Efficacy of Autologous Adult Live-Cultured Osteoblast (AALCO) Implantation in Avascular Necrosis of the Femoral Head: A Mid-Term Outcome Analysis.

Introduction: Avascular Necrosis (AVN) of the femoral head, a condition characterized by the interruption of blood supply leading to bone tissue death, presents significant therapeutic challenges. Recent advancements in orthobiologics, including the use of Autologous Adult Live-Cultured Osteoblasts (AALCO), combined with core decompression, offer a novel approach for managing AVN. This study assesses the efficacy of this treatment modality in improving functional outcomes and hindering disease progression. Materials and methods: This retrospective observational study encompassed 30 patients treated between 2020 and 2023 for idiopathic AVN of the femoral head, grades I to III, who had not responded to conservative treatment. Patients were excluded based on specific criteria including age, secondary AVN causes, and certain health conditions. The treatment involved a two-stage surgical procedure under spinal anesthesia with OSSGROW® for AALCO generation. Post-operative care emphasized early mobilization, DVT prevention, and avoidance of NSAIDs. Outcome measures were evaluated using the Visual Analog Scale (VAS) for pain, modified Harris Hip Score, and annual MRI imaging for up to 36 months. Results: Among 26 patients (41 hips) completing the study, statistically significant improvements in pain and hip functionality were documented, alongside positive radiological signs of osteogenesis in the majority of cases. However, four instances required advancement to total hip replacement due to disease progression. Conclusion: The combination of core decompression and AALCO implantation shows promise as an effective treatment for AVN of the femoral head, with notable improvements in functional and radiological outcomes. This study supports the potential of orthobiologic approaches in AVN treatment, warranting further investigation through comprehensive randomized controlled trials.

RUNX2 regulation in osteoblast differentiation: A possible therapeutic function of the lncRNA and miRNA-mediated network.

Osteogenic differentiation is a crucial process in the formation of the skeleton and the remodeling of bones. It relies on a complex system of signaling pathways and transcription factors, including Runt-related transcription factor 2 (RUNX2). Non-coding RNAs (ncRNAs) control the bone-specific transcription factor RUNX2 through post-transcriptional mechanisms to regulate osteogenic differentiation. The most research has focused on microRNAs (miRNAs) and long ncRNAs (lncRNAs) in studying how they regulate RUNX2 for osteogenesis in both normal and pathological situations. This article provides a concise overview of the recent advancements in understanding the critical roles of lncRNA/miRNA/axes in controlling the expression of RUNX2 during bone formation. The possible application of miRNAs and lncRNAs as therapeutic agents for the treatment of disorders involving the bones and bones itself is also covered.

Periodontal Ligament Cell Apoptosis Activates Lepr+ Osteoprogenitors in Orthodontics.

Alveolar bone (AB) remodeling, including formation and absorption, is the foundation of orthodontic tooth movement (OTM). However, the sources and mechanisms underlying new bone formation remain unclear. Therefore, we aimed to understand the potential mechanism of bone formation during OTM, focusing on the leptin receptor+ (Lepr+) osteogenitors and periodontal ligament cells (PDLCs). We demonstrated that Lepr+ cells activated by force-induced PDLC apoptosis served as distinct osteoprogenitors during orthodontic bone regeneration. We investigated bone formation both in vivo and in vitro. Single-cell RNA sequencing analysis and lineage tracing demonstrated that Lepr represents a subcluster of stem cells that are activated and differentiate into osteoblasts during OTM. Targeted ablation of Lepr+ cells in a mouse model disrupted orthodontic force-guided bone regeneration. Furthermore, apoptosis and sequential fluorescent labeling assays revealed that the apoptosis of PDLCs preceded new bone deposition. We found that PDL stem cell-derived apoptotic vesicles activated Lepr+ cells in vitro. Following apoptosis inhibition, orthodontic force-activated osteoprogenitors and osteogenesis were significantly downregulated. Notably, we found that bone formation occurred on the compression side during OTM; this has been first reported here. To conclude, we found a potential mechanism of bone formation during OTM that may provide new insights into AB regeneration.

