Active Bone Formation Research Articles

Type-H endothelial cell protein Clec14a orchestrates osteoblast activity during trabecular bone formation and patterning

Type-H capillary endothelial cells control bone formation during embryogenesis and postnatal growth but few signalling mechanisms underpinning this influence have been characterised. Here, we identify a highly expressed type-H endothelial cell protein, Clec14a, and explore its role in coordinating osteoblast activity. Expression of Clec14a and its ligand, Mmrn2 are high in murine type-H endothelial cells but absent from osteoblasts. Clec14a−/− mice have premature condensation of the type-H vasculature and expanded distribution of osteoblasts and bone matrix, increased long-bone length and bone density indicative of accelerated skeletal development, and enhanced osteoblast maturation. Antibody-mediated blockade of the Clec14a-Mmrn2 interaction recapitulates the Clec14a−/− phenotype. Endothelial cell expression of Clec14a regulates osteoblast maturation and mineralisation activity during postnatal bone development in mice. This finding underscores the importance of type-H capillary control of osteoblast activity in bone formation and identifies a novel mechanism that mediates this vital cellular crosstalk.

Micro-arc oxidation for balancing antibacterial activity and bone formation on titanium surface

Ti surfaces must balance bone formation and antibacterial activity to achieve bone reconstruction and prevent infections. In this study, a Ti surface was electrochemically modified via micro-arc oxidation in an electrolyte containing calcium ions, phosphate ions, and either Ag, or Cu ions to form an antibacterial porous oxide layer on Ti surface. The resultant porous oxide layers consisted of both electrolyte and substrate elements. The Ag- and Cu-incorporated porous oxide layers exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Furthermore, animal experiments were performed to reveal the bone-formation on the Ag- and Cu-incorporated porous oxide layers. Both porous oxide layers promoted bone formation more effectively than untreated Ti. Thus, the Ag- and Cu-incorporated porous oxide layers formed on the Ti surface by micro-arc oxidation balanced bone formation and antibacterial activity owing to their structural and compositional features. Our findings will be useful for the surface design of Ti-based medical devices and can provide a simple electrochemical treatment to form porous surfaces to balance antibacterial activity and bone formation on Ti surfaces.

LINC01133 promotes the osteogenic differentiation of bone marrow mesenchymal stem cells by upregulating CTNNB1 by acting as a sponge for miR-214-3p

BackgroundOsteoporosis results from decreased bone mass and disturbed bone structure. Human bone marrow mesenchymal stem cells (hBMSCs) demonstrate robust osteogenic differentiation, a critical process for bone formation. This research was designed to examine the functions of LINC01133 in osteogenic differentiation.MethodsDifferentially expressed lncRNAs affecting osteogenic differentiation in hBMSCs were identified from the GEO database. A total of 74 osteoporosis patients and 70 controls were enrolled. hBMSCs were stimulated to undergo osteogenic differentiation using an osteogenic differentiation medium (OM). RT-qPCR was performed to evaluate LINC01133 levels and osteogenesis-related genes such as osteocalcin, osteopontin, and RUNX2. An alkaline phosphates (ALP) activity assay was conducted to assess osteogenic differentiation. Cell apoptosis was detected using flow cytometry. Dual luciferase reporter assay and RIP assay were employed to investigate the association between miR-214-3p and LINC01133 or CTNNB1. Loss or gain of function assays were conducted to elucidate the impact of LINC01133 and miR-214-3p on osteogenic differentiation of hBMSCs.ResultsLINC01133 and CTNNB1 expression decreased in osteoporotic patients but increased in OM-cultured hBMSCs, whereas miR-214-3p showed an opposite trend. Depletion of LINC01133 suppressed the expression of genes associated with bone formation and ALP activity triggered by OM in hBMSCs, leading to increased cell apoptosis. Nevertheless, this suppression was partially counteracted by the reduced miR-214-3p levels. Mechanistically, LINC01133 and CTNNB1 were identified as direct targets of miR-214-3p.ConclusionsOur study highlights the role of LINC01133 in positively regulating CTNNB1 expression by inhibiting miR-214-3p, thereby promoting osteogenic differentiation of BMSCs. These findings may provide valuable insights into bone regeneration in osteoporosis.

