Role of NEL­like molecule­1 in osteogenesis/chondrogenesis (Review).

A dynamic balance exists between osteogenesis and osteoclastogenesis in bone tissue, which can lead to several bone diseases, such as osteoporosis, osteoarthritis, bone necrosis and bone defects, in cases of insufficient osteogenesis or excessive osteoclastogenesis. NEL­like molecule­1 (NELL­1) was first discovered in 1999 as an osteogenic factor that can prevent or treat bone diseases by increasing osteogenic levels. To date, research has identified multiple signaling pathways involved in improving osteogenic levels. Furthermore, to apply NELL­1 in clinical practice, researchers have optimized its osteogenic effect by combining it with other molecules, changing its molecular structure and performing bone tissue engineering. Currently, research on NELL­1 is gaining increasing attention. In the near future, it will definitely be applied in clinical practice to eliminate diseases. Thus, the present study provides a comprehensive review of NELL­1 in enhancing osteogenic levels from the perspectives of the molecular mechanism, interactions with other molecules/cells, molecular­level changes, applications in bone tissue engineering and its expression in tumors, providing a solid theoretical basis for its clinical application.

Quercetin ameliorates senescence and promotes osteogenesis of BMSCs by suppressing the repetitive element­triggered RNA sensing pathway.

Cell senescence impedes the self­renewal and osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs), thus limiting their application in tissue regeneration. The present study aimed to elucidate the role and mechanism of repetitive element (RE) activation in BMSC senescence and osteogenesis, as well as the intervention effect of quercetin. In an H2O2­induced BMSC senescence model, quercetin treatment alleviated senescence as shown by a decrease in senescence­associated ß­galactosidase (SA­ß­gal)­positive cell ratio, increased colony formation ability and decreased mRNA expression of p21 and senescence­associated secretory phenotype genes. DNA damage response marker γ­H2AX increased in senescent BMSCs, while expression of epigenetic markers methylation histone H3 Lys9, heterochromatin protein 1α and heterochromatin­related nuclear membrane protein lamina­associated polypeptide 2 decreased. Quercetin rescued these alterations, indicating its ability to ameliorate senescence by stabilizing heterochromatin structure where REs are primarily suppressed. Transcriptional activation of REs accompanied by accumulation of cytoplasmic double­stranded (ds)RNA, as well as triggering of the RNA sensor retinoic acid­inducible gene I (RIG­I) receptor pathway in H2O2­induced senescent BMSCs were shown. Similarly, quercetin treatment inhibited these responses. Additionally, RIG­I knockdown led to a decreased number of SA­ß­gal­positive cells, confirming its functional impact on senescence. Induction of senescence or administration of dsRNA analogue significantly hindered the osteogenic capacity of BMSCs, while quercetin treatment or RIG­I knockdown reversed the decline in osteogenic function. The findings of the current study demonstrated that quercetin inhibited the activation of REs and the RIG­I RNA sensing pathway via epigenetic regulation, thereby alleviating the senescence of BMSCs and promoting osteogenesis.

Transcriptomic profiling of human mesenchymal stem cells using a pulsed electromagnetic-wave motion bioreactor system for enhanced osteogenic commitment and therapeutic potentials.

Traditional bioreactor systems involve the use of three-dimensional (3D) scaffolds or stem cell aggregates, limiting the accessibility to the production of cell-secreted biomolecules. Herein, we present the use a pulse electromagnetic fields (pEMFs)-assisted wave-motion bioreactor system for the dynamic and scalable culture of human bone marrow-derived mesenchymal stem cells (hBMSCs) with enhanced the secretion of various soluble factors with massive therapeutic potential. The present study investigated the influence of dynamic pEMF (D-pEMF) on the kinetic of hBMSCs. A 30-min exposure of pEMF (10V-1Hz, 5.82 G) with 35 oscillations per minute (OPM) rocking speed can induce the proliferation (1 × 105 â†’ 4.5 × 105) of hBMSCs than static culture. Furthermore, the culture of hBMSCs in osteo-induction media revealed a greater enhancement of osteogenic transcription factors under the D-pEMF condition, suggesting that D-pEMF addition significantly boosted hBMSCs osteogenesis. Additionally, the RNA sequencing data revealed a significant shift in various osteogenic and signaling genes in the D-pEMF group, further suggesting their osteogenic capabilities. In this research, we demonstrated that the combined effect of wave and pEMF stimulation on hBMSCs allows rapid proliferation and induces osteogenic properties in the cells. Moreover, our study revealed that D-pEMF stimuli also induce ROS-scavenging properties in the cultured cells. This study also revealed a bioactive and cost-effective approach that enables the use of cells without using any expensive materials and avoids the possible risks associated with them post-implantation.

