expressions. Also, miR-148a-3p mimic and inhibitor reversed the effects of Wnt1 overexpression and siWnt1. Conclusions MiR-148a-3p modulated the invasion and odontoblastic differentiation of hDPSCs through the Wnt1/β-catenin pathway.

Phlomis umbrosa has been traditionally used for bone diseases in traditional Korean Medicine. Sweroside (SOS), marker compounds of P. umbrosa, has been known to promote osteoblast differentiation. In this study, ameliorative effects of SOS on osteoporosis and potential target pathway were investigated. Ovariectomized mice were administered three doses of SOS three times a week for 4 weeks after inducing osteoporosis. Bone mineral content (BMC) and bone mineral density (BMD) were analyzed by dual energy X-ray absorptiometry. A human osteosarcoma cell line (SaOS-2) was differentiated to clarify the promoting effects of SOS on osteoblast differentiation and bone formation. Osteoblastic bone-forming markers were evaluated in lumbar vertebrae (LV) and mineralized SaOS-2 cells. SOS markedly elevated BMC and BMD levels and attenuated the bone marrow adipocytes in the femoral shaft. SOS increased the formation of bone matrix in SaOS-2 cells. Bone morphogenetic protein-2 (BMP2) and runt-related transcription factor 2 (CBFA1) in LV and SaOS-2 cells were up-regulated by SOS. SOS increased alkaline phosphatase (ALPL), osteopontin (SPP1), and bone sialoprotein-1 (BSPH1). In conclusion, SOS induced the formation of mineralized bone matrix by regulating BMP2/CBFA1-mediated molecules. Therefore, SOS could be a therapeutic compound of treatment for osteoporosis by producing the new bone matrix.

Dental pulp cells are a source of multipotent mesenchymal stem cells with a high proliferation rate and multilineage differentiation potential. This study investigated the photobiomodulated bioenergetic effects of mitochondria in osteoblasts that differentiated from human pulp stem cells. The systematic review followed PRISMA guidelines. The PICO question was formulated. Criteria for inclusion and exclusion were established prior to searches being performed on the PubMed/MEDLINE, Embase, and Scopus. Articles were identified and included if published in English within last 10 years; photobiomodulation or low-level laser therapy were discussed; the delivery parameters for dose and time were included and the studies focused on bioenergetics of osteoblast mitochondria. Studies excluded were non-human dental pulp tissue and in vivo studies. A total number of 110 articles were collated, 106 were excluded leaving a total of 4 articles. These studies demonstrated that in vitro use of photobiomodulation was performed using different laser and LED types; InGaAlP; InGaN; and InGaAsP with average wavelengths of 630 to 940 nm. Primary human osteoblastic STRO-1 and mesenchymal stem cell lineages were studied. Three out of four articles confirmed positive bioenergetic effects of photobiomodulation on mitochondria of osteoblasts derived from human pulp cells. This systematic review demonstrated a lack of adequate reporting of bioenergetics of osteoblast mitochondria after photobiomodulation treatment.

SNP rs3755955 (major/minor allele: G/A) located in Iduronidase-Alpha-L- (IDUA) gene was reported to be significant for human bone mineral density (BMD). This follow-up study was to uncover the underlying association mechanism through molecular and cellular functional assays relevant to bone. We tested the effects of single nucleotide polymorphisms (SNP) rs3755955 (defined allele G as wild-type and allele A as variant-type) on osteoblastic and osteoclastic functions, as well as protein phosphorylation in stably transfected human fetal osteoblast (hFOB) cell and mononuclear-macrophage (RAW264.7) cell. In hFOB cells, transfection with variant-type IDUA significantly decreased osteoblastic gene expression (OPN, COL1A1 and RANKL) (p

Excessive ethanol consumption is a risk factor for osteopenia. Since a previous study showed that transgenic female mice with overexpression of catalase are partially protected from ethanol-mediated trabecular bone loss, we investigated the role of endogenous catalase in skeletal ethanol toxicity comparing catalase knockout to wild-type mice. We hypothesized that catalase depletion would exacerbate ethanol effects. The mice were tested in a newly designed binge ethanol model, in which 12-week-old mice were exposed to 4 consecutive days of gavage with ethanol at 3, 3, 4, and 4.5 g ethanol/kg body weight. Binge ethanol decreased the concentration of serum osteocalcin, a marker of bone formation. The catalase genotype did not affect the osteocalcin levels. RNA sequencing of femoral shaft RNA from males was conducted. Ethanol exposure led to significant downregulation of genes expressed in cells of the osteoblastic lineage with a role in osteoblastic function and collagen synthesis, including the genes encoding major structural bone proteins. Binge ethanol further induced a smaller set of genes with a role in osteoclastic differentiation. Catalase depletion affected genes with expression in erythroblasts and erythrocytes. There was no clear interaction between binge ethanol and the catalase genotype. In an independent experiment, we confirmed that the binge ethanol effects on gene expression were reproducible and occurred throughout the skeleton in males. In conclusion, the binge ethanol exposure, independently of endogenous catalase, reduces expression of genes involved in osteoblastic function and induces expression of genes involved in osteoclast differentiation throughout the skeleton in males.