Developing long bones respond to surrounding tissues by trans-pairing of periosteal osteoclasts and endocortical osteoblasts.

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone-forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles, and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury.

Sclerostin and Wnt Signaling in Idiopathic Juvenile Osteoporosis Using High-Resolution Confocal Microscopy for Three-Dimensional Analyses.

Idiopathic juvenile osteoporosis (IJO) is a rare condition characterized by low bone mass that can increase the risk of fractures in children. Treatment options for these patients are limited as the molecular mechanisms of disease initiation and progression are incompletely understood. Sclerostin inhibits canonical Wnt signaling, which is important for the bone formation activity of osteoblasts, and elevated sclerostin has been implicated in adult osteoporosis. To evaluate the role of sclerostin in IJO, high-resolution confocal microscopy analyses were performed on bone biopsies collected from 13 pediatric patients. Bone biopsies were stained with sclerostin, and β-catenin antibodies showed elevated expression across osteocytes and increased sclerostin-positive osteocytes in 8 of the 13 total IJO patients (62%). Skeletal sclerostin was associated with static and dynamic histomorphometric parameters. Further, colocalization analyses showed that bone sclerostin colocalized with phosphorylated β-catenin, a hallmark of Wnt signaling that indicates Wnt inhibition. In contrast, sclerostin-positive osteocytes were not colocalized with an "active" unphosphorylated form of β-catenin. These results support a model that altered levels of sclerostin and Wnt signaling activity occur in IJO patients.

3D Printed Calcium Phosphate Physiochemically Dual-Regulating Pro-Osteogenesis and Antiosteolysis for Enhancing Bone Tissue Regeneration.

Osteoblasts and osteoclasts are two of the most important types of cells in bone repair, and their bone-forming and bone-resorbing activities influence the process of bone repair. In this study, we proposed a physicochemical bidirectional regulation strategy via ration by physically utilizing hydroxyapatite nanopatterning to recruit and induce MSCs osteogenic differentiation and by chemically inhibiting osteolysis activity through the loaded zoledronate. The nanorod-like hydroxyapatite coating was fabricated via a modified hydrothermal process while the zoledronic acid was loaded through the chelation within the calcium ions. The fabrication of a hydroxyapatite/zoledronic acid composite biomaterial. This biomaterial promotes bone tissue regeneration by physically utilizing hydroxyapatite nanopatterning to recruit and induce MSCs osteogenic differentiation and by chemically inhibiting osteolysis activity through the loaded zoledronate. The nanorod-like hydroxyapatite coating was fabricated via a modified hydrothermal process while the zoledronic acid was loaded through the chelation within the calcium ions. The in vitro results tested on MSCs and RAW 246.7 indicated that the hydroxyapatite enhanced cells' physical sensing system, therefore enhancing the osteogenesis. At the same time the zoledronic acid inhibited osteolysis by downregulating the RANK-related genes. This research provides a promising strategy for enhancing bone regeneration and contributes to the field of orthopedic implants.

Investigation of the Effects of Thymoquinone and Dental Pulp-Derived Mesenchymal Stem Cells on Tibial Bone Defect Models.

