Stimulates Osteoblast Differentiation Research Articles

Hydroxychloroquine and a low antiresorptive activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects

Osteoporosis remains incurable. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation, while the anabolic agent, teriparatide, does not inhibit bone resorption, and thus they have limited efficacy in preventing osteoporotic fractures and cause some side effects. Thus, there is an unmet need to develop dual antiresorptive and anabolic agents to prevent and treat osteoporosis. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited marrow fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

Relaxin-2 promotes osteoblastic differentiation mediated by epidermal growth factor and epidermal growth factor receptor signaling.

Loss of osteogenic differentiation potential of osteoblasts has been associated with the pathogenesis of osteoporosis. Thus, stimulation of osteoblastic differentiation is a therapeutic strategy for osteoporosis. Relaxin-2 is a peptide hormone with potent biological functions. However, the effects of Relaxin-2 in osteoblastic differentiation and osteoporosis have not been reported before. Here, we report a novel physiological role of Relaxin-2 in promoting osteoblastic differentiation and mineralization of MC3T3-E1 cells. Our results indicate that exposure to Relaxin-2 upregulated the expression, and elevated the activity of alkaline phosphatase (ALP) when MC3T3-E1 cells were cultured in osteogenic differentiation medium (OM). Additionally, Relaxin-2 upregulated the mRNA levels of osteocalcin (ocn), osteopontin (opn), and collagen type I alpha 1 (Col1a1). The alizarin red S staining assay revealed that Relaxin-2 promoted the mineralization of MC3T3-E1 cells. We also found that Relaxin-2 increased the expression of Runx-2 as well as the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). Importantly, silencing of EGF abolished the effects of Relaxin-2 in osteoblastic differentiation and related gene expression. These findings suggest that Relaxin-2 stimulates osteogenic differentiation through activating EGF/EGFR signaling.

GREM2 inactivation increases trabecular bone mass in mice

Osteoporosis is a common skeletal disease affecting millions of individuals world-wide, with an increased risk of fracture, and a decreased quality of life. Despite its well-known consequences, the etiology of osteoporosis and optimal treatment methods are not fully understood. Human genetic studies have identified genetic variants within the FMN2/GREM2 locus to be associated with trabecular volumetric bone mineral density (vBMD) and vertebral and forearm fractures, but not with cortical bone parameters. GREM2 is a bone morphogenetic protein (BMP) antagonist. In this study, we employed Grem2-deficient mice to investigate whether GREM2 serves as the plausible causal gene for the fracture signal at the FMN2/GREM2 locus. We observed that Grem2 is moderately expressed in bone tissue and particularly in osteoblasts. Complete Grem2 gene deletion impacted mouse survival and body growth. Partial Grem2 inactivation in Grem2+/− female mice led to increased trabecular BMD of femur and increased trabecular bone mass in tibia due to increased trabecular thickness, with an unchanged cortical thickness, as compared with wildtype littermates. Furthermore, Grem2 inactivation stimulated osteoblast differentiation, as evidenced by higher alkaline phosphatase (Alp), osteocalcin (Bglap), and osterix (Sp7) mRNA expression after BMP-2 stimulation in calvarial osteoblasts and osteoblasts from the long bones of Grem2−/− mice compared to wildtype littermates. These findings suggest that GREM2 is a possible target for novel osteoporotic treatments, to increase trabecular bone mass and prevent osteoporotic fractures.

A Zn2+ cross-linked sodium alginate/epigallocatechin gallate hydrogel scaffold for promoting skull repair

