Impact of Bone Morphogenetic Protein 7 and Prostaglandin receptors on osteoblast healing and organization of collagen.

PURPOSE: This study seeks to investigate the impact of co-administering either a Prostaglandin EP2 receptor agonist or an EP1 receptor antagonist alone with a low dose BMP7 on in vitro healing process, collagen content and maturation of human osteoblasts. METHODOLOGY: Human osteoblast cells were used in this study. These cells were cultured and subjected to different concentrations of Prostaglandin EP2 receptor agonist, EP1 receptor antagonist, BMP7, Control (Ct) (Vehicle alone), and various combinations treatments. Cell viability at 24, 48 and 72 hours (h) was evaluated using the XTT assay. A wound healing assay was conducted to observe the migration ability of human osteoblast cells. Additionally, Sirius red staining and Fourier-Transform Infrared Spectroscopy Imaging (FT-IR) was employed to analyze various parameters, including total protein concentration, collagen production, mature collagen concentration, and mineral content. RESULTS: The combination of low dose BMP7 and Prostaglandin EP2 receptor agonist resulted to the lowest cell viability when compared to both the Ct and individual treatments. In contrast, the Prostaglandin EP1 receptor antagonist alone showed the highest cellular viability at 72 h. In the wound healing assay, the combined treatment of low dose BMP7 with the Prostaglandin EP2 receptor agonist and EP1 receptor antagonist showed a decrease in human osteoblast healing after 24 h. Analysis of FT-IR data indicated a reduction in total protein content, collagen maturity, collagen concentration and mineral content in combination treatment compared to the single or Ct treatments. CONCLUSION: The combination of a Prostaglandin EP2 receptor agonist or an EP1 receptor antagonist when combined with low dose BMP7 significantly hinders both human osteoblast healing and collagen maturity/concentration in comparison to low dose BMP7 treatment alone.

Evaluation of the Effects of Whey Protein and Moringa Oleifera Leaves Extract Mixture on Osseointegration in Rabbits.

Background: Osteogenic, antioxidant and anti-inflammatory effects of Whey protein and M. oleifera gel prompted us to evaluate their role alone or in combination on osseointegration in rabbits. Methods: In this study, 24 titanium implants were inserted in the femurs of six rabbits. One implant was placed without treatment, and another one was coated with a mixture of whey protein and M. oleifera gel for each side. The animals were divided into two groups of 2- and 6-week intervals and evaluated using histopathological and immunohistochemical techniques. Results: Histological evaluation revealed a significant difference between the experimental and the control groups after two weeks in osteoblast and osteocyte counts. The experimental group had mature bone development after six weeks of implantation, while the control group had a woven bone. Immunohistochemical results showed that the experimental group, compared to the control group, exhibited early positive expression of osteoblast cells at two weeks after the experiment. Based on histopathological observations, the experimental group showed a tiny area of collagenous fiber in 6th week after the implantation. Conclusion: A mixture of whey protein and M. oleifera could accelerate osseointegration and healing processes.

Modulation of the microRNA-6089/E2F transcription factor2 axis by querceting: implications for osteoblast viability, proliferation, migration, and osteogenic differentiation in fracture healing.