The thymoquinone obtained from Nigella sativa increases osteoblastic activity and significantly reduces the number of osteoclasts, thereby accelerating bone healing. In addition, mesenchymal stem cells isolated from various tissues are considered a potential cell source for bone regenerative therapies. The aim of this study is to investigate the effectiveness of thymoquinone, a current and novel agent, in combination with mesenchymal stem cells derived from the dental pulp in promoting bone healing. In the study, 28 male Sprague Dawley rats were used. The rats were divided into 4 groups, each consisting of 7 rats: the control group (group 1) (n=7), thymoquinone group (group 2) (n=7), stem cell group (group 3) (n=7), stem cell+thymoquinone group (group 4) (n=7). A bone defect of 4mm in diameter and 5mm in length was created in the left tibial bones of all rats with a trephine bur. In group 1, no procedure was applied to the defect area. Group 2 was applied thymoquinone (10mg/kg) with oral gavage. In group 3, stem cells were used locally to the defect area. In group 4, stem cells and thymoquinone (10mg/kg) was applied to the defect area. All rats were killed on the 28th day of the experiment. Tibia tissues extracted during sacrifice were histomorphologically examined in a fixative solution. Significant differences were found in terms of new bone formation and osteoblastic activity values in the "thymoquinone" ( P <0.05), "stem cell" ( P <0.05), and "stem cell+thymoquinone" ( P <0.05) groups compared to the "control" group. In addition, while there was no significant difference in the "thymoquinone" group compared to these stem cell+thymoquinone group in terms of osteoblastic activity ( P >0.05), the difference in terms of new bone formation was found to be significantly lower. No significant differences among the other groups were observed in new bone formation and osteoblastic activity ( P >0.05). According to the results of our study, stem cell+thymoquinone treatment for bone defects is not only more effective than thymoquinone or stem cell treatment alone but also induces greater development of bone trabeculae, contributes to the matrix and connective tissue formation, and increases the number of osteoblasts and osteocytes involved in bone formation.

Bone-to-Implant Contact in Implants with Plasma-Treated Nanostructured Calcium-Incorporated Surface (XPEEDActive) Compared to Non-Plasma-Treated Implants (XPEED): A Human Histologic Study at 4 Weeks

Titanium implants undergo an aging process through surface hydrocarbon deposition, resulting in decreased wettability and bioactivity. Plasma treatment was shown to significantly reduce surface hydrocarbons, thus improving implant hydrophilicity and enhancing the osseointegration process. This study investigates the effect of plasma surface treatment on bone-to-implant contact (BIC) of implants presenting a nanostructured calcium-incorporated surface (XPEED®). Following a Randomized Controlled Trial (RCT) design, patients undergoing implant surgery in the posterior maxilla received additional plasma-treated (n = 7) or -untreated (n = 5) 3.5 × 8 mm implants that were retrieved after a 4-week healing period for histological examination. Histomorphometric analysis showed that plasma-treated implants exhibited a 38.7% BIC rate compared to 22.4% of untreated implants (p = 0.002), indicating enhanced osseointegration potential. Histological images also revealed increased bone formation and active osteoblastic activity around plasma-treated implants when compared to untreated specimens. The findings suggest that plasma treatment improves surface hydrophilicity and biological response, facilitating early bone formation around titanium implants. This study underscores the importance of surface modifications in optimizing implant integration and supports the use of plasma treatment to enhance osseointegration, thereby improving clinical outcomes in implant dentistry and offering benefits for immediate and early loading protocols, particularly in soft bone conditions.

A comparative investigation of the effects of Resveratrol and dental pulp delivered mesenchimal stem cells on rat tibia bone defect healing

Resveratrol (3,4,5–trihydroxystilbene), an antioxidant compound, has a natural phytoalexin structure and also has many properties such as anti–inflammatory, antineoplastic and antiplatelet. In addition, mesenchymal stem cells isolated from various tissues are considered as a potential cell source for bone regenerative therapies. The present study aims to examine the effects of Resveratrol and dental pulp–derived mesenchymal stem cells on new bone formation in rats, both isolated and combined, by immunohistochemical methods. Twenty eigth Spraque Dawley male rats were used in the study. The rats were divided into four groups with seven rats in each group; the control group (Group 1) (n=7), the Systemic Resveratrol group (Group 2) (n=7), the Stem cell group (Group 3) (n=7), the Stem cell + Systemic Resveratrol group (Group 4) (n=7). A defect was opened on the tibia bones of the rats in all groups with a trephane bur (diameter of 3 mm and a length of 4 mm). After the 4–week experiment, all rats were sacrificed following the experimental protocols specific to each group. The specimens of tibia were subjected to histomorphological examination in fixative solutions. Values of inflammation, connective tissue formation, osteoclastic activity, osteoblast values, new bone formation, BMP2 and BMP4 expression levels obtained for all groups were evaluated by statistical analysis. Compared to the control group, new bone formation and osteoblastic activity were found to be significantly higher in the Stem cell group and Stem cell + Systemic Resveratrol group. (P=0.001) Additionally, new bone formation in the Systemic Resveratrol group was found to be significantly lower than in the Stem cell + Systemic Resveratrol group. (P=0.006) No significant difference was observed between other groups. (P&gt;0.05) According to the results of the study, it was observed that Stem cell + Resveratrol treatment was more effective than isolated Resveratrol or isolated stem cell treatment applications, it induced the development of more bone trabeculae, decrease inflammation and increased the number of osteoblasts involved in bone formation. In the light of these data, it was concluded that the combined use of Resveratrol and Stem cells is more effective on the healing of bone defects than their isolated use.