Stiff extracellular matrix drives the differentiation of mesenchymal stem cells toward osteogenesis by the multiscale 3D genome reorganization.

Extracellular matrix (ECM) stiffness is a major driver of stem cell fate. However, the involvement of the three-dimensional (3D) genomic reorganization in response to ECM stiffness remains unclear. Here, we generated comprehensive 3D chromatin landscapes of mesenchymal stem cells (MSCs) exposed to various ECM stiffness. We found that there were more long-range chromatin interactions, but less compartment A in MSCs cultured on stiff ECM than those cultured on soft ECM. However, the switch from compartment B in MSCs cultured on soft ECM to compartment A in MSCs cultured on stiff ECM included genes encoding proteins primarily enriched in cytoskeleton organization. At the topologically associating domains (TADs) level, stiff ECM tends to have merged TADs on soft ECM. These merged TADs on stiff ECM include upregulated genes encoding proteins enriched in osteogenesis, such as SP1, ETS1, and DCHS1, which were validated by quantitative real-time polymerase chain reaction and found to be consistent with the increase of alkaline phosphatase staining. Knockdown of SP1 or ETS1 led to the downregulation of osteogenic marker genes, including COL1A1, RUNX2, ALP, and OCN in MSCs cultured on stiff ECM. Our study provides an important insight into the stiff ECM-mediated promotion of MSC differentiation towards osteogenesis, emphasizing the influence of mechanical cues on the reorganization of 3D genome architecture and stem cell fate.

Precision pore structure optimization of additive manufacturing porous tantalum scaffolds for bone regeneration: A proof-of-concept study.

Currently, the treatment of bone defects in arthroplasty is a challenge in clinical practice. Nonetheless, commercially available orthopaedic scaffolds have shown limited therapeutic effects for large bone defects, especially for massiveand irregular defects. Additively manufactured porous tantalum, in particular, has emerged as a promising material for such scaffolds and is widely used in orthopaedics for its exceptional biocompatibility, osteoinduction, and mechanical properties. Porous tantalum has also exhibited unique advantages in personalised rapid manufacturing, which allows for the creation of customised scaffolds with complex geometric shapes for clinical applications at a low cost and high efficiency. However, studies on the effect of the pore structure of additively manufactured porous tantalum on bone regeneration have been rare. In this study, our group designed and fabricated a batch of precision porous tantalum scaffolds via laser powder bed fusion (LPBF) with pore sizes of 250 µm (Ta 250), 450 µm (Ta 450), 650 µm (Ta 650), and 850 µm (Ta 850). We then performed a series of in vitro experiments and observed that all four groups showed good biocompatibility. In particular, Ta 450 demonstrated the best osteogenic performance. Afterwards, our team used a rat bone defect model to determine the in vivo osteogenic effects. Based on micro-computed tomography and histology, we identified that Ta 450 exhibited the best bone ingrowth performance. Subsequently, sheep femur and hip defect models were used to further confirm the osteogenic effects of Ta 450 scaffolds. Finally, we verified the aforementioned in vitro and in vivo results via clinical application (seven patients waiting for revision total hip arthroplasty) of the Ta 450 scaffold. The clinical results confirmed that Ta 450 had satisfactory clinical outcomes up to the 12-month follow-up. In summary, our findings indicate that 450 µm is the suitable pore size for porous tantalum scaffolds. This study may provide a new therapeutic strategy for the treatment of massive, irreparable, and protracted bone defects in arthroplasty.