Quercetin is widely distributed in plants as a flavonol compound with multiple biological activities. It has been found that quercetin can regulate bone homeostasis through multiple pathways and targets. This study investigated the role and specific molecular mechanisms of quercetin in regulating osteoblast viability, proliferation, migration and osteogenic differentiation. A mouse model of traumatic fracture was established and then 100 mg/kg quercetin corn oil suspension was gavaged at the same time every day for 28 days. miR-6089 and E2F transcription factor 2 (E2F2) expression levels in mice were measured. Fracture healing in mice was observed. MC3T3-E1 cells were transfected with plasmids targeting miR-6089 and E2F2, and cell viability, proliferation, migration, apoptosis, and osteogenic differentiation were determined. The targeting relationship between miR-6089 and E2F2 was verified. In vivo experiments showed that quercetin significantly increased osteocalcin (OCN) expression (P<0.05) and promoted fracture healing in traumatic fracture (TF) mice. miR-6089 expression was down-regulated (P<0.05) and E2F2 expression was up-regulated (P<0.05) in TF mice. Quercetin promoted miR-6089 expression and inhibited E2F2 expression (both P<0.05). In vitro results showed that quercetin promoted miR-6089 expression and inhibited E2F2 expression in a dose-dependent manner (both P<0.05). Quercetin dose-dependently promoted MC3T3-E1 cell viability, proliferation, migration, and osteogenic differentiation, and inhibited MC3T3-E1 cell apoptosis (all P<0.05). Up-regulating miR-6089 further promoted MC3T3-E1 cell viability, proliferation, migration and osteogenic differentiation, and inhibited MC3T3-E1 cell apoptosis (all P<0.05). miR-6089 targeted and regulated E2F2 expression. Up-regulating E2F2 attenuated the promoting effect of up-regulated miR-6089 on MC3T3-E1 cell viability, proliferation, migration, osteogenic differentiation, and inhibition of apoptosis (all P<0.05). We conclude that quercetin enhances osteoblast viability, proliferation, migration, and osteogenic differentiation by modulating the miR-6089/E2F2 axis, thereby promoting fracture healing.

Hyperthyroidism-driven bone loss depends on BMP receptor Bmpr1a expression in osteoblasts.

Hyperthyroidism is a well-known trigger of high bone turnover that can lead to the development of secondary osteoporosis. Previously, we have shown that blocking bone morphogenetic protein (BMP) signaling systemically with BMPR1A-Fc can prevent bone loss in hyperthyroid mice. To distinguish between bone cell type-specific effects, conditional knockout mice lacking Bmpr1a in either osteoclast precursors (LysM-Cre) or osteoprogenitors (Osx-Cre) were rendered hyperthyroid and their bone microarchitecture, strength and turnover were analyzed. While hyperthyroidism in osteoclast precursor-specific Bmpr1a knockout mice accelerated bone resorption leading to bone loss just as in wildtype mice, osteoprogenitor-specific Bmpr1a deletion prevented an increase of bone resorption and thus osteoporosis with hyperthyroidism. In vitro, wildtype but not Bmpr1a-deficient osteoblasts responded to thyroid hormone (TH) treatment with increased differentiation and activity. Furthermore, we found an elevated Rankl/Opg ratio with TH excess in osteoblasts and bone tissue from wildtype mice, but not in Bmpr1a knockouts. In line, expression of osteoclast marker genes increased when osteoclasts were treated with supernatants from TH-stimulated wildtype osteoblasts, in contrast to Bmpr1a-deficient cells. In conclusion, we identified the osteoblastic BMP receptor BMPR1A as a main driver of osteoporosis in hyperthyroid mice promoting TH-induced osteoblast activity and potentially its coupling to high osteoclastic resorption.

Vitamin D Attenuates Fibrotic Properties of Fibrous Dysplasia-Derived Cells for the Transit towards Osteocytic Phenotype.

Fibrous dysplasia (FD) poses a therapeutic challenge due to the dysregulated extracellular matrix (ECM) accumulation within affected bone tissues. In this study, we investigate the therapeutic potential of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in managing FD by examining its effects on FD-derived cells in vitro. Our findings demonstrate that 1,25(OH)2D3 treatment attenuates the pro-fibrotic phenotype of FD-derived cells by suppressing the expression of key pro-fibrotic markers and inhibiting cell proliferation and migration. Moreover, 1,25(OH)2D3 enhances mineralization by attenuating pre-osteoblastic cellular hyperactivity and promoting maturation towards an osteocytic phenotype. These results offer valuable insights into potential treatments for FD, highlighting the role of 1,25(OH)2D3 in modulating the pathological properties of FD-derived cells.