Treatment of non-hypertrophic pseudoarthrosis of long bones with a Tissue Engineered Product loaded with autologous bone marrow-derived Mesenchymal Stromal Cells: Results from a phase IIa, prospective, randomized, parallel, pilot clinical trial comparing to iliac crest autograft

BackgroundAtrophic pseudoarthrosis is a serious complication with an incidence of 5–10 % of bone fractures located in the diaphysis of long bones. Standard treatments involve aggressive surgical procedures and re-interventions requiring the use of autografts from the iliac crest as a source of bone-forming biological activity (Standard of Care, SoC). In this context, regenerative ex vivo expanded osteogenic cell-based medicines could be of interest. Particularly, Mesenchymal Stromal Cells (MSC) offer new prospects to promote bone tissue repair in pseudoarthrosis by providing biological activity in an osteoconductive and osteoinductive environment. MethodsWe conducted a phase IIa, prospective, randomised, parallel, two-arms, open-label with blinded assessor pilot clinical trial to compare SoC vs. a tissue-engineered product (TEP), composed of autologous bone marrow (BM)-derived MSCs loaded onto allogeneic decellularised, lyophilised spongy bone cubes, in a cohort of 20 patients with non-hypertrophic pseudoarthrosis of long bones. Patients were followed up for 12 months. Radiological bone healing was evaluated by standard X-ray and computed tomography (CT) scanning. Quality of life was measured using the EUROQOL-5D questionnaire. ResultsTen patients were randomized to TEP and 10 to SoC with iliac crest autograft. Manufacturing of TEP was feasible and reproducibly achieved. TEP implantation in the bone defect was successful in all cases and none of the 36 adverse events (AE) reported were related to the treatment. Efficacy analyses were performed in the Full Analysis Set (FAS) population, which included 17 patients after 3 patients withdrew from the study. The degree of consolidation, estimated by measuring Hounsfield units (HU) on CT, showed no significant differences between the two treatment groups at 12 months post treatment (main efficacy variable) (p = 0.4835) or at 6 months. ConclusionsAlthough only a small number of patients were included in our study, it is notable that no significant differences were observed between the experimental treatment and SoC, thus suggesting TEP as an alternative where autograft is not available or contraindicated.

Combining raloxifene and mechanical loading improves bone composition and mechanical properties in a murine model of chronic kidney disease (CKD)