Natural biomaterials with intrinsic biomineralization potential are of utmost interest in bone tissue engineering (BTE) applications. Here, we report the biomimetic mineralization potential of a natural fiber derived from Cissus quadrangularis (CQ) stem, CQF, with inherent biomineralization and osteoinductive potential and is proposed for regenerative BTE. CQF has an organized microstructure characterized by an array of submicron size fibrils (SEM analysis) saturated with –OH groups of cellulose on the surface (FTIR spectrum), which provides a better platform for cell adhesion/proliferation and play a key role in the early nucleation of calcium phosphate, the inorganic phase of bone. A single unit of CQF has a tensile strength of 89.75 ± 5.25 MPa, which is in line with the mechanical property requirement of bone. CQF depicts signs of surface erosion (SEM analysis) with a weight loss of 11% after 30 days incubation in physiological conditions, supporting its intended stability required during the bone regeneration phase. CQF demonstrated excellent cytocompatibility (MTT assay and cell adhesion) and elicited primary nucleation and growth of apatite crystals (Ca/P 1.30; day 7), progressed further to secondary growth (Ca/P 1.51; day 14) upon incubation in simulated body fluid (SBF; 1.5X). Biomineralization of CQF was further confirmed by Alizarin Red S staining (day 14) in MG-63 cells. The osteoinductive potential of CQF was validated by quantifying early osteoblast differentiation marker alkaline phosphatase activity in Rat Bone marrow Mesenchymal Cells (BMC). The results, therefore, confer that CQF could be proposed for regenerative BTE.

Bone marrow (BM) is a heterogeneous niche where bone marrow stromal cells (BMSCs), osteoblasts, osteoclasts, adipocytes, hematopoietic cells, and immune cells coexist. The cellular composition of BM changes with various pathophysiological states. A reduction in osteoblast number and a concomitant increase in adipocyte number in aging and pathological conditions put bone marrow adipose tissue (BMAT) into spotlight. Accumulating evidence strongly supports that an overwhelming production of BMAT is a major contributor to bone loss disorders. Therefore, BMAT-targeted therapy can be an efficient and feasible intervention for osteoporosis. However, compared to blocking bone-destroying molecules produced by BMAT, suppressing BMAT formation is theoretically a more effective and fundamental approach in treating osteoporotic bone diseases. Thus, a deep insight into the molecular basis underlying increased BM adiposity during pathologic bone loss is critical to formulate strategies for therapeutically manipulating BMAT. In this review, we comprehensively summarize the molecular mechanisms involved in adipocyte differentiation of BMSCs as well as the interaction between bone marrow adipocytes and osteoclasts. More importantly, we further discuss the potential clinical implications of therapeutically targeting the upstream of BMAT formation in bone loss diseases.

The primary cilium is a nonmotile microtubule-based organelle in most vertebrate cell types. The primary cilium plays a critical role in tissue development and homeostasis by sensing and transducing various signaling pathways. Ciliary proteins such as intraflagellar transport (IFT) proteins as well as ciliary motor proteins, kinesin and dynein, comprise a bidirectional intraflagellar transport system needed for cilia formation and function. Mutations in ciliary proteins that lead to loss or dysfunction of primary cilia cause ciliopathies such as Jeune syndrome and Ellis–van Creveld syndrome and cause abnormalities in tooth development. These diseases exhibit severe skeletal and craniofacial dysplasia, highlighting the significance of primary cilia in skeletal development. Cilia are necessary for the propagation of hedgehog, transforming growth factor β, platelet-derived growth factor, and fibroblast growth factor signaling during osteogenesis and chondrogenesis. Ablation of ciliary proteins such as IFT80 or IFT20 blocks cilia formation, which inhibits osteoblast differentiation, osteoblast polarity, and alignment and reduces bone formation. Similarly, cilia facilitate chondrocyte differentiation and production of a cartilage matrix. Cilia also play a key role in mechanosensing and are needed for increased bone formation in response to mechanical forces.