The optimal material for repairing skull defects should exhibit outstanding biocompatibility and mechanical properties. Specifically, hydrogel scaffolds that emulate the microenvironment of the native bone extracellular matrix play a vital role in promoting osteoblast adhesion, proliferation, and differentiation, thereby yielding superior outcomes in skull reconstruction. In this study, a composite network hydrogel comprising sodium alginate (SA), epigallocatechin gallate (EGCG), and zinc ions (Zn2+) was developed to establish an ideal osteogenic microenvironment for bone regeneration. Initially, physical entanglement and hydrogen bonding between SA and EGCG resulted in the formation of a primary network hydrogel known as SA-EGCG. Subsequently, the inclusion of Zn2+ facilitated the creation of a composite network hydrogels named SA-EGCG-Zn2+ via dynamic coordination bonds with SA and EGCG. The engineered SA-EGCG2 %-Zn2+ hydrogels offered an environment mimicking the native extracellular matrix (ECM). Moreover, the sustained release of Zn2+ from the hydrogel effectively enhanced cell adhesion, promoted proliferation, and stimulated osteoblast differentiation. In vitro experiments have shown that SA-EGCG2 %-Zn2+ hydrogels greatly enhance the attachment and growth of osteoblast precursor cells (MC3T3-E1), while also increasing the expression of genes related to osteogenesis in these cells. Additionally, in vivo studies have confirmed that SA-EGCG2 %-Zn2+ hydrogels promote new bone formation and accelerate the regeneration of bone in situ, indicating promising applications in the realm of bone tissue engineering.

Design, Synthesis, and Biological Evaluation of β-Trifluoroethoxydimethyl Selenides as Potent Antiosteoporosis Agents.

An efficient protocol for the synthesis of β-trifluoroethoxydimethyl selenides was achieved under mild reaction conditions, and 39 compounds were prepared. All compounds were evaluated for their abilities to inhibit RANKL-induced osteoclastogenesis, compound 4aa exhibited the most potent activity. Further investigations revealed that 4aa could inhibit F-actin ring generation, bone resorption, and osteoclast-specific gene expression in vitro. Western blot analyses demonstrated that compound 4aa abrogated the RANKL-induced mitogen-activated protein kinase and NF-kB-signaling pathways. In addition, 4aa also displayed a notable impact on the osteoblastogenesis of MC3T3-E1 preosteoblasts. In vivo experiments revealed that compound 4aa significantly ameliorated bone loss in an ovariectomized (OVX) mice model. Furthermore, the surface plasmon resonance experiment results revealed that 4aa probably bound to RANKL. Collectively, the above-mentioned findings suggested that compound 4aa as a potential RANKL inhibitor averted OVX-triggered osteoporosis by regulating the inhibition of osteoclast differentiation and stimulation of osteoblast differentiation.

Silk sericin-based functional food for osteoporosis prevention and therapy

The development of new functional foods that can potentially reduce the risk of diseases, such as osteoporosis, has received increasing attention in recent years. Here we demonstrated that silk sericin-based functional food (SSBF) exerted preventive and therapeutic effects against bone loss. Sericin isolated at 70 °C for 60 min from the cocoon of Bombyx mori stimulated osteoblast differentiation by increasing osteoblastic gene expression while inhibiting receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in vitro. Micro-computed tomography analysis revealed that pre- and post-oral administration of SSBF increased bone mass in ovariectomized mouse model in vivo. Furthermore, dynamic bone histomorphometry analysis revealed that SSBF enhanced osteoblast activity and suppressed bone resorption by reducing the number of osteoclasts. These findings indicated the potential role of SSBF in preventing and being a therapeutic candidate for osteoporosis.

Incretins Beyond the Pancreas: Exploring the Impact of GIP and GLP-1/2 on Bone Remodeling.

Incretin hormones, such as glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 and 2 (GLP-1, 2), belong to the group of gastrointestinal hormones. Their actions occur through interaction with GIP and GLP-1/2 receptors, which are present in various target tissues. Apart from their well-established roles in pancreatic function and insulin regulation, incretins elicit significant effects that extend beyond the pancreas. Specifically, these hormones stimulate osteoblast differentiation and inhibit osteoclast activity, thereby promoting bone anabolism. Moreover, they play a pivotal role in bone mineralization and overall bone quality and function, making them potentially therapeutic for managing bone health. Thus, this review provides a summary of the crucial involvement of incretins in bone metabolism, influencing both bone formation and resorption processes. While existing evidence is persuasive, further studies are necessary for a comprehensive understanding of the therapeutic potential of incretins in modifying bone health.

Abstract 2920: In vitro assays for investigating the effects of cancer therapeutics on bone

Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2920.