IntroductionPatients with chronic kidney disease (CKD) are at an alarming risk of fracture compared to age and sex-matched non-CKD individuals. Clinical and preclinical data highlight two key factors in CKD-induced skeletal fragility: cortical porosity and reduced matrix-level properties including bone hydration. Thus, strategies are needed to address these concerns to improve mechanical properties and ultimately lower fracture risk in CKD. We sought to evaluate the singular and combined effects of mechanical and pharmacological interventions on modulating porosity, bone hydration, and mechanical properties in CKD. MethodsSixteen-week-old male C57BL/6J mice underwent a 10-week CKD induction period via a 0.2 % adenine-laced casein-based diet (n = 48) or remained as non-CKD littermate controls (Con, n = 48). Following disease induction (26 weeks of age), n = 7 CKD and n = 7 Con were sacrificed (baseline cohort) to confirm a steady-state CKD state was achieved prior to the initiation of treatment. At 27 weeks of age, all remaining mice underwent right tibial loading to a maximum tensile strain of 2050 μƐ 3× a week for five weeks with the contralateral limb as a non-loaded control. Half of the mice (equal number CKD and Con) received subcutaneous injections of 0.5 mg/kg raloxifene (RAL) 5× a week, and the other half remained untreated (UN). Mice were sacrificed at 31 weeks of age. Serum biochemistries were performed, and bi-lateral tibiae were assessed for microarchitecture, whole bone and tissue level mechanical properties, and composition including bone hydration. ResultsRegardless of intervention, BUN and PTH were higher in CKD animals throughout the study. In CKD, the combined effects of loading and RAL were quantified as lower cortical porosity and improved mechanical, material, and compositional properties, including higher matrix-bound water. Loading was generally responsible for positive impacts in cortical geometry and structural mechanical properties, while RAL treatment improved some trabecular outcomes and material-level mechanical properties and was responsible for improvements in several compositional parameters. While control animals responded positively to loading, their bones were less impacted by the RAL treatment, showing no deformation, toughness, or bound water improvements which were all evident in CKD. Serum PTH levels were negatively correlated with matrix-bound water. DiscussionAn effective treatment program to improve fracture risk in CKD ideally focuses on the cortical bone and considers both cortical porosity and matrix properties. Loading-induced bone formation and mechanical improvements were observed across groups, and in the CKD cohort, this included lower cortical porosity. This study highlights that RAL treatment superimposed on active bone formation may be ideal for reducing skeletal complications in CKD by forming new bone with enhanced matrix properties.

Perbedaan Jumlah Hitung Osteoblas pada Pergerakan Ortodontik Gigi Setelah Pemberian Jintan Hitam

Osteoblasts are important for orthodontic tooth movement because they influence the activity of bone formation on the tension area. The administration of natural ingredients such as black cumin is proven to increase the number of osteoblasts because it contains thymoquinone as antioxidants to accelerate the process of tooth movement. The study aims to determine the differences in osteoblast count and their effect on the orthodontic movement of teeth after oral administration of black cumin. The method used in this research is experimental laboratory in vivo with a randomized post-test only control group design using 16 male Sprague-Dawley rat samples that were devided into four groups: the control group (K) with distilled water for 7 and 14 days, also the treatment group (P) with black cumin (Nigella sativa) for 7 and 14 days Elastomeric separators were placed on the upper jaw central incisors (right central incisor) using a separator applier to provide orthodontic mechanical force conditions. Alveolar bone tissue samples were taken after treatment then the number of osteoblasts was calculated histologically. Data were anaylzed using One Way Anova Test and LSD (p&lt;0.05). Result of this research indicated that there was an increase in the number of osteoblasts in orthodontic movement after oral administration of black cumin.

The roles of extracellular vesicles released by mechanically stimulated osteocytes in regulating osteoblast and osteoclast functions

Bone adapts to mechanical loading by changing its shape and mass. Osteocytes, as major mechanosensors, are critical for bone modeling/remodeling in response to mechanical stimuli. Intracellular calcium oscillation is one of the early responses in osteocytes, and this further facilitates bone cell communication through released biochemical signals. Our previous study has found that mechanically induced calcium oscillations in osteocytes enhance the release of extracellular vesicles (EVs), and those released EVs can elevate bone formation activity. However, the mechanism of mechanically stimulated EVs’ regulation of bone formation and resorption is still unclear. Here, using in vitro studies, we exposed OCY454 cells, with relatively high sclerostin expression, to steady fluid flow (SFF) and characterized the functions of rapidly released EVs in osteoblast and osteoclast regulation. Our study demonstrates that SFF stimulates intracellular calcium response in OCY454 cells and further induces sclerostin, osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) inside or outside EVs to regulate osteoblast and osteoclast activities. This load-induced protein and EVs release is load-duration dependent. Moreover, stimulated osteocytes rapidly regulate osteoclast maturation through EVs capsulated RANKL. In contrast, other regulating proteins, OPG, and sclerostin, are mainly released directly into the medium without EV capsulation.