Reticulocalbin1 (RCN1) is implicated in tumorigenesis and tumour progression. However, whether RCN1‐mediated bone metastasis of non‐small cell lung cancer (NSCLC) cells was elusive. Here, we assessed the effect of osteoblast‐conditioned medium (CM) on proliferation and migration of NSCLC cell line, NCI‐H1299 and NCI‐H460 cells, and identified the soluble mediators in CMs from osteoblasts and NSCLC cells using MTT, Clonogenicity, Transwell, wound healing, RT‐PCR, and Western blotting assays, and LC‐MS/MS analysis, respectively. Furthermore, the role of RCN1 was investigated in NSCLC cells cultured with or without osteoblast‐CM. Tumour growth and bone resorption were measured in a nude mouse model bearing NCI‐H1299 cells transduced with shRNA/RCN1 vector using in vivo imaging technique and micro‐CT. The results showed that RCN1 with a higher abundance in osteoblast‐CM, which was present in extracellular vesicles (EVs), enhanced RCN1 expression in NSCLC cells. Osteoblast‐CM partially offset the inhibitory effect of RCN1 depletion on proliferation and migration of NSCLC cells. RCN1 depletion‐induced endoplasmic reticulum (ER) stress caused by increasing GRP78, CHOP, IRE1α, p‐IRE1α, p‐PERK and p‐JNK, which was positively regulated by self‐induced autophagy, contributed to suppression of proliferation and migration in NCI‐H1299 cells. Therefore, osteoblasts produced RCN1 to transfer into NSCLC cells partially through EVs, facilitating proliferation and migration of NSCLC cells via blocking ER stress. RCN1 could be required for proliferation and migration of NSCLC cells regulated by osteoblast‐CM.

Bone marrow is a spongy tissue that contains stem cells that are found inside some bones, including the hip and femur. Bone marrow cancer is a type of cancer that is caused by stem cells that make up the blood cells in the bone marrow. Sometimes these cells grow too fast or abnormally, which is called bone marrow cancer. Bone tissue cells are mainly composed of osteoblasts and osteoclasts. Osteoblast cells constantly build new bone throughout the life of each bone, and other cells called osteoclasts constantly absorb pieces of bone, so the bone is constantly being renewed. In this paper, mathematical models of tumors, the effect of the body on the drug, and the drug on the body are introduced, and then the appropriate dose of the drug to reduce tumor density is calculated using the model predictive control (MPC) algorithm. To obtain an adaptive MPC strategy, the extended least squares (ELS) method developed to learn the parameters of the tumor growth model is used. Finally, the simulation in MATLAB, assuming the model is correct, shows that the tumor is gone, and the bone mass improves over a period of time. The results demonstrate that the proposed method is effective for the treatment of bone marrow cancer.

Endocrine and paracrine fibroblast growth factor 23 (FGF23) is a protein predominantly produced by bone cells with strong impact on phosphate and vitamin D metabolism by targeting the kidney. Plasma FGF23 concentration early rises in kidney and cardiovascular diseases correlating with progression and outcome. Lactic acid is generated in anaerobic glycolysis. Lactic acidosis is the consequence of various physiological and pathological conditions and may be fatal. Since FGF23 production is stimulated by inflammation and lactic acid induces pro-inflammatory signaling, we investigated whether and how lactic acid influences FGF23. Experiments were performed in UMR106 osteoblast-like cells, Fgf23 mRNA levels estimated from quantitative real-time polymerase chain reaction, and FGF23 protein determined by enzyme-linked immunosorbent assay. Lactic acid dose-dependently induced Fgf23 gene expression and up-regulated FGF23 synthesis. Also, Na+-lactate as well as formic acid and acetic acid up-regulated Fgf23. The lactic acid effect was significantly attenuated by nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB) inhibitors wogonin and withaferin A. Lactic acid induces FGF23 production, an effect at least in part mediated by NFκB. Lactic acidosis may, therefore, be paralleled by a surge in plasma FGF23.

Phytoestrogens are strongly recommended for treating osteoporosis. Our previous study showed that naringin, a citrus flavonoid, can enhance the bone mass in ovariectomized rats. In this study, we further elucidated the mechanisms of naringin-induced osteoblast maturation and bone healing. Treatment of human osteoblasts with naringin increased cell viability and proliferation. In parallel, exposure to naringin enhanced translocation of estrogen receptor alpha (ERα) to nuclei and its transactivation activity. Sequentially, naringin induced alkaline phosphatase (ALP) mRNA and protein expression and its enzyme activity. Pretreatment with methylpiperidinopyrazole (MPP), a specific inhibitor of ERα, attenuated naringin-induced augmentations in ERα transactivation activity, ALP gene expression, and cell mineralization. The beneficial effects of naringin were also confirmed in mouse MC3T3-E1 cells. Moreover, administration of mice with a bone defect with naringin increased levels of ERα and ALP in damaged sites and simultaneously enhanced the healing rate and bone strength. Nevertheless, treatment with MPP weakened naringin-triggered expression of ERα and ALP and improved bone healing and mass. Therefore, naringin could improve osteoblast mineralization and bone healing via regulating ERα-dependent ALP gene expression. Naringin can be clinically applied for treatment of osteoporosis-related bone diseases.