Preparation of black phosphorus@sodium alginate microspheres with bone matrix vesicle structure via electrospraying for bone regeneration

Bone matrix vesicles are commonly acknowledged as the primary site of biomineralization in human skeletal tissue. Black phosphorus has exhibited favorable properties across various chemical and physical domains. In this investigation, a novel composite microsphere was synthesized through the amalgamation of sodium alginate (ALG) with black phosphorus nanosheets (BP) utilizing the electrospray (ES) technique. These microspheres were tailored to mimic the regulatory function of matrix vesicles (MV) upon exposure to a biomimetic mineralization fluid (SBF) during the biomineralization process. Results revealed that black phosphorus nanosheets facilitated the generation of hydroxyapatite (HA) on the microsphere surface. Live-dead assays and cell proliferation experiments showcased a cell survival rate exceeding 85 %. Moreover, wound healing assessments unveiled that M-ALG-BP microspheres exhibited superior migration capacity, with a migration rate surpassing 50 %. Furthermore, after 7 days of osteogenic induction, M-ALG-BP microspheres notably stimulated osteoblast differentiation. Particularly noteworthy, M-ALG-BP microspheres significantly enhanced osteogenic differentiation of osteoblasts and induced collagen production in vitro. Additionally, experiments involving microsphere implantation into mouse skeletal muscle demonstrated the potential for ectopic mineralization by ALG-BP microspheres. This investigation underscores the outstanding mineralization properties of ALG-BP microspheres and their promising clinical prospects in bone tissue engineering.

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.

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1β and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Naked cuticle homolog 2 controls the differentiation of osteoblasts and osteoclasts and ameliorates bone loss in ovariectomized mice

Naked cuticle homolog 2 (NKD2) has been recognized as an antagonist of Wnt/β-catenin signaling and a tumor suppressor. The role of NKD2 in osteoblast and osteoclast differentiation and the mechanism are not fully understood. In this study, we identified the up-regulation of NKD2 during osteoblast and adipocyte differentiation. Functional experiments revealed that NKD2 stimulated osteoblast differentiation and suppressed adipocyte formation. Furthermore, NKD2 down-regulated the expression of receptor activator of nuclear factor-κB ligand in bone marrow mesenchymal stem cells and inhibited osteoclast formation from osteoclast precursor cells. Mechanistic investigations revealed that the regulation of osteoblast and adipocyte differentiation by NKD2 involved Wnt/β-catenin and tuberous sclerosis complex subunit 1 (TSC1)/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathways. Unlike in undifferentiated mesenchymal cells where NKD2 promoted Dishevelled-1 degradation, in the cells differentiating toward osteoblasts or adipocytes NKD2 down-regulated secreted frizzled related protein 1/4 expression and failed to destabilize Dishevelled-1, thereby activating Wnt/β-catenin signaling. Moreover, NKD2 bound to TSC1 and inhibited mTORC1 signaling. Further investigation uncovered an interplay between TSC1/mTORC1 and Wnt/β-catenin signaling pathways. Finally, transplantation of NKD2-overexpressing bone marrow mesenchymal stem cells into the marrow of mice increased osteoblasts, reduced osteoclasts and marrow fat, and partially prevented bone loss in ovariectomized mice. This study provides evidence that NKD2 in mesenchymal stem/progenitor cells reciprocally regulates the differentiation of osteoblasts and adipocytes by modulating Wnt/β-catenin and mTORC1 pathways and inhibits osteoclast formation by down-regulating receptor activator of nuclear factor-κB ligand. It suggests that NKD2 up-regulation may ameliorate postmenopausal bone loss.

Effect of Strontium-Substituted Calcium Phosphate Coatings Prepared by One-Step Electrodeposition at Different Temperatures on Corrosion Resistance and Biocompatibility of AZ31 Magnesium Alloys.

As potential degradable biomaterials, magnesium (Mg) alloys have development prospects in the field of orthopedic load-bearing, whereas the clinical application has encountered a bottleneck due to a series of problems caused by its rapid corrosion. In this study, strontium-substituted calcium phosphate (CaP) coatings with different structures were prepared on the surface of the Mg matrix by a simple one-step electrodeposition method at different temperatures, which enhanced the poor corrosion resistance of the Mg matrix. The coated sample prepared at 65 °C reduced the corrosion current density by 3 orders of magnitude and increased the impedance by nearly 2 orders of magnitude compared with bare Mg alloy, thanks to its dense fibrous structure similar to that of natural bones. Although the coating composition varies with different preparation temperatures, CaP, as an inorganic component similar to natural bone, has good cytocompatibility. Doping the right amount of strontium, which is a trace element in human bones, is beneficial to stimulate osteoblast differentiation, inhibit the activity of osteoclasts, and induce the formation of bone tissues. This provides a new option for modifying the Mg alloy with CaP coatings as a base.