Knockdown of SMYD3 by RNA Interference Regulates the Expression of Autophagy-Related Proteins and Inhibits Bone Formation in Fluoride-Exposed Osteoblasts.

This study aimed to explore the role of histone methyltransferase SET and MYND domain containing 3 (SMYD3) in bone metabolism of osteoblasts exposed to fluoride. The levels of urine fluoride, BALP, and OC and the mRNA expression of SMYD3 were determined in patients with skeletal fluorosis and non-fluoride-exposed people on informed consent. The expression of SMYD3 protein, OC contents, and BALP activities were detected in human osteoblast-like MG63 cells and rat primary osteoblasts treated with sodium fluoride (NaF) for 48 h. The autophagosomes were observed by transmission electron microscopy. Then, we knocked down SMYD3 to confirm whether it was involved in the regulation of bone formation and related to autophagy and Wnt/ß-catenin pathway. We observed that OC and BALP levels in patients with skeletal fluorosis significantly increased, while the mRNA expression of SMYD3 significantly decreased in the skeletal fluorosis groups. In vitro, the OC contents, BALP activities, and expression of SMYD3 significantly increased, and many autophagosomes were observed in NaF treated osteoblasts. The downregulation of SMYD3 significantly inhibited OC contents, BALP activities, and expression of autophagy-related proteins, but with no significant changes in the Wnt/ß-catenin pathway. Our results demonstrated that fluoride exposure with coal-burning pollution caused orthopedic injuries and abnormalities in the levels of OC and BALP and hindered normal bone metabolism. Silencing the SMYD3 gene could significantly reduce OC and BALP levels via inhibiting the increase in autophagy induced by fluoride.

In Vitro and In Vivo Evaluation of Toxicity of Structurally Different Diamond-Like Carbon Wear Debris in Joint Replacements.

Diamond-like carbon (DLC) wear debris, which is often composed of different types of structures, is generated from DLC-modified artificial joints in the human body, and its biocompatibility evaluation is especially important to prevent wear-debris-induced implant failure. Here, RAW 264.7 macrophages (inflammatory-reaction assay) and primary mouse osteoblasts (osteoblastogenesis assay) were employed to investigate the toxicity of DLC wear particles (DWPs) by evaluation of cell viability and morphology, enzyme-linked immunosorbent assays, and quantitative reverse-transcription polymerase chain reaction (PCR). Relevant histopathological analysis of rat joints was also performed in vivo. We found that DWPs with a relatively high sp2/sp3 ratio (graphite-phase tendency) manifested a higher cytotoxicity and significant inhibition of osteoblastogenesis. DWPs with a relatively low sp2/sp3 ratio (diamond-phase tendency) showed good biocompatibility in vivo. The DWPs exhibiting a low sp2/sp3 ratio demonstrated reduced secretion of TNF-α and IL-6, along with increased secretion of TIMP-1, resulting in the downregulation of MMP-2 and MMP-9 and upregulation of interleukin-10 (IL-10), thereby attenuating the inflammatory response. Moreover, coculturing osteoblasts with DWPs exhibiting a low sp2/sp3 ratio resulted in an elevated OPG/RANKL ratio and increased expression of OPG mRNA. Because of the absence of electrostatic repulsion, DWPs with a relatively low sp2/sp3 ratio enhanced bovine serum albumin adsorption, which favored cellular activities. Cytotoxicity assessment of DWPs can help establish an evaluation system for particle-related joint disease and can facilitate the clinical application of DLC-coated prostheses.

In vitro osteoclast differentiation enhanced by hepatocyte supernatants from high-fat diet mice.