Natural Compounds for Bone Remodeling: A Computational and Experimental Approach Targeting Bone Metabolism-Related Proteins.

Osteoporosis, characterized by reduced bone density and increased fracture risk, affects over 200 million people worldwide, predominantly older adults and postmenopausal women. The disruption of the balance between bone-forming osteoblasts and bone-resorbing osteoclasts underlies osteoporosis pathophysiology. Standard treatment includes lifestyle modifications, calcium and vitamin D supplementation and specific drugs that either inhibit osteoclasts or stimulate osteoblasts. However, these treatments have limitations, including side effects and compliance issues. Natural products have emerged as potential osteoporosis therapeutics, but their mechanisms of action remain poorly understood. In this study, we investigate the efficacy of natural compounds in modulating molecular targets relevant to osteoporosis, focusing on the Mitogen-Activated Protein Kinase (MAPK) pathway and the gut microbiome's influence on bone homeostasis. Using an in silico and in vitro methodology, we have identified quercetin as a promising candidate in modulating MAPK activity, offering a potential therapeutic perspective for osteoporosis treatment.

Aspirin use and changes in circulating tumor DNA levels in patients with metastatic colorectal cancer.

The study aimed to investigate the effects of aspirin on patients with metastatic colorectal cancer, focusing on circulating tumor DNA levels and bone tissue. Two groups (A and B) of ten patients with osteoporosis were selected for the study. Bone tissue samples were obtained from the patients and cultured under sterile conditions. The aspirin group showed a significant decrease in circulating tumor DNA levels and an increase in bone tissue density compared to the control group. Additionally, osteoblast apoptosis was reduced, while proliferation was enhanced in the aspirin group. The protein pAkt related to the PI3K/Akt signaling pathway was upregulated in the aspirin group. These results indicate that aspirin can effectively lower circulating tumor DNA levels, promote bone tissue proliferation, inhibit apoptosis, and activate the PI3K/Akt signaling pathway, thereby influencing bone cell function. These findings provide a basis for aspirin's potential application in treating metastatic colorectal cancer and encourage further research on its mechanism and clinical use.

Short- and long-term exposure to high glucose induces unique transcriptional changes in osteoblasts in vitro.

Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10 days, and prior to analysis, they were exposed to hyperglycemia (25 mM) for the short-term (1 or 3 days) or long-term (10 days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3 days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.

Circadian Regulation of Bone Remodeling.

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments.

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1.

The silent information regulator T1 (SIRT1) is linked to longevity and is a crucial mediator of osteoblast function. We investigated the direct role of Sirt1 during bone modeling and remodeling stages in vivo using Tamoxifen-inducible osteoblast-specific Sirt1 conditional knockout (cKO) mice. cKO mice exhibited lower trabecular and cortical bone mass in the distal femur. These phenotypes were coupled with lower bone formation and bone resorption. Metabolomics analysis revealed that the metabolites involved in glycolysis were significantly decreased in cKO mice. Further analysis of the quantitative acetylome revealed 11 proteins with upregulated acetylation levels in both the femur and calvaria of cKO mice. Cross-analysis identified four proteins with the same upregulated lysine acetylation site in both the femur and calvaria of cKO mice. A combined analysis of the metabolome and acetylome, as well as immunoprecipitation, gene knockout, and site-mutation experiments, revealed that Sirt1 deletion inhibited glycolysis by directly binding to and increasing the acetylation level of Glutamine oxaloacetic transaminase 1 (GOT1). In conclusion, our study suggested that Sirt1 played a crucial role in regulating osteoblast metabolism to maintain bone homeostasis through its deacetylase activity on GOT1. These findings provided a novel insight into the potential targeting of osteoblast metabolism for the treatment of bone-related diseases.

The screened compounds from Ligustri Lucidi Fructus using the immobilized calcium sensing receptor column exhibit osteogenic activity in vitro.