Visualization of bone formation in sheep’s middle ear by using fluorochrome sequential labelling (FSL)

One factor for the lacking integration of the middle ear stapes footplate prosthesis or the missing healing of stapes footplate fractures could be the known osteogenic inactivity. In contrast, it was recently demonstrated that titanium prostheses with an applied collagen matrix and immobilised growth factors stimulate osteoblastic activation and differentiation on the stapes footplate. Regarding those findings, the aim of this study was to evaluate the potential of bone regeneration including bone remodeling in the middle ear. Ten one-year-old female merino sheep underwent a middle ear surgery without implantation of middle ear prostheses or any other component for activating bone formation. Post-operatively, four fluorochromes (tetracycline, alizarin complexion, calcein green and xylenol orange) were administered by subcutaneous injection at different time points after surgery (1 day: tetracycline, 7 days: alizarin, 14 days: calcein, 28 days: xylenol). After 12 weeks, the temporal bones including the lateral skull base were extracted and histologically analyzed. Fluorescence microscopy analysis of the entire stapes with the oval niche, but in particular stapes footplate and the Crura stapedis revealed evidence of new bone formation. Calcein was detected in all and xylenol in 60% of the animals. In contrast, tetracycline and alizarin could only be verified in two animals. The authors were able to demonstrate the osseoregenerative potential of the middle ear, in particular of the stapes footplate, using fluorescence sequence labelling.

Distraction osteogenesis in combined sequential use of external fixation and nailing (lengthening and then nailing): An experimental study in rabbits.

Limb lengthening relies on the process of distraction osteogenesis. The active periosteal bone formation has been detected in clinical practice with a lengthening and then nail (LATN) technique but has not been confirmed by experimental studies to date. The aim of this study is to compare the tissue regeneration of the distraction regenerate during tibial lengthening in rabbits using a LATN technique. This study was performed on 54 mature rabbits of the Soviet Chinchilla breed, which were divided into three groups of 18 animals. In group 1 (control), the tibia was lengthened in an external fixator. In group 2, the LATN technique was modeled and in group 3, lengthening over nail (LON) was modeled. The total duration of the experiment was 45 days. On the 10th, 15th, 20th, 30th, and 45th day X-ray, computed tomography and morphological studies were performed. In the experimental groups (2 and 3), a more pronounced periosteal bone formation in the area of regenerate was noted when compared to group 1. In group 2 (LATN), wide cortical plates were formed from the intermediate and periosteal areas. In this group, the maximum densitometric density values were noted. Endosteal bone formation was preserved in all groups. The LON and LATN techniques, when compared with the classical Ilizarov lengthening, do not demonstrate any deficiency in the tissue regeneration of the bone tissue at the regenerate sites. The most powerful bone structures are formed with the sequential use of the external fixation and nailing (LATN).

Long non-coding RNA-NONMMMUT004552.2 regulates the unloading-induced bone loss through the miRNA-15b-5p/Syne1 in mice

Exercise-induced mechanical loading can increase bone strength whilst mechanical unloading enhances bone-loss. Here, we investigated the role of lncRNA NONMMUT004552.2 in unloading-induced bone-loss. Knockout of lncRNA NONMMUT004552.2 in hindlimb-unloaded mice caused an increase in the bone formation and osteoblast activity. The silencing of lncRNA NONMMUT004552.2 also decreased the osteoblast apoptosis and expression of Bax and cleaved caspase-3, increased Bcl-2 protein expression in MC3T3-E1 cells. Mechanistic investigations demonstrated that NONMMUT004552.2 functions as a competing endogenous RNA (ceRNA) to facilitate the protein expression of spectrin repeat containing, nuclear envelope 1 (Syne1) by competitively binding miR-15b-5p and subsequently inhibits the osteoblast differentiation and bone formation in the microgravity unloading environment. These data highlight the importance of the lncRNA NONMMUT004552.2/miR-15b-5p/Syne1 axis for the treatment of osteoporosis.