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 μm pores at a density of (2 ± 0.4) × 10(6) pores per cm(2). Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ~ 1,400 ohms•cm(2) at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.

Hydroxyapatite (HA) is a highly regarded synthetic bone graft material. Porous HA ceramics blocks are used to substitute harvested natural bone grafts. Being similar to bone mineral, HA material integrates with the host bone through surface osteointegration and slowly resorb along with the natural bone remodeling process. The blocks in use currently have random and tortuous pore structures. The present work explores the usefulness of cage-like HA ceramic design with end-to-end open pores, with the help of in vitro cell culture methods. Such a structure, on implantation, will take up the blood factors and cells and host the bone remodeling process inside the bulk of the cage, leading to early healing. In the study, HA samples with aligned through-pores were prepared and explored in vitro, with a focus on how the pores host the cells inside and to what level the cells maintain their activity. Human osteoblast-like cells (HOS) were used, at different seeding and culturing approaches. Cell seeding was done through (i) conventional large volume cell suspension, (ii) a confined mini chamber with a limited volume of cell suspension, and (iii) placing a concentrated drop of cell suspension directly on top of the scaffold. The third approach gave the best cell adhesion and proliferation, and hence used for further explorations. A dynamic culture system was designed in-house by bifurcating the cell culture wells using vertical inserts, holding the samples horizontally with their ends open to both sides, and making the media flow across using a rocker platform. The HOS cell adhesion, viability and proliferation were tested in the HA cages, in static and dynamic culture conditions, with conventional porous ceramics as the control. The cell infiltration was deeper and the cell viability over a period of 7 days was significantly higher in dynamic culture conditions in the test samples.

Vitamin D3, vitamin K2, and Mg (10%, 1.25%, and 5%, w/w, respectively)-loaded PLA (12%, w/v) (TCP (5%, w/v))/PCL (12%, w/v) 1:1 (v/v) composite nanofibers (DKMF) were produced by electrospinning method (ES) and their osteoinductive effects were investigated in cell culture test. Neither pure nanofibers nor DKMF caused a significant cytotoxic effect in fibroblasts. The induction of the stem cell differentiation into osteogenic cells was observed in the cell culture with both DKMF and pure nanofibers, separately. Vitamin D3, vitamin K2, and magnesium demonstrated to support the osteogenic differentiation of mesenchymal stem cells by expressing Runx2, BMP2, and osteopontin and suppressing PPAR-γ and Sox9. Therefore, the Wnt/β-catenin signaling pathway was activated by DKMF. DKMF promoted large axonal sprouting and needle-like elongation of osteoblast cells and enhanced cellular functions such as migration, infiltration, proliferation, and differentiation after seven days of incubation using confocal laser scanning microscopy. The results showed that DKMF demonstrated sustained drug release for 144 h, tougher and stronger structure, higher tensile strength, increased water up-take capacity, decreased degradation ratio, and slightly lower Tm and Tg values compared to pure nanofibers. Consequently, DKMF is a promising treatment approach in bone tissue engineering due to its osteoinductive effects.

The gingival medicinal signaling cells conditioned medium (GMSCs-CM) is a biocompatible material which possessed beneficial cytokine, anti-microbial peptide, growth factor that can be collected after culture. GMSCs- CM may inhibit bone resorption in order to improve the patient’s quality of life. In this study, the potential effect of GMSCs-CM on the number of osteoclasts and osteoblasts in Lipopolysaccharide (LPS)-induced calvaria bone resorption in wistar rats (Rattus novergicus) has been analyzed. Twenty-eight male and healthy wistar rats (R. novergicus) at the age of 1-2 months old with 250-300 grams body weight were divided into 4 groups, namely PBS group: 100μg PBS day 1-7; LPS group: 100μg LPS day 1-7; LPS and GMSCs group: 100μg LPS + 100μg GMSCS-CM day 1 1-7, GMSCs group: 100μg M-GMSCs day 1-7. Escherichia Coli LPS was used to induce the bone resorption on the calvaria with subcutaneous injection. GMSCs-CM was collected after passage 4-5 then injected subcutaneously on the calvaria. All samples were examined on the. 8th day through cervical dislocation. The number of osteoblasts and osteoclasts in calvaria was then observed under 400x magnification. One Way ANOVA and Tukey HSD were conducted to analyze differences between groups (p