Non-alcoholic fatty liver disease (NAFLD) is associated with abnormal bone metabolism, potentially mediated by elevated levels of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-ɑ) and interleukin 6 (IL-6). This study aims to investigate the direct regulatory effects of liver tissues on osteoblast and osteoclast functions in vitro, focusing on the liver-bone axis in NAFLD. Twelve-week-old C57BL/6 mice were fed either a control diet or a high-fat diet (HFD) for 12 weeks. Bone structural parameters were assessed using microCT. Primary hepatocyte cultures were established from control and HFD-fed C57BL/6 mice, as well as IL-6-/- and TNF-α-/- mice. The supernatants from these hepatocyte cultures were used to induce differentiation in bone marrow cell-derived osteoblasts and osteoclasts in vitro. Results showed that mice on a HFD exhibited increased lipid infiltration in liver and bone marrow tissues, alongside reduced bone mass. Moreover, the supernatants from hepatocyte cultures from mice on a HFD displayed elevated TNF-α and IL-6 levels. These supernatants, particularly those derived from HFD-fed and IL-6-/- mice, significantly enhanced osteoclast differentiation in vitro. In contrast, supernatants from TNF-α-/- mice did not significantly affect osteoblast or osteoclast differentiation in vitro. In conclusions, this current study suggested that fatty liver tissues may negatively impact bone metabolism. Additionally, knockout of TNF-α and IL-6 genes revealed distinct influence on osteoblast and osteoclast functions, highlighting the complex interplay between live pathology and bone health.

Macro-Calcium Carbonate-Hydrogel Hybrid Spheroids: Design and Biomedical Applications.

In advancing tissue engineering, we introduce a particle system combining the strength of calcium carbonate with the flexibility of hydrogels enhanced with alkaline phosphatase (ALP) for improved bone regeneration. Our innovation lies in creating large hybrid macrospheroids by bonding mineral nanostructured microparticles loaded with ALP through hydrogel polymerization. These composite macrospheroids address critical challenges in cell seeding, growth, and handling within three-dimensional (3D) environments. We conducted extensive characterization of these particles using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), mechanical property assessment, and fluorescence microscopy. The results demonstrate that the hybrid macrospheroids significantly enhance cell manipulation and growth in three-dimensional structures. Specifically, ALP-loaded macrospheroids showed a marked improvement in osteogenic activity, promoting effective bone tissue regeneration. This study not only showcases a unique approach to overcoming the limitations of traditional hydrogels in tissue engineering but also opens pathways for bone tissue regeneration. Our findings offer a promising tool for cell seeding and growth in 3D structures, potentially revolutionizing practices in tissue engineering and regenerative medicine.

Mussel shell-derived biogenic hydroxyapatite as reinforcement on chitosan-loaded gentamicin composite for antibacterial activity and bone regeneration.

In this study, hydroxyapatite (HAp) was synthesized from natural biowaste materials, specifically mussel shells, and combined with chitosan (CS) and gentamicin sulfate antibiotic (GA) using an in-situ method. The resulting composite material, designated HAp/CS-GA, has its physicochemical and structural properties characterized by Fourier transform infrared spectroscopy (FTIR) analysis. The drug-loaded structure was confirmed by UV-visible absorption spectroscopy (UV-Vis) and X-ray diffraction (XRD) analysis. Additionally, field emission scanning electron microscopy (FE-SEM) equipped with the energy dispersive X-ray spectroscopic (EDX) technique was used to determine the surface topography and main components. The composite of HAp/CS-GA was analyzed using a drug release profile and UV-visible spectroscopy (UV-Vis). The fabricated composites antimicrobial behavior was examined against bone infection-causing Gram-positive and Gram-negative bacteria, showing potential activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus compared to Escherichia coli, respectively. Simultaneously, the cytotoxicity of the composite was evaluated by MTT assay using an MG-63 osteoblast-like cell line that exhibited no toxicity in the prepared composite. After a 24 h incubation period, the MG-63 cells on the HAp/CS-GA composite showed good proliferation, according to Hoechst 33258 fluorescence staining results. The results suggested that the composite had excellent biocompatibility and antibacterial activity and enhanced the osteoblast cell proliferation. Therefore, the designed HAp/CS-GA composite would be a promising candidate for bone tissue engineering.

Molecular landscape of prostate cancer bone metastasis.