LRP1-deficient leptin receptor-positive cells in periodontal ligament tissue reduce alveolar bone mass by inhibiting bone formation

ObjectiveLeptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells. DesignBone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers. ResultsBone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly. ConclusionLRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue.

Impact of Mechanical Strain and Nicotinamide on RUNX2-Deficient Osteoblast Mimicking Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is a rare genetic defect caused by a heterozygous mutation of runt-related transcription factor 2 (RUNX2), which is important for osteoblast and skeletal development. RUNX2-deficiency causes extra- and intra-oral malformations that often require orthodontic treatment. Nicotinamide (NAM) affects bone remodelling processes. As these are crucial for orthodontic therapy, NAM could improve orthodontic treatment in CCD patients. This study investigates the effect of NAM in control and RUNX2-deficient osteoblasts under mechanical strain mimicking orthodontic treatment. First, the optimal NAM concentration and the differences in the expression profile of control and RUNX2-deficient osteoblasts were determined. Subsequently, osteoblasts were exposed to tensile and compressive strain with and without NAM, and the expression of genes critically involved in bone remodelling was investigated. NAM increased the expression of bone remodelling genes. RUNX2-deficient osteoblasts expressed more receptor activator of NFkB ligand (RANKL) and interleukin-6 (IL6), but less colony-stimulating factor-1 (CSF1). Most of the positive effects of NAM on bone remodelling genes were impaired by mechanical loading. In conclusion, NAM stimulated osteoblast differentiation by increasing the expression of RUNX2 and regulated the expression of osteoclastogenic factors. However, the positive effects of NAM on bone metabolism were impaired by mechanical loading and RUNX2 deficiency.

Gamma-aminobutyric acid (GABA)-mediated bone formation and its implications for anti-osteoporosis strategies: Exploring the relation between GABA and GABA receptors

Osteoporosis is a significant global health concern, linked to reduced bone density and an increased fracture risk, with effective treatments still lacking. This study explored the potential of gamma-aminobutyric acid (GABA) and its receptors as a novel approach to promote osteogenesis and address osteoporosis. GABA concentrations up to 10 mM were well-tolerated by MC3T3-E1 preosteoblast, stimulating osteoblast differentiation and mineralization in a concentration- and time-dependent manner. In vivo experiments with zebrafish larvae demonstrated the ability of GABA to improve vertebral formation and enhanced bone density, indicating the potential therapeutic value for osteoporosis. Notably, GABA countered the adverse effects of prednisolone on vertebral formation, bone density, and osteogenic gene expression in zebrafish larvae, suggesting a promising therapeutic solution to counteract corticosteroid-induced osteoporosis. Moreover, our study highlighted the involvement of GABA receptors in mediating the observed osteogenic effects. By using GABAA, GABAB, and GABAC receptor antagonists, we demonstrated that blocking these receptors attenuated GABA-induced osteoblast differentiation and vertebral formation in both MC3T3-E1 cells and zebrafish larvae, underscoring the importance of GABA receptor interactions in promoting bone formation. In conclusion, these findings underscore the osteogenic potential of GABA and its ability to mitigate the detrimental effects of corticosteroids on bone health. Targeting GABA and its receptors could be a promising strategy for the development of novel therapeutic interventions to address osteoporosis. However, further investigations are warranted to fully elucidate the underlying molecular mechanism of GABA and its clinical applications in treating osteoporosis.

Advanced Progress of the Relationship Between Antihypertensive Drugs and Bone Metabolism.