Lactoferrin, as the main component of milk, can maintain osteoblast formation, which is conducive to the prevention and treatment of osteoporosis. Lactoferrin also serves as an autophagy regulator, especially in osteoblasts. This study aimed to explore the significance of autophagy in osteoblast formation regulated by lactoferrin and the internal mechanism. In this study, we firstly explored the roles of lactoferrin in the autophagy activity of primary osteoblasts (LC3 transformation rate, autophagosome formation). Subsequently, we further investigated the effects of lactoferrin on the BCL2 expression and BCL2-Beclin1 complex. Ultimately, the significance of BCL2 overexpression and Beclin1 silencing on lactoferrin-regulated osteoblast autophagy and osteogenic parameters (ALP activity and mRNA expression of PCNA, Col1, BGLAP and OPN) was observed by gene processing, respectively. Our results showed that lactoferrin enhanced the autophagy activity of osteoblasts. Importantly, lactoferrin inhibited BCL2 expression and the co-immunoprecipitation of BCL2 and Beclin1 in osteoblasts. Moreover, lactoferrin-promoted autophagy and osteogenic parameters was reversed by BCL2 overexpression or Beclin1 silencing in osteoblasts. In conclusion, lactoferrin can inhibit BCL2 expression in osteoblasts, further enhancing Beclin1-dependent autophagy activation.

The use of BMP-2 in orthopedic surgery is limited by uncertainty surrounding its effects on the differentiation of mesenchymal stem cells (MSCs) and how this is affected by cellular aging. This study compared the effects of recombinant human BMP-2 (rhBMP-2) on osteogenic and adipogenic differentiation between senescent and non-senescent MSCs. Senescent and non-senescent MSCs were cultured in osteogenic and adipogenic differentiation medium containing various concentrations of rhBMP-2. The phenotypes of these cells were compared by performing a calcium assay, adipogenesis assay, staining, real-time PCR, western blotting, and microarray analysis. rhBMP-2 induced osteogenic differentiation to a lesser extent (P

Background The long-term use of dexamethasone (Dex), a well-known immunosuppressant, leads to an imbalance in bone metabolism and rapid decline of bone mineral density due to apoptosis of osteoblasts. The molecular mechanisms by which Dex induces osteoblast apoptosis remain unclear. Materials and methods MC3T3-E1 cells were treated with 0, 10−8, 10−6, and 10−4 M Dex for 24 h. ATF6, phosphorylated PERK, PERK, phosphorylated IRE1, and IRE1 expression, cell apoptosis, and caspase-12 and caspase-3 activity were measured. CHOP expression and calcium ion influx rate were measured in cells treated with 0 and 10−4 M Dex for 24 h. The effect of 2-APB treatment was assessed in cells treated with 0 or 10−4 M Dex. Results Levels of ATF6 and phosphorylated PERK and IRE1 increased in a dose-dependent manner in MC3T3-E1 cells treated with 10−8, 10−6, and 10−4 M Dex, compared to the control group (P −6 and 10−4 M Dex had significantly increased apoptotic rates and caspase-12 and caspase-3 activities (P −4 M Dex had significantly increased CHOP levels and calcium ion influx rates (P −4 M Dex and 2-APB abrogated the observed increases in cell apoptosis and caspase-12 and caspase-3 activities (P Conclusions High doses of Dex induce CHOP expression by promoting calcium ion influx-dependent induction of ATF6, phosphorylated PERK and phosphorylated IRE1, which induce endoplasmic reticulum stress-mediated apoptosis in osteoblasts. 2-APB protects the osteoblasts from the effects of Dex, preventing endoplasmic reticulum stress-mediated apoptosis.

BACKGROUND: Bone defect difficult to manage clinically and it is a big challenge to repair it. Secondary metabolites source from herb has shown potential for the treatment of bone defect. METHODS: Mesenchymal stem cells (MSCs) were isolated from mice and incubated with urolithin A (UA) (10, 25, and 50 µg/mL). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to estimate apoptosis and mineralisation was evaluated by alkaline phosphatase assay and alizarin red S staining. A middle femoral defect was induced in mice and bone tissue was prepared for endochondral ossification by treating with UA. The effect of UA was estimated by determining markers of osteoblast proliferation in serum and micro-computed tomography to analyse bone defects. RESULTS: UA enhanced mineralisation of MSCs and osteogenic gene markers in MSCs in vitro. Also, the bone defect score and bone mineral density were improved by UA. Moreover, UA ameliorated the altered Wnt3a protein and histopathological changes in bone defect mice. CONCLUSION: Presented report conclude that UA enhances osteoblast proliferation in bone-defect mice by activating the Wnt pathway.