Prostate cancer (PC) has a high propensity to develop bone metastases, causing severe pain and pathological fractures that profoundly impact a patients' normal functions. Current clinical intervention is mainly palliative focused on pain management, and tumor progression is refractory to standard therapeutic regimens. This limited treatment efficacy is at least partially due to a lack of comprehensive understanding of the molecular landscape of the disease pathology, along with the intensive overlapping of physiological and pathological molecular signaling. The niche is overwhelmed with diverse cell types with inter- and intra-heterogeneity, along with growth factor-enriched cells that are supportive of invading cell proliferation, providing an additional layer of complexity. This review seeks to provide molecular insights into mechanisms underlying PC bone metastasis development and progression.

Effect of primary osteoblast-derived extracellular vesicles on osteoclast differentiation. / åŽŸä»£æˆéª¨ç»†èƒžæ¥æºçš„ç»†èƒžå¤–å›Šæ³¡å¯¹ç ´éª¨ç»†èƒžåˆ†åŒ–çš„å½±å“.

OBJECTIVES: To investigate the effect of osteoblast-derived extracellular vesicles (OB-EVs) on the proliferation and differentiation of osteoclasts, and to explore the possible molecular mechanism of extracellular vesicles involved in the communication between osteoblasts and osteoclasts, and to elucidate the specific mechanism of extracellular vesicles interfering with alveolar bone homeostasis. METHODS: Primary osteoblasts were isolated from newborn mouse calvarial bone and induced by dexamethasone, ß glycerin phosphate and ascorbic acid. Osteogenic feature was tested by alkaline phosphatase (ALP) and alizarin red S staining. Extracellular vesicles were isolated by ultracentrifugation from the supernatant of cell culture. The vesicle morphology was observed by transmission electron microscopy, and the characteristic markers of tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (Alix) and cluster of differentiation 9 (CD9) on the surface of extracellular vesicles were identified by Western blotting. Cell counting kit 8 (CCK-8) assay was used to determine the proliferation effect of OB-EVs on mouse mononuclear macrophage RAW264.7 cells. Furthermore, the expression level of specific markers of osteoclast differentiation in RAW264.7 cells was detected by Western blotting after the combined effect of OB-EVs and nuclear factor kappa B receptor activating factor ligand (RANKL). The number of osteoclasts was observed and compared with OB-EVs-treated mouse bone marrow-derived macrophages (BMMs) by tartrate-resistant acid phosphatase (TRAP) staining, and the effect of OB-EVs on osteoclast differentiation was determined. RESULTS: The extracted OB-EVs showed a double-layer cup-like structure with a diameter of 30-150 nm, and TSG101, Alix and CD9 were positively expressed. RAW264.7 cells were stimulated with OB-EVs, and the results of CCK-8 assay showed that high concentration of OB-EVs (more than 20 µg/mL) inhibited cell proliferation (P<0.05). Western blot analysis showed that the expression of osteoclast differentiation marker proteins such as c-Fos, activated T cell nuclear factor (NFATc1) and c-Jun N-terminal kinase (JNK) in RAW264.7 cells was significantly increased, and the promoting effect was enhanced with the increase of OB-EVs concentration (P<0.05). In addition, the combination of OB-EVs and RANKL on BMM showed that the number of TRAP-positive cells was significantly higher than that of the RANKL induction group alone (P<0.05). CONCLUSIONS: High concentration of OB-EVs can inhibit the proliferation of RAW264.7 cells, and OB-EVs can promote the differentiation of osteoclast precursor cells into osteoclasts.

Experimental long bone fracture healing in goats with cockle shell-based calcium carbonate bone paste.