Calcium sensing receptor (CaSR) has become the novel target of treating osteoporosis with herbal medicine Ligustri Lucidi Fructus (LLF), however, the bioactive compounds responsible for anti-osteoporosis are hard to clarify due to the complexity and diversity of chemical constituents in it. Herein, the immobilized CaSR column was packed with stationary phase materials, which were derived from integrating CLIP-tagged CaSR directly out of crude cell lysates onto the surface of silica gels (5.83 mg/g) in a site-specific covalent manner. The column had a great specificity of recognizing agonists and kept a good stability for at least 3 weeks. The two compounds from LLF extract were screened and identified as olenuezhenoside and ligustroflavone using the immobilized CaSR column in conjunction with mass spectrometry. Molecular docking predicted that both compounds were bound in venus flytrap (VFT) domain of CaSR by the formation of hydrogen bonds. Cellular results showed that both compounds exhibited the distinct osteogenic activity by enhancing the proliferation, differentiation and mineralization of osteoblastic cells. Our study demonstrated that, the immobilized protein column enables to screen the bioactive compounds rapidly from herbal extract, and the newly discovered natural product ligands towards CaSR, including olenuezhenoside and ligustroflavone, will be the candidates for the treatment of osteoporosis.

Sclerostin blockade inhibits bone resorption through PDGF receptor signaling in osteoblast lineage cells.

While sclerostin-neutralizing antibodies (Scl-Abs) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Abs control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anticatabolic effect of Scl-Abs could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effects on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin coactivates PDGFRs independently of Wnt/ß-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in preosteoblasts. In turn, Scl-Ab prevents sclerostin-mediated coactivation of PDGFR signaling and consequent M-CSF upregulation in preosteoblast cultures, thereby inhibiting osteoclast activity in preosteoblast/osteoclast coculture assays. These results provide a potential mechanism explaining the dissociation between anabolic and antiresorptive effects of long-term Scl-Ab.

Immunohistochemical and Morphometric Assessment on the Biological Function and Vascular Endothelial Cells in the Initial Process of Cortical Porosity in Mice With PTH Administration.

To clarify the cellular mechanism of cortical porosity induced by intermittent parathyroid hormone (PTH) administration, we examined the femoral cortical bone of mice that received 40 µg/kg/day (four times a day) human PTH (hPTH) (1-34). The PTH-driven cortical porosity initiated from the metaphyseal region and chronologically expanded toward the diaphysis. Alkaline phosphatase (ALP)-positive osteoblasts in the control mice covered the cortical surface, and endomucin-positive blood vessels were distant from these osteoblasts. In PTH-administered mice, endomucin-reactive blood vessels with TRAP-positive penetrated the ALP-positive osteoblast layer, invading the cortical bone. Statistically, the distance between endomucin-positive blood vessels and the cortical bone surface abated after PTH administration. Transmission electron microscopic observation demonstrated that vascular endothelial cells often pass through the flattened osteoblast layer and accompanied osteoclasts in the deep region of the cortical bone. The cell layers covering mature osteoblasts thickened with PTH administration and exhibited ALP, α-smooth muscle actin (αSMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-κB ligand (RANKL). Within these cell layers, osteoclasts were found near endomucin-reactive blood vessels. In PTH-administered femora, osteocytes secreted Dkk1, a Wnt inhibitor that affects angiogenesis, and blood vessels exhibited plasmalemma vesicle-associated protein, an angiogenic molecule. In summary, endomucin-positive blood vessels, when accompanied by osteoclasts in the ALP/αSMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1.

Loading rutin on surfaces by the layer-by-layer assembly technique to improve the oxidation resistance and osteogenesis of titanium implants in osteoporotic rats.