Evaluation of functioning and associated factors in children and adolescents with osteogenesis imperfecta / Avaliação da funcionalidade e fatores associados de crianças e adolescentes com osteogênese imperfeita

ABSTRACT Objective: The aim of this study was to evaluate the functioning and associated factors in children and adolescents with osteogenesis imperfecta (OI). Methods: This is a cross-sectional study conducted on 30 children and adolescents with OI. Medical records, use of bisphosphonates, socioeconomic status, handgrip strength, balance, joint hypermobility, ambulatory level, and the Pediatric Evaluation of Disability Inventory—Computer Adaptative Test (PEDI-CAT) scores were assessed. Data is presented as mean and standard deviation and Student's t-test or Mann-Whitney U test. Categorical data is presented as frequency and analyzed using Fisher's exact test. Within-group analyses were conducted using ANCOVA or Wilcoxon signed-rank test. Correlations used Kendall's Tau-b test. Results: The participants involved in this study were 6-18 years old. The sample was separated into two groups according to disease severity. The moderate/severe OI group (n=10) presented a lower height and muscular strength than the mild group (n=20). Muscle weakness was observed in all participants with OI when compared with the normal population. No differences were observed between the groups in the PEDI-CAT scores except for the mobility domain. There were correlations between the PEDI-CAT mobility domain and the number of fractures, OI type, weight, and balance; there was also a correlation between the PEDI-CAT daily activities, mobility, responsibility, and social/cognitive domains. Conclusions: The findings suggest that children with moderate/severe forms of OI can achieve the same function levels as children with mild OI. Fractures can have a major influence on the functional level, and treatment should focus on the prevention and rehabilitation of these events when they occur.

RESUMO Objetivo: Avaliar a funcionalidade e fatores associados em crianças e adolescentes com osteogênese imperfeita (OI). Métodos: Estudo transversal com 30 crianças e adolescentes com OI. Foram avaliados prontuários médicos, uso de bisfosfonatos, características socioeconômicas, dinamometria de preensão palmar, equilíbrio, hipermobilidade articular, nível de deambulação e escores do Pediatric Evaluation of Disability Inventory - Computer Adaptative Test (PEDI-CAT). Os dados foram apresentados em média e desvio padrão e comparados por teste t por Mann-Whitney, enquanto os categóricos foram apresentados em frequência e comparados pelo teste exato de Fisher. Análises intragrupos foram realizadas por análise de covariância (ANCOVA) ou Teste de Wilcoxon para postos sinalizados. O teste Tau-b de Kendall foi usado para correlações. Resultados: A idade variou de 6 a 18 anos. A amostra foi dividida em dois grupos de acordo com a gravidade da doença. Casos moderados/graves (n=10) apresentaram menor estatura e força muscular comparadas às dos leves (n=20). Fraqueza muscular foi observada em todos os casos de OI quando comparados à população normal. Não houve diferença nos domínios do PEDI-CAT com exceção do domínio mobilidade. Houve correlação entre o número de fraturas, tipo de OI, peso e equilíbrio e o domínio mobilidade; e entre os domínios Atividades Diárias e Mobilidade e Responsabilidade e Social/cognitivo do PEDI-CAT. Conclusões: Nossos achados sugerem que crianças com OI moderada/severa podem atingir o mesmo nível de funcionalidade que crianças com a forma leve. Fraturas podem ter grande influência no nível de funcionalidade e o tratamento deve enfocar a prevenção e a reabilitação desses eventos.

Respiratory insufficiency in an infant with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a rare presentation in the pediatric population. Whilst orthopedic manifestations are well-publicised, the multiple respiratory complications and mechanisms of respiratory failure in more severe cases are less well described. We report the clinical, radiological and histopathological details of the case of an infant with genetically-confirmed OI (Type 2) and associated respiratory insufficiency, as well as summarise the relevant existing literature. This case highlights the importance of the recognition of clinical challenges associated with the management of respiratory complications in a patient with OI.