Long bone fractures are common orthopedic conditions. There are numerous ways to repair these fractures. Bone grafting becomes necessary when a broken bone has a significant gap. However, due to insufficient donor volume and donor site morbidity, substitutes are required. In veterinary orthopaedics, calcium carbonate from cockle shells could be used as a bone biomaterial. We investigated its efficacy as a bone biomaterial repair for goat femoral fractures. The study included 10 healthy adult male Black Bengal goats weighing 8 kg and aged 12-13 months. The study includes control and treatment groups. Intramedullary pinning stabilized an 8-mm right femur diaphyseal fracture in the treatment and control groups. The treated group received 2 ml of bone paste in the fractured gap, whereas the control group left it empty. We examined all goats with X-rays on the 7th, 45th, and 60th days, followed by gross and histological findings. Due to callus bridging, radiographs revealed faster bone growth in the treated group than in the control group. Gross examination demonstrates the treated group had a larger fracture callus than the control group. Histopathology showed that bone formed faster and included more osteocytes, osteoblasts, osteoclasts, and bony spicules than in the control group. The treated group had more periosteum osteoblasts, while the control group had fibroblasts. These results showed that the treated group had more osteogenic activity than the control group. This study demonstrates the potential of cockle shell-based calcium carbonate bone paste as a synthetic biomaterial for healing long bone fractures in goats.

Enhancing postmenopausal osteoporosis: a study of KLF2 transcription factor secretion and PI3K-Akt signaling pathway activation by PIK3CA in bone marrow mesenchymal stem cells.

Introduction: Mesenchymal stem cells can develop into osteoblasts, making them a promising cell-based osteoporosis treatment. Despite their therapeutic potential, their molecular processes are little known. Bioinformatics and experimental analysis were used to determine the molecular processes of bone marrow mesenchymal stem cell (BMSC) therapy for postmenopausal osteoporosis (PMO). Material and methods: We used weighted gene co-expression network analysis (WGCNA) to isolate core gene sets from two GEO microarray datasets (GSE7158 and GSE56815). GeneCards found PMO-related genes. GO, KEGG, Lasso regression, and ROC curve analysis refined our candidate genes. Using the GSE105145 dataset, we evaluated KLF2 expression in BMSCs and examined the link between KLF2 and PIK3CA using Pearson correlation analysis. We created a protein-protein interaction network of essential genes involved in osteoblast differentiation and validated the functional roles of KLF2 and PIK3CA in BMSC osteoblast differentiation in vitro. Results: We created 6 co-expression modules from 10 419 differentially expressed genes (DEGs). PIK3CA, the key gene in the PI3K-Akt pathway, was among 197 PMO-associated DEGs. KLF2 also induced PIK3CA transcription in PMO. BMSCs also expressed elevated KLF2. BMSC osteoblast differentiation involved the PI3K-Akt pathway. In vitro, KLF2 increased PIK3CA transcription and activated the PI3K-Akt pathway to differentiate BMSCs into osteoblasts. Conclusions: BMSCs release KLF2, which stimulates the PIK3CA-dependent PI3K-Akt pathway to treat PMO. Our findings illuminates the involvement of KLF2 and the PI3K-Akt pathway in BMSC osteoblast development, which may lead to better PMO treatments.

Deletion of the transcription factor EBF1 in perivascular stroma disrupts skeletal homeostasis and precipitates premature aging of the marrow microenvironment.

Early B cell factor 1 (EBF1) is a transcription factor expressed by multiple lineages of stromal cells within the bone marrow. While cultures of Ebf1-deficient cells have been demonstrated to have impaired differentiation into either the osteoblast or adipogenic lineage in vitro by several groups, in vivo there has been a nominal consequence of the loss of EBF1 on skeletal development. In this study we used Prx-cre driven deletion of Ebf1 to eliminate EBF1 from the entire mesenchymal lineage of the skeleton and resolve this discrepancy. We report here that EBF1 is expressed primarily in the Mesenchymal Stem and Progenitor Cell (MSPC)-Adipo, MSPC-Osteo, and the Early Mesenchymal Progenitors, and that loss of EBF1 has a plethora of consequences to maintenance of the skeleton throughout adulthood. Stroma from the Prx-cre;Ebf1fl/fl bones had impaired osteogenic differentiation, an age-dependent loss of CFU-F, and elevated senescence accompanying Ebf1-deletion. New bone formation was reduced after 3 months, and resulted in a quiescent bone environment with fewer osteoblasts and an accompanied reduction in osteoclast-mediated remodeling. Consequently, bones were less ductile at a younger age, and deletion of EBF1 dramatically impaired fracture repair. Disruption of EBF1 in perivascular populations also rearranged the vascular network within these bones and disrupted cytokine signaling from key hematopoietic niches resulting in anemia, reductions in B cells, and myeloid skewing of marrow hematopoietic lineages. Mechanistically we observed disrupted BMP signaling within Ebf1-deficient progenitors with reduced SMAD1-phosphorylation, and elevated secretion of the soluble BMP-inhibitor Gremlin from the MSPC-Adipo cells. Ebf1-deficient progenitors also exhibited posttranslational suppression of glucocorticoid receptor expression. Together, these results suggest that EBF1 signaling is required for mesenchymal progenitor mobilization to maintain the adult skeleton, and that the primary action of EBF1 in the early mesenchymal lineage is to promote proliferation, and differentiation of these perivascular cells to sustain a healthy tissue.