ObjectiveThis study aimed to investigate the effect of small molecules incorporated into the engineered nanofibrous scaffold to enhance the osteoblast differentiationMaterials and methodsPoly-ε-caprolactone (PCL) nanofiber matrices with lithium chloride (LiCl) were fabricated using the electrospinning technique. Scaffolds were characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX). Scaffolds were seeded with MC3T3-E1 cells and assessed using Western blots (β-catenin), alamarBlue assay (proliferation), qPCR (osteoblast differentiation), and mineralization (Alizarin Red staining).ResultsWe observed LiCl nanofiber scaffolds induced concentration-dependent cell proliferation that correlated with an increased β-catenin expression indicating sustained Wnt signaling. Next, we examined osteoblast differentiation markers such as osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) and noted increased expression in LiCl nanofiber scaffolds. We also noted increased bone morphogenetic protein (BMP-2, 4, and 7) expressions suggesting activated Wnt can promote cures to further osteogenic differentiation. Finally, Alizarin Red staining demonstrated increased mineral deposition in LiCl-incorporated nanofiber scaffolds.ConclusionsTogether, these results indicated that LiCl-incorporated nanofiber scaffolds enhance osteoblast differentiation.Clinical relevanceSmall molecule-incorporated nanofibrous scaffolds are an innovative clinical tool for bone tissue engineering.

Osteoporosis is a systemic bone metabolism disorder, which increases the risk of fractures, and in severe cases it may cause disability or even death. An important factor contributing to osteoporosis is the imbalance between bone formation and resorption. Naringin was reported to promote osteoblast differentiation, thus enhancing bone formation and alleviating osteoporosis development. However, the signalling pathways related to the regulatory mechanism of naringin in osteoporosis development are not clear. Proliferation of bone mesenchymal stem cells (BMSCs) treated with naringin in vitro was detected by CCK-8. An osteogenesis differentiation medium supplemented with naringin was applied to explore the effects of naringin on BMSC osteogenic differentiation, as detected by Alizarin red staining. Ovariectomy (OVX)-induced postmenopausal osteoporosis (PMOP) rats were orally administered with naringin. Dual-energy X-ray absorptiometry (DEXA) and micro-CT were applied to measure bone mineral density (BMD), bone volume/total volume (BV/TV), trabecula thickness (Tb.Th), trabecula number (Tb.N), trabecular separation (Tb.Sp) and bone surface/bone volume (BS/BV). H&E staining was performed to show pathological changes of the femur in PMOP rats after naringin treatment. Bone metabolism indicators were assessed by ELISA. We found that naringin suppressed the activation of the JAK2/STAT3 pathway. Naringin promoted BMSC proliferation and osteogenic differentiation. Furthermore, naringin alleviates bone loss and improves abnormal bone metabolism of PMOP rats. Collectively, naringin promotes BMSC osteogenic differentiation to ameliorate osteoporosis development by targeting JAK2/STAT3 signalling.

Daily walking or exercise of the bone implant recipients may generate particles due to wear and tear. Reports have mentioned that particles could circulate in the human body and trigger aseptic loosening, inflammation, and other potential complications. The mechanism of these phenomena remains mostly unclear. This study is to investigate the cytotoxicity of titanium (Ti), stainless steel 316 (SS316), and magnesium (Mg) particles due to these materials are the most commonly used biomaterials based on their adequate mechanical properties and excellent biocompatibility. Human osteoblasts (SAOS2 cells) were exposed directly to different concentrations of Ti/SS316/Mg particle during the direct cytotoxicity test. Together with the previous study, we found out that Ti particles showed cytotoxicity to osteoblasts at different dosages and times, while SS316 particles and Mg particles (low dosage) can reduce the cytotoxicity induced by Ti particles and boost cell viability. Mg particles can be toxic to osteoblast at a higher dosage, while SS316 particles are “safer” than Mg particles at higher dosages. Cell viability and cell morphology of SAOS2 cells under different treatments were observed at 2/3/5 days. This study found out that cell viability could be enhanced with certain combinations of Ti/SS316/Mg particles. This can give us certain guideline on how to design and fabricate a hybrid bone implant. However, how to quantify the particles inside the human body in real-time, and the exact interaction among particles, cells, tissues, and even organs require further research.