Controlled domain gels with a wide stiffness gradient simultaneously promote bone regeneration and suppress tumor recurrence through DAPK activity

The reconstruction of bone defects following bone tumor resection pose significant challenges, including high risks for tumor recurrence. To address these challenges, we designed controlled-domain gels that possessed exceptional anti-tumorigenic effectiveness and osteogenic activity. This was achieved through the selection of an amino-acid-based low-molecular-weight gels (LMWGs) with superior biocompatibility and antitumor efficacy, and further construction of three-domain structure with a large-range stiffness gradient, through the assembly/disassembly of LMWGs inside a stable methacrylated alginate (AlgMA) network using photopolymerization. Such layered design not only constructed a top anti-tumorigenic domain at 0.6 kPa and a bottom osteogenic domain at 500 kPa, allowing for selectively inducing tumor cell apoptosis and promoting osteogenic differentiation of mesenchymal stem cells (MSCs), but also generated a gradient selectively confining the migration of tumor cells along soft-to-hard direction, without affecting the recruiting of MSCs toward the injury sites. In particular, the critical involvement of the DAPK-mediated signaling cascades might lead to the selected apoptosis of tumor cells on LMWG domain. The in-vivo results further suggested that multi-domain gel effectively inhibited tumor growth and displayed excellent bone-forming activity. Overall, such strategies could advance our fundamental understanding of the bone tumor postoperative treatment, in addition to offering new therapeutic opportunities.

2,4'-Dihydroxybenzophenone Exerts Bone Formation and Antiosteoporotic Activity by Stimulating the β-Catenin Signaling Pathway.

2,4'-Dihydroxybenzophenone (DHP) is an organic compound derived from Garcinia xanthochymus, but there have been no reports on its biochemical functions and bioavailability. In this study, we evaluated whether DHP affects osteoblast differentiation and activation in MC3T3-E1 preosteoblast cells, as well as antiosteoporotic activity in zebrafish larvae. Nontoxic concentrations of DHP-treated MC3T3-E1 preosteoblast cells increased alkaline phosphatase (ALP) activation and mineralization in a concentration-dependent manner, accompanied by higher expression of osteoblast-specific markers, including Runt-related transcription factor 2 (RUNX2), osterix, and ALP. Consistent with the data in MC3T3-E1 preosteoblast cells, DHP upregulated osteoblast-specific marker genes in zebrafish larvae and simultaneously enhanced vertebral formation. We also revealed that DHP increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 and the total expression of β-catenin in the cytosol and markedly increased the localization of β-catenin into the nucleus. Furthermore, DHP restored the prednisolone (PDS)-induced marked decrease in ALP activity and mineralization, as well as osteoblast-specific marker expression. In PDS-treated zebrafish, DHP also alleviated PDS-induced osteoporosis by restoring vertebral formation and osteoblast-related gene expression. Taken together, these results suggest that DHP is a potential osteoanabolic candidate for treating osteoporosis by stimulating osteoblast differentiation.

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

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

RNA-binding proteins in bone pathophysiology.

Bone remodelling is a highly regulated process that maintains mineral homeostasis and preserves bone integrity. During this process, intricate communication among all bone cells is required. Indeed, adapt to changing functional situations in the bone, the resorption activity of osteoclasts is tightly balanced with the bone formation activity of osteoblasts. Recent studies have reported that RNA Binding Proteins (RBPs) are involved in bone cell activity regulation. RBPs are critical effectors of gene expression and essential regulators of cell fate decision, due to their ability to bind and regulate the activity of cellular RNAs. Thus, a better understanding of these regulation mechanisms at molecular and cellular levels could generate new knowledge on the pathophysiologic conditions of bone. In this Review, we provide an overview of the basic properties and functions of selected RBPs, focusing on their physiological and pathological roles in the bone.