Bone Fracture Healing under the Intervention of a Stretchable Ultrasound Array.

Ultrasound treatment has been recognized as an effective and noninvasive approach to promote fracture healing. However, traditional rigid ultrasound probe is bulky, requiring cumbersome manual operations and inducing unfavorable side effects when functioning, which precludes the wide application of ultrasound in bone fracture healing. Here, we report a stretchable ultrasound array for bone fracture healing, which features high-performance 1-3 piezoelectric composites as transducers, stretchable multilayered serpentine metal films in a bridge-island pattern as electrical interconnects, soft elastomeric membranes as encapsulations, and polydimethylsiloxane (PDMS) with low curing agent ratio as adhesive layers. The resulting ultrasound array offers the benefits of large stretchability for easy skin integration and effective affecting region for simple skin alignment with good electromechanical performance. Experimental investigations of the stretchable ultrasound array on the delayed union model in femoral shafts of rats show that the callus growth is more active in the second week of treatment and the fracture site is completely osseous healed in the sixth week of treatment. Various bone quality indicators (e.g., bone modulus, bone mineral density, bone tissue/total tissue volume, and trabecular bone thickness) could be enhanced with the intervention of a stretchable ultrasound array. Histological and immunohistochemical examinations indicate that ultrasound promotes osteoblast differentiation, bone formation, and remodeling by promoting the expression of osteopontin (OPN) and runt-related transcription factor 2 (RUNX2). This work provides an effective tool for bone fracture healing in a simple and convenient manner and creates engineering opportunities for applying ultrasound in medical applications.

The impact of copper on bone metabolism.

Copper is an essential trace element for the human body. Abnormalities in copper metabolism can lead to bone defects, mainly by directly affecting the viability of osteoblasts and osteoclasts and their bone remodeling function, or indirectly regulating bone metabolism by influencing enzyme activities as cofactors. Copper ions released from biological materials can affect osteoblasts and osteoclasts, either directly or indirectly by modulating the inflammatory response, oxidative stress, and rapamycin signaling. This review presents an overview of recent progress in the impact of copper on bone metabolism. Translational potential of this article: The impact of copper on bone metabolism can provide insights into clinical application of copper-containing supplements and biomaterials.

Halofuginone Inhibits Osteoclastogenesis and Enhances Osteoblastogenesis by Regulating Th17/Treg Cell Balance in Multiple Myeloma Mice with Bone Lesions.