The purpose of this study was to construct a rutin-controlled release system on the surface of Ti substrates and investigate its effects on osteogenesis and osseointegration on the surface of implants. The base layer, polyethylenimine (PEI), was immobilised on a titanium substrate. Then, hyaluronic acid (HA)/chitosan (CS)-rutin (RT) multilayer films were assembled on the PEI using layer-by-layer (LBL) assembly technology. We used scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and contact angle measurements to examine all Ti samples. The drug release test of rutin was also carried out to detect the slow-release performance. The osteogenic abilities of the samples were evaluated by experiments on an osteoporosis rat model and MC3T3-E1 cells. The results (SEM, FTIR and contact angle measurements) all confirmed that the PEI substrate layer and HA/CS-RT multilayer film were effectively immobilised on titanium. The drug release test revealed that a rutin controlled release mechanism had been successfully established. Furthermore, thein vitrodata revealed that osteoblasts on the coated titanium matrix had greater adhesion, proliferation, and differentiation capacity than the osteoblasts on the pure titanium surface. When MC3T3-E1 cells were exposed to H2O2-induced oxidative stressin vitro, cell-based tests revealed great tolerance and increased osteogenic potential on HA/CS-RT substrates. We also found that the HA/CS-RT coating significantly increased the new bone mass around the implant. The LBL-deposited HA/CS-RT multilayer coating on the titanium base surface established an excellent rutin-controlled release system, which significantly improved osseointegration and promoted osteogenesis under oxidative stress conditions, suggesting a new implant therapy strategy for patients with osteoporosis.

Mechanically strained osteocyte-derived exosomes contained miR-3110-5p and miR-3058-3p and promoted osteoblastic differentiation.

BACKGROUND: Osteocytes are critical mechanosensory cells in bone, and mechanically stimulated osteocytes produce exosomes that can induce osteogenesis. MicroRNAs (miRNAs) are important constituents of exosomes, and some miRNAs in osteocytes regulate osteogenic differentiation; previous studies have indicated that some differentially expressed miRNAs in mechanically strained osteocytes likely influence osteoblastic differentiation. Therefore, screening and selection of miRNAs that regulate osteogenic differentiation in exosomes of mechanically stimulated osteocytes are important. RESULTS: A mechanical tensile strain of 2500 µÎµ at 0.5 Hz 1 h per day for 3 days, elevated prostaglandin E2 (PGE2) and insulin-like growth factor-1 (IGF-1) levels and nitric oxide synthase (NOS) activity of MLO-Y4 osteocytes, and promoted osteogenic differentiation of MC3T3-E1 osteoblasts. Fourteen miRNAs differentially expressed only in MLO-Y4 osteocytes which were stimulated with mechanical tensile strain, were screened, and the miRNAs related to osteogenesis were identified. Four differentially expressed miRNAs (miR-1930-3p, miR-3110-5p, miR-3090-3p, and miR-3058-3p) were found only in mechanically strained osteocytes, and the four miRNAs, eight targeted mRNAs which were differentially expressed only in mechanically strained osteoblasts, were also identified. In addition, the mechanically strained osteocyte-derived exosomes promoted the osteoblastic differentiation of MC3T3-E1 cells in vitro, the exosomes were internalized by osteoblasts, and the up-regulated miR-3110-5p and miR-3058-3p in mechanically strained osteocytes, were both increased in the exosomes, which was verified via reverse transcription quantitative polymerase chain reaction (RT-qPCR). CONCLUSIONS: In osteocytes, a mechanical tensile strain of 2500 µÎµ at 0.5 Hz induced the fourteen differentially expressed miRNAs which probably were in exosomes of osteocytes and involved in osteogenesis. The mechanically strained osteocyte-derived exosomes which contained increased miR-3110-5p and miR-3058-3p (two of the 14 miRNAs), promoted osteoblastic differentiation.

Buccal Mesenchymal Stromal Cells as a Source of Osseointegration of Titanium Implants.