Hypertension and osteoporosis are common comorbidities among elderly individuals. Drug therapy has been widely used in clinical practice as the preferred antihypertensive treatment. Therefore, antihypertensive drugs have become some of the most commonly prescribed drugs in healthcare settings. However, antihypertensive drugs have different effects on bone metabolism. The results of animal and clinical studies on the effects of antihypertensive drugs on osteoporosis or fracture risk are controversial and have aroused widespread concern among clinicians. Recent studies found that angiotensin receptor blockers, selective β-adrenergic receptor blockers, and thiazide diuretics might improve bone trabecular number and bone mineral density by stimulating osteoblast differentiation, reducing osteoclast generation, and other mechanism. Furthermore, nonselective β-adrenergic receptor blockers and dihydropyridine calcium channel blockers were found to have no significant relationship with bone mineral density or bone strength, and α-adrenergic receptor blockers and loop diuretics might increase fracture risk by decreasing bone mineral density. This article aimed to review previous animal experiments, clinical studies, and meta-analyses focusing on the effects of different antihypertensive drugs on bone metabolism, and to provide a new approach for the prevention and treatment of osteoporosis.

Novel bone Morphogenetic Protein (BMP)-2/4 Consensus Peptide (BCP) for the Osteogenic Differentiation of C2C12 Cells.

Despite the promising clinical potential of bone morphogenetic protein (BMP)-related therapies for bone formation, their side effects warrant the need for alternative therapeutic peptides. BMP family members can aid in bone repair; however, peptides derived from BMP2/ 4 have not yet been investigated. In this study, three candidates BMP2/4 consensus peptide (BCP) 1, 2, and 3 were identified and their ability to induce osteogenesis in C2C12 cells was analyzed. First, an alkaline phosphatase (ALP) staining assay was performed to evaluate the osteogenic effects of BCPs. Next, the effects of BCPs on RNA expression levels and protein abundances of osteogenic markers were explored. Furthermore, the transcriptional activity of ALP by BCP1 and in silico molecular docking model on BMP type IA receptor (BRIA) were performed. BCP1-3 induced higher RUNX2 expression than BMP2. Interestingly, among them, BCP1 significantly promoted osteoblast differentiation more than BMP2 in ALP staining with no cytotoxicity. BCP1 significantly induced the osteoblast markers, and the highest RUNX2 expression was observed at 100 ng/mL compared to other concentrations. In transfection experiments, BCP1 stimulated osteoblast differentiation via RUNX2 activation and the Smad signaling pathway. Finally, in silico molecular docking suggested the possible binding sites of BCP1 on BRIA. These results show that BCP1 promotes osteogenicity in C2C12 cells. This study suggests that BCP1 is the most promising candidate peptide to replace BMP2 for osteoblast differentiation.

Synthetic peptides activating discoidin domain receptor 2 and collagen-binding integrins cooperate to stimulate osteoblast differentiation of skeletal progenitor cells.

Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. STATEMENT OF SIGNIFICANCE: A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration.

Combination of human platelet lysate and 3D gelatin scaffolds to enhance osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells

Bone disorders are major health issues requiring specialized care; however, the traditional bone grafting method had several limitations. Thus, bone tissue engineering has become a potential alternative. In therapeutic treatments, using fetal bovine serum (FBS) as a culture supplement may result in the risk of contamination and host immunological response; therefore, human platelet lysate (hPL) has been considered a viable alternative source. This study attempted to compare the effectiveness and safety of different culture supplements, either FBS or hPL, on the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid (hAF-MSCs) under a three-dimensional gelatin scaffold. The results indicate that hAF-MSCs have the potential to be used in clinical applications as they meet the criteria for mesenchymal stem cells based on their morphology, the expression of a particular surface antigen, their proliferation ability, and their capacity for multipotent differentiation. After evaluation by MTT and Alamar blue proliferation assay, 10% of hPL was selected. The osteogenic differentiation of hAF-MSCs under three-dimensional gelatin scaffold using osteogenic-induced media supplemented with hPL was achievable and markedly stimulated osteoblast differentiation. Moreover, the expressions of osteoblastogenic related genes, including OCN, ALP, and COL1A1, exhibited the highest degree of expression under hPL-supplemented circumstances when compared with the control and the FBS-supplemented group. The induced cells under hPL-supplemented conditions also presented the highest ALP activity level and the greatest degree of calcium accumulation. These outcomes would indicate that hPL is a suitable substitute for animal derived serum. Importantly, osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells using hPL-supplemented media and three-dimensional scaffolds may open the door to developing an alternative construct for repairing bone defects.