Alendronate-functionalized polymeric micelles target icaritin to bone for mitigating osteoporosis in a rat model.

Formulating drugs into nanoparticles that target sites of disease can lead to strong therapeutic effects with lower doses of drugs and lower rates of off-target adverse effects. Few ways to target drugs to bone have been described, hampering the treatment of osteoporosis. Here we exploit the ability of alendronate to bind tightly to hydroxyapatite in bone as a tactic to target polymeric micelles loaded with the plant flavonoid icaritin to osteoporotic lesions. The traditional Chinese medicine icaritin, from Herba Epimedii, has previously been shown to inhibit adipogenesis and enhance osteogenesis by bone mesenchymal stem cells, but the compound on its own persists only briefly in the bloodstream. Our delivery system led to stronger inhibition of adipogenesis and activation of osteogenesis in a rat model of osteoporosis than when the icaritin-loaded micelles lacked alendronate. These results establish the feasibility of using alendronate to target osteogenic phytomolecules to sites of bone injury, which may guide the development of effective therapies against osteoporosis and, by extension, other bone disorders.

The impact of the physicochemical properties of calcium phosphate ceramics on biocompatibility and osteogenic differentiation of mesenchymal stem cells.

OBJECTIVE: In this study we have focused on biocompatibility and osteoinductive capacity analysis of self-manufactured single-phase (HAP) and two-phase (HAP and ß-ТСР) bioactive ceramics with various chemical modifications (Fig. 1). RESULTS: We demonstrate a reduction in solubility for all analyzed composite after the treatment with H2O and H2O2, accompanied by an enhancement in adsorption activity. This modification also resulted in an increase in micro- and macroporosity, along with a rise in the open porosity. Adipose-derived mesenchymal stromal cells demonstrated excellent cell adhesion and survival when cultured with these ceramics. Calcium phosphate ceramics (H-500, HT-500, and HT-1 series) stimulated alkaline phosphatase expression, promoted calcium deposition, and enhanced osteopontin expression in ADSCs, independently inducing osteogenesis without additional osteogenic stimuli. These findings underscore the promising potential of HAP-based bioceramics for bone regeneration/reconstruction.

Graphene Oxide Quantum Dots-Preactivated Dental Pulp Stem Cells/GelMA Facilitates Mitophagy-Regulated Bone Regeneration.

Background: In bone tissue engineering (BTE), cell-laden scaffolds offer a promising strategy for repairing bone defects, particularly when host cell regeneration is insufficient due to age or disease. Exogenous stem cell-based BTE requires bioactive factors to activate these cells. Graphene oxide quantum dots (GOQDs), zero-dimensional derivatives of graphene oxide, have emerged as potential osteogenic nanomedicines. However, constructing biological scaffolds with GOQDs and elucidating their biological mechanisms remain critical challenges. Methods: We utilized GOQDs with a particle size of 10 nm, characterized by a surface rich in C-O-H and C-O-C functional groups. We developed a gelatin methacryloyl (GelMA) hydrogel incorporated with GOQDs-treated dental pulp stem cells (DPSCs). These constructs were transplanted into rat calvarial bone defects to estimate the effectiveness of GOQDs-induced DPSCs in repairing bone defects while also investigating the molecular mechanism underlying GOQDs-induced osteogenesis in DPSCs. Results: GOQDs at 5 µg/mL significantly enhanced the osteogenic differentiation of DPSCs without toxicity. The GOQDs-induced DPSCs showed active osteogenic potential in three-dimensional cell culture system. In vivo, transplantation of GOQDs-preactivated DPSCs/GelMA composite effectively facilitated calvarial bone regeneration. Mechanistically, GOQDs stimulated mitophagy flux through the phosphatase-and-tensin homolog-induced putative kinase 1 (PINK1)/Parkin E3 ubiquitin ligase (PRKN) pathway. Notably, inhibiting mitophagy with cyclosporin A prevented the osteogenic activity of GOQDs. Conclusion: This research presents a well-designed bionic GOQDs/DPSCs/GelMA composite scaffold and demonstrated its ability to promote bone regeneration by enhancing mitophagy. These findings highlight the significant potential of this composite for application in BTE and underscore the crucial role of mitophagy in promoting the osteogenic differentiation of GOQDs-induced stem cells.