Evidences shows that T helper 17 (Th17) and regulatory T (Treg) cells imbalance plays a critical role in bone lesions of MM patients. Therefore, regulating the Th17/Treg imbalance may be beneficial for bone lesions in MM. Ten MM mice complicated with bone lesions were established and divided into the halofuginone (HF) group and the PBS group. After treatment, tibia and fibula from both groups were scanned by micro-CT. Osteoclasts and osteoblasts were validated by histochemical staining and ELISA. Th17 and Treg cells were tested by flow cytometry. The correlations between Th17/Treg cell ratio and osteoclasts, osteoblasts and bone remodeling were analyzed using the Spearman relative analysis. After treatment, mice in the HF group had an increase in trabecular bone volume fraction and thickened cortex, but a decrease in trabecular separation compared to mice in the PBS group.Tartrate-resistant acid phosphase (TRAP) + osteoclasts and its biomarker TRACP5b in serum were reduced, while alkaline phosphatase (ALP) + osteoblasts and its biomarker N-terminal propeptide of type 1precollagen (P1NP) in serum were accreted in the HF group. Th17/Treg cell ratio in halofuginone-treated mice was 0.85 ± 0.05, and was significantly lower than that in PBS-treated mice, which was 1.51 ± 0.03. In addition, it showed that the Th17/Treg cell ratio was significantly and positively associated with osteoclasts, but was significantly and negatively associated with osteoblasts and bone remodeling. Halofuginone plays a critical role in the amelioration bone lesions in MM, as it can inhibit osteoclastogenesis and enhance osteoblastogenesis by regulating the Th17/Treg cell balance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-024-01756-4.

Tailoring zirconia surface topography via femtosecond laser-induced nanoscale features: effects on osteoblast cells and antibacterial properties.

The performance and long-term durability of dental implants hinge on the quality of bone integration and their resistance to bacteria. This research aims to introduce a surface modification strategy for zirconia implants utilizing femtosecond laser ablation techniques, exploring their impact on osteoblast cell behavior and bacterial performance, as well as the integral factors influencing the soft tissue quality surrounding dental implants. Ultrafast lasers were employed to craft nanoscale groove geometries on zirconia surfaces, with thorough analyses conducted using x-ray diffraction, scanning electron microscopy, atomic force microscopy, and water contact angle measurements. The study evaluated the response of human fetal osteoblastic cell lines to textured zirconia ceramics by assessing alkaline phosphatase activity, collagen I, and interleukin 1ßsecretion over a 7 day period. Additionally, the antibacterial behavior of the textured surfaces was investigated usingFusobacterium nucleatum, a common culprit in infections associated with dental implants. Ciprofloxacin (CIP), a widely used antibacterial antibiotic, was loaded onto zirconia ceramic surfaces. The results of this study unveiled a substantial reduction in bacterial adhesion on textured zirconia surfaces. The fine biocompatibility of these surfaces was confirmed through the MTT assay and observations of cell morphology. Moreover, the human fetal osteoblastic cell line exhibited extensive spreading and secreted elevated levels of collagen I and interleukin 1ßin the modified samples. Drug release evaluations demonstrated sustained CIP release through a diffusion mechanism, showcasing excellent antibacterial activity against pathogenic bacteria, includingStreptococcus mutans, Pseudomonas aeruginosa, andEscherichia coli.

Roles of Sp7 in osteoblasts for the proliferation, differentiation, and osteocyte process formation.