Novel multifunctional biomimetic injectable hybrid systems were synthesized. The physicochemical as well as biological in vitro and in vivo tests demonstrated that they are promising candidates for bone tissue regeneration. The hybrids are composed of a biopolymeric collagen/chitosan/hyaluronic acid matrix and amine group-functionalized silica particles decorated with apatite to which the alendronate molecules were coordinated. The components of these systems were integrated and stabilized by cross-linking with genipin, a compound of natural origin. They can be precisely injected into the diseased tissue in the form of a viscous sol or a partially cross-linked hydrogel, where they can serve as scaffolds for locally controlled bone tissue regeneration/remodeling by supporting the osteoblast formation/proliferation and maintaining the optimal osteoclast level. These materials lack systemic toxicity. They can be particularly useful for the repair of small osteoporotic bone defects.

Dysfunctional changes and remodeling of adipose tissue (АT) are associated with the formation of microcalcifications in the vascular wall. Biologically active substances synthesized by АT (adipocytokines) can act as promoters and inhibitors of vascular calcification development. The few available experimental and clinical studies do not fully explain the possible mechanisms of these effects.Aim. To study the relationships between the adipocytokine profiles of adipocytes in epicardial and perivascular AT with the severity of coronary artery calcification in patients with coronary artery disease (CAD).Material and Methods. A total of 125 patients with CAD aged 59 (53; 66) years were examined. The isolated adipocytes of subcutaneous adipose tissue (SAT), epicardial adipose tissue (EAT), and perivascular adipose tissue (PVAT), obtained during coronary artery bypass grafting, were used to determine gene expression and secretion of adipocytokines (adiponectin, leptin, and IL-6). Expression of adipocytokine genes was assessed using quantitative PCR with detection of products in real time (real-time qPCR); the concentration of adipocytokines in the culture medium was determined by enzyme-linked immunosorbent assay using R&D Systems kits (Canada). Coronary artery (CA) calcification degree was assessed by multislice spiral computed tomography (MSCT) method. The calcium index of CA was determined by the Agatston method using the Syngo Calcium Scoring software package (Siemens AG Medical Solution, Germany).Results. Massive coronary calcification (CC) had the highest prevalence (58.8%) in patients with CAD. The highest level of expression of the ADIPOQ gene in all types of fat stores was observed in patients with moderate/medium CС compared to those with massive CС; the maximum expression of ADIPOQ was observed in the culture of PVAT adipocytes. Expression of the LEP and IL6 genes in massive CC was higher, with the maximum values in the culture of EAT adipocytes relative to SAT and PVAT adipocytes. Decreases in the levels of ADIPOQ mRNA and its secretion, increases in the levels of mRNA of LEP and IL6 and their secretion in adipocytes of the EAT and PVAT were associated with the development of СС in patients with CAD.Conclusion. Proinflammatory adipokines produced by adipocytes of patients with CAD during hypoxia induced vascular calcification by stimulating oxidative stress, osteoblast differentiation, apoptosis, and proliferation of smooth muscle cells. Endothelial cells, when stimulated with proinflammatory adipocytokines, tended to transform into osteoblasts, which further aggravated the degree of vascular inflammation and calcification.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

Objectives: Osteoporosis is a metabolic bone disease caused by various factors. As a prosthetic group of haemoglobin, heme can promote the formation of blood vessels and the regeneration of related cells by increasing the proliferation level of endothelial cells and reticulocytes. This study observed the anti-osteoporosis effect of heme on preosteoblast (POB) cells and rat models. Methods: Heme mimics were transfected into POB cells of patients in stable culture, and the effect of heme transfection on the activity of POB cells was detected via the cell scratch test and cell migration assay. 45 SD female rats were randomly divided into group A, the rats were only treated with surgery (n = 15); group B, the rats were given 30 mg/kg of heme chloride by tail vein injection on the 1st, 7th, and 14th days after surgery (n = 15); group C, rats were injected with the same amount of saline in the tail vein (n = 15). The mRNA and protein expressions of alkaline phosphatase (ALP), bone-specific alkaline phosphatase (B-ALP), and type I collagen C-terminal peptide (CTX) in rats serum were detected via western blot and real-time PCR (real-time polymerase chain reaction). The expressions of IL-6 and TNF-α in rats serum were detected via Elisa. The pathological changes in the morphology of distal femur were observed by Hematoxylin-eosin staining. Results: After transfected with heme mimics, the proliferation, migration and invasion ability of the patient’s cells increased significantly, and the apoptosis rate of the cells decreased significantly. The relative expression levels of ALP and CTX mRNA and protein in serum of osteoporosis patients and rat significantly decreased 24 hours after transfection of heme mimics (p < 0.05), while B-ALP significantly increased. The expression of IL-6 and TNF-α in the rat model of osteoporosis group significantly increased (p < 0.05), but after transfection with heme mimic, the expression of IL-6 and TNF-α in the rat reduced significantly. HE showed that, after transfection, the femoral trabeculae were substantially broader and thicker, and the number also increased significantly. The number of trabecular fractures and fat cells was decreased, the trabecular bones were smoother and close to the control group. Conclusion: Heme can significantly promote the proliferation, migration and invasion of POB cells in the body, and also can achieve the anti-osteoporosis effect by adjusting the expression of ALP, B-ALP, and CTX, IL-6 and TNF-α in osteoporosis model of rat.