NEDD4L affects stability of the CHEK2/TP53 axis through ubiquitination modification to enhance osteogenic differentiation of periodontal ligament stem cells.

BACKGROUND: Checkpoint kinase 2 (CHEK2) and its regulated tumor protein p53 (TP53) have been correlated with osteogenic differentiation of osteoblast-like cells. Based on bioinformatics predictions, this study aims to investigate the effect of the CHEK2/TP53 axis on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to explore the regulatory mechanism. METHODS: PDLSCs were isolated from human impacted wisdom teeth, and they were cultured in normal medium (NM) or osteogenic medium (OM). Protein levels of CHEK2 and TP53 were examined using western blot analysis. Osteogenic differentiation ability of PDLSCs was analyzed by measuring marker proteins (RUNX2, OCN, and OSX), ALP activity, and ALP staining. Molecular interaction between NEDD4 like E3 ubiquitin protein ligase (NEDD4L) and CHEK2 was examined by ubiquitination and co-immunoprecipitation assays. Gain- and loss-of function assays of NEDD4L, CHEK2, and TP53 were performed to analyze their function in osteogenic differentiation of PDLSCs. A rat model of mandibular bone defect was generated for in vivo validation. RESULTS: NEDD4L was upregulated, while CHEK2 and TP53 were downregulated in PDLSCs cultured in OM. CHEK2 protected TP53 from degradation, while NEDD4L reduced CHEK2 protein level by ubiquitination modification. NEDD4L silencing reduced osteogenic differentiation ability of PDLSCs both in vitro and in vivo, which was restored by CHEK2 silencing. By contrast, CHEK2 overexpression blocked the osteogenic differentiation of PDLSCs in vitro. CONCLUSION: This study demonstrates that NEDD4L affects protein stability of the CHEK2/TP53 axis through ubiquitination modification, thus increasing osteogenic differentiation of PDLSCs.

Promotion of mandibular distraction osteogenesis by parathyroid hormone via macrophage polarization induced through iNOS downregulation.

Objective: To investigate whether Parathyroid hormone (PTH) can promote mandibular distraction osteogenesis by regulating macrophage polarization and the underlying mechanisms of this phenomenon. Methods: Forty-eight Rabbits were used to establish the mandibular distraction osteogenesis experimental model, randomly divided into 2 groups. Intermittent post-operative injections of 20 µg/kg PTH and normal saline were administered to the experimental and control groups, respectively. Regenerated new bone was examined using HE staining, osteoclast numbers were determined through tartrate-resistant acid phosphatase (TRAP) staining, and macrophage polarization markers arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) expressions were elucidated using immunohistochemistry (IHC), the mRNA expression of CD206, CD11C, Arg1 and iNOS were detected using qPCR. Results: The bone trabeculae in the experimental group were thicker, with a more homogeneous structure and more new osteoid than in the control group. In the area of distraction osteogenesis, the osteoclast count in the experimental group was higher than in the control group (P < 0.05). IHC results indicated differential expressions of Arg1 and iNOS in the experimental group compared to the control group (P < 0.05). Relative mRNA expressions of CD11c and iNOS were lower in the experimental group than in the control group (P < 0.05), whereas the expressions of CD206 and Arg1 mRNA were higher in the experimental group compared to the control group (P < 0.05). Conclusion: Intermittent PTH injections increased macrophage quantity in the mandible generated by distraction osteogenesis, downregulated iNOS, upregulated Arg1, and promoted macrophage polarization from M1 to M2 phenotype, thereby promoting mandibular distraction osteogenesis.