Background: Zinc finger-containing transcription factor Osterix/Specificity protein-7 (Sp7) is an essential transcription factor for osteoblast differentiation. However, its functions in differentiated osteoblasts remain unclear and the effects of osteoblast-specific Sp7 deletion on osteocytes have not been sufficiently studied. Methods: Sp7 floxneo/floxneo mice, in which Sp7 expression was 30 % of that in wild-type mice because of disturbed splicing by neo gene insertion, and osteoblast-specific knockout (Sp7 fl/fl;Col1a1-Cre) mice using 2.3-kb Col1a1 enhanced green fluorescent protein (EGFP)-Cre were examined by micro-computed tomography (micro-CT), bone histomorphometry, serum markers, and histological analyses. The expression of osteoblast and osteocyte marker genes was examined by real-time reverse transcription (RT)-PCR analysis. Osteoblastogenesis, osteoclastogenesis, and regulation of the expression of collagen type I alpha 1 chain (Col1a1) were examined in primary osteoblasts. Results: Femoral trabecular bone volume was higher in female Sp7 floxneo/floxneo and Sp7 fl/fl;Col1a1-Cre mice than in the respective controls, but not in males. Bromodeoxyuridine (BrdU)-positive osteoblastic cells were increased in male Sp7 fl/fl;Col1a1-Cre mice, and osteoblast number and the bone formation rate were increased in tibial trabecular bone in female Sp7 fl/fl;Col1a1-Cre mice, although osteoblast maturation was inhibited in female Sp7 fl/fl;Col1a1-Cre mice as shown by the increased expression of an immature osteoblast marker gene, secreted phosphoprotein 1 (Spp1), and reduced expression of a mature osteoblast marker gene, bone gamma-carboxyglutamate protein/bone gamma-carboxyglutamate protein 2 (Bglap/Bglap2). Furthermore, alkaline phosphatase activity was increased but mineralization was reduced in the culture of primary osteoblasts from Sp7 fl/fl;Col1a1-Cre mice. Therefore, the accumulated immature osteoblasts in Sp7 fl/fl;Col1a1-Cre mice was likely compensated for the inhibition of osteoblast maturation at different levels in males and females. Vertebral trabecular bone volume was lower in both male and female Sp7 fl/fl;Col1a1-Cre mice than in the controls and the osteoblast parameters and bone formation rate in females were lower in Sp7 fl/fl;Col1a1-Cre mice than in Sp7 fl/fl mice, suggesting differential regulatory mechanisms in long bones and vertebrae. The femoral cortical bone was thin and porous in Sp7 floxneo/floxneo and Sp7 fl/fl;Col1a1-Cre mice of both sexes, the number of canaliculi was reduced, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive lacunae and the osteoclasts were increased, whereas the bone formation rate was similar in Sp7 fl/fl;Col1a1-Cre and Sp7 fl/fl mice. The serum levels of total procollagen type 1 N-terminal propeptide (P1NP), a marker for bone formation, were similar, while those of tartrate-resistant acid phosphatase 5b (TRAP5b), a marker for bone resorption, were higher in Sp7 fl/fl;Col1a1-Cre mice. Osteoblasts were less cuboidal, the expression of Col1a1 and Col1a1-EGFP-Cre was lower in Sp7 fl/fl;Col1a1-Cre mice, and overexpression of Sp7 induced Col1a1 expression. Conclusions: Our studies indicated that Sp7 inhibits the proliferation of immature osteoblasts, induces osteoblast maturation and Col1a1 expression, and is required for osteocytes to acquire a sufficient number of processes for their survival, which prevents cortical porosity. The translational potential of this article: This study clarified the roles of Sp7 in differentiated osteoblasts in proliferarion, maturation, Col1a1 expression, and osteocyte process formation, which are required for targeting SP7 in the development of therapies for osteoporosis.

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-1ß 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.

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. Bone formation is increased by removing the PI3K inhibitor PTEN, but the effect of direct PI3K in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 weeks of age. Both sexes had smaller marrow areas from 6 weeks of age. Female mice also exhibited greater cross sectional area, which continued to increase until 24 weeks of age, resulting in a further increase in bone strength. While both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was a greater extent of lamellar bone formation with highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Patients with genetic activation of an enzyme called phosphoinositide-3 kinase (PI3K) have tissue overgrowth syndromes, where parts of the body become enlarged, sometimes including the skeleton. There are two types of mutations that cause these problems: one that directly causes the PI3K enzyme to be more active, or one that removes the normal brake on PI3K signaling (called PTEN). We studied the effect of directly activating PI3K enzyme specifically in osteoblasts (the cells that form bone) and osteocytes (osteoblasts that make a network inside the bone tissue itself). We found mice with these mutations formed normally shaped bones that were very strong because the outer shell was thicker than usual. In both male and female mice, it became thicker on the inside of the shell, but in female mice it also became thicker on the outside, making the bones even stronger over time. The new bone was well-organized bone, which likely helped make the increase in bone strength so profound. This is very different to what has previously been shown in mice with the other type of mutation in their bone forming cells; those mice had a shell that contained many large holes (pores). This indicates that directly stimulating PI3K enzyme is more beneficial for bone than removing the PTEN brake.