Aim Demineralized dentin material membrane (DDMM) is a novel bioresorbable guided bone regeneration (GBR) which is derived from the demineralization process of bovine dentin. This material/process could be an alternative to resolve musculoskeletal dysfunction that harms the quality of human life. Purpose To evaluate the cytotoxic effect of DDMM as GBR membrane on MC3T3-E1 osteoblast cell line. Methods Cytotoxic effect was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteoblast MC3T3-E1 cell culture was used as a parameter of cell viability after reacting with GBR materials. The absorbance values were examined at each treatment to determine the percentage of cell viability. There were four groups created in the present study: two treatment groups and two control groups. The treatment groups consisted of a DDMM group and a bovine pericardium collagen membrane (BPCM) group. The control groups comprised a group containing cell culture medium as a negative control group and another positive control group that contained cell cultures. Results The results revealed no significant difference in MC3T3-E1 cell viability between the treatment and control groups (p < 0.05). Moreover, as observed in the DDMM group, there was an increase in the number of osteoblast cells. Conclusion DDMM is a suitable alternative biomaterial for GBR as it is non-cytotoxic and could potentially increase the rate of repair of craniofacial defects., Video Abstract Point your SmartPhone at the code above. If you have a QR code reader the video abstract will appear. Or use: https://youtu.be/-9wbabBPZIo.

Objective The present study aimed to evaluate the effect of a high water-soluble curcuminoids-rich extract (CRE) in a solid dispersion form (CRE-SD) using polyvinylpyrrolidone K30 on osteogenic induction of MC3T3-E1 cells. Methods CRE was pre-purified using a microwave assisted extraction couple with a Diaion® HP-20 column chromatography. The osteoblastic cell proliferation and differentiation potentials of CRE-SD in MC3T3-E1 cells were tested by cell viability, alkaline phosphatase (ALP) activity, and Alizarin red S activity assays. The mRNA expressions of osteoblast-specific genes and underline mechanisms were assessed by a real time PCR and western blot analysis. Results CRE-SD 50 µg/mL increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts in both MC3T3-E1 cells and non-osteogenic mouse pluripotent cell line, C3H10T1/2, indicating the action of CRE-SD was not cell-type specific. Alizarin red S activity showed a significant amount of calcium deposition in cells treated with CRE-SD. CRE-SD also upregulated the mRNA expression levels of transcription factors that favor osteoblast differentiation including Bmp-2, Runx2 and Collagen 1a, in a dose dependent manner. Western blot analysis revealed that noggin attenuated CRE-SD-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt/β-catenin signaling pathway also annulled the influence of CRE-SD, indicating Wnt/β-catenin dependent activity. Inhibition of the different signaling pathways abolished the influence of CRE-SD on ALP activity, confirming that CRE-SD induced MC3T3-E1 cells into osteoblasts through Wnt/β-catenin and BMP signaling pathway. Conclusion These results collectively demonstrate that CRE-SD may be a potential therapeutic agent for the treatment of osteoporosis.

Made available in DSpace on 2022-04-29T08:30:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process. Universidade Federal de São Paulo - UNIFESP Escola Paulista de Medicina - EPM Departamento de Morfologia e Genética Disciplina de Histologia e Biologia Estrutural Universidade Federal de São Paulo - UNIFESP Escola Paulista de Medicina - EPM Departamento de Ginecologia São Paulo State University (UNESP) School of Dentistry Araraquara - Department of Morphology Genetics Orthodontics and Pediatric Dentistry - Laboratory of Histology and Embryology São Paulo State University (UNESP) School of Dentistry Araraquara - Department of Morphology Genetics Orthodontics and Pediatric Dentistry - Laboratory of Histology and Embryology CAPES: 001 FAPESP: 2012/22666-8