We studied the interaction of human buccal mesenchymal stem cells (MSCs) and osteoblasts differentiated from them with the surface of titanium samples. MSCs were isolated by enzymatic method from buccal fat pads. The obtained cell culture was presented by MSCs, which was confirmed by flow cytometry and differentiation into adipocytes and osteoblasts. Culturing of buccal MSCs on titanium samples was accompanied by an increase in the number of cells for 15 days and the formation of a developed network of F-actin fibers in the cells. The viability of buccal MSCs decreased by 8 days, but was restored by 15 days. Culturing of osteoblasts obtained as a result of buccal MSC differentiation on the surface of titanium samples was accompanied by a decrease in their viability and proliferation. Thus, MSCs from buccal fat pads can be used to coat implants to improve osseointegration during bone reconstruction in craniofacial surgery and dentistry. To improve the integration of osteoblasts, modification of the surface of titanium samples is required.

Influence of metallic particles and TNF on the transcriptional regulation of NLRP3 inflammasome-associated genes in human osteoblasts.

Introduction: The release of mature interleukin (IL-) 1ß from osteoblasts in response to danger signals is tightly regulated by the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome. These danger signals include wear products resulting from aseptic loosening of joint arthroplasty. However, inflammasome activation requires two different signals: a nuclear factor-kappa B (NF-κB)-activating priming signal and an actual inflammasome-activating signal. Since human osteoblasts react to wear particles via Toll-like receptors (TLR), particles may represent an inflammasome activator that can induce both signals. Methods: Temporal gene expression profiles of TLRs and associated intracellular signaling pathways were determined to investigate the period when human osteoblasts take up metallic wear particles after initial contact and initiate a molecular response. For this purpose, human osteoblasts were treated with metallic particles derived from cobalt-chromium alloy (CoCr), lipopolysaccharides (LPS), and tumor necrosis factor-alpha (TNF) alone or in combination for incubation times ranging from one hour to three days. Shortly after adding the particles, their uptake was observed by the change in cell morphology and spectral data. Results: Exposure of osteoblasts to particles alone increased NLRP3 inflammasome-associated genes. The response was not significantly enhanced when cells were treated with CoCr + LPS or CoCr + TNF, whereas inflammation markers were induced. Despite an increase in genes related to the NLRP3 inflammasome, the release of IL-1ß was unaffected after contact with CoCr particles. Discussion: Although CoCr particles affect the expression of NLRP3 inflammasome-associated genes, a single stimulus was not sufficient to prime and activate the inflammasome. TNF was able to prime the NLRP3 inflammasome of human osteoblasts.

Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed "Cre-only" Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

Mechanically robust and personalized silk fibroin-magnesium composite scaffolds with water-responsive shape-memory for irregular bone regeneration.

The regeneration of critical-size bone defects, especially those with irregular shapes, remains a clinical challenge. Various biomaterials have been developed to enhance bone regeneration, but the limitations on the shape-adaptive capacity, the complexity of clinical operation, and the unsatisfied osteogenic bioactivity have greatly restricted their clinical application. In this work, we construct a mechanically robust, tailorable and water-responsive shape-memory silk fibroin/magnesium (SF/MgO) composite scaffold, which is able to quickly match irregular defects by simple trimming, thus leading to good interface integration. We demonstrate that the SF/MgO scaffold exhibits excellent mechanical stability and structure retention during the degradative process with the potential for supporting ability in defective areas. This scaffold further promotes the proliferation, adhesion and migration of osteoblasts and the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. With suitable MgO content, the scaffold exhibits good histocompatibility, low foreign-body reactions (FBRs), significant ectopic mineralisation and angiogenesis. Skull defect experiments on male rats demonstrate that the cell-free SF/MgO scaffold markedly enhances bone regeneration of cranial defects. Taken together, the mechanically robust, personalised and bioactive scaffold with water-responsive shape-memory may be a promising biomaterial for clinical-size and irregular bone defect regeneration.