Effect of the polyphenol flavonoids fisetin and quercetin on the adipogenic differentiation of human mesenchymal stromal cells.

Fisetin and quercetin, polyphenol flavonoids, have been shown to have a wide range of beneficial pharmacological effects including anti-inflammatory, antioxidative, and anti-cancer. Our previous work shows that fisetin also affects the specification of the adipogenic-osteogenic lineage of human mesenchymal stem cells (hMSCs) by modulating the Hippo-YAP signaling pathway. Although quercetin has a structure similar to that of fisetin, its effects on the functional properties of hMSCs have not yet been investigated. The objective of the present study is to determine the effects of quercetin on the various properties of hMSCs, including proliferation, migration, and differentiation capacity toward adipogenic and osteogenic lineages. The results show that while fisetin increases hMSC adipogenic differentiation, quercetin inhibited adipogenic differentiation of hMSCs. The inhibition is mediated, at least in part, by the activation of hippo signaling and up-regulation of miR-27b, which inhibits the expression of genes involved in all critical steps of lipid droplet biogenesis, resulting in a decrease in the number of lipid droplets in hMSCs. It is possible that the lack of hydroxylation of the 5 position on the A ring of quercetin could be responsible for its different effect on the adipogenic-osteogenic lineage specification of hMSCs compared with fisetin. Molecular docking and molecular dynamics simulation suggested that fisetin and quercetin possibly bind to serine / threonine protein kinases 4 (STK4/MST1), which is an upstream kinase responsible for LATS phosphorylation. Taken together, our results demonstrate more insight into the mechanism underlying the role of flavonoid fisetin and quercetin in the regulation of adipogenesis.

Dual scalable osteogenic microtissue engineering via GelMA microsphere-inspired mechanical training and autonomous assembling of dental pulp stem cell.

Large bone tissue defects present a significant clinical challenge due to the lack of stem cells and an osteogenic microenvironment, leading to fibrotic healing and impaired bone regeneration. Microsphere-based cell-on three-dimensional (3D) culture systems show great promise for constructing osteogenic microtissues. However, the underlying mechanisms require further investigation. In this study, we propose a simple, scalable framework for highly efficient osteogenic microtissue construction, utilizing gelatin methacryloyl (GelMA) microspheres and dental pulp stem cells (DPSCs). The GelMA microspheres provide an extensive, scalable 3D framework for the autonomous adhesion, migration, and proliferation of DPSCs. Within the enormous 3D space created by the microspheres, DPSCs anchor to the microspheres and neighboring cells, inducing intrinsic tensile stress and simulating a mechanical force akin to "rock climbing training". Transcriptomic sequencing results reveal that the 3D spatial and mechanical microenvironment modulates biological processes involved in cell adhesion, extracellular matrix organization, and the positive regulation of cell migration. Further investigations demonstrate that triggering the FAK/YAP pathway mediate mechanical driven differentiation of DPSCs into the osteoblastic lineage in the excellent osteogenic microtissues. Moreover, this simple scalable 3D framework strategy is expected to enable the efficient and large-scale preparation of stem cell-based microtissues.

IMPC-based screening revealed that ROBO1 can regulate osteoporosis by inhibiting osteogenic differentiation.

Introduction: The utilization of denosumab in treating osteoporosis highlights promising prospects for osteoporosis intervention guided by gene targets. While omics-based research into osteoporosis pathogenesis yields a plethora of potential gene targets for clinical transformation, identifying effective gene targets has posed challenges. Methods: We first queried the omics data of osteoporosis clinical samples on PubMed, used International Mouse Phenotyping Consortium (IMPC) to screen differentially expressed genes, and conducted preliminary functional verification of candidate genes in human Saos2 cells through osteogenic differentiation and mineralization experiments. We then selected the candidate genes with the most significant effects on osteogenic differentiation and further verified the osteogenic differentiation and mineralization functions in mouse 3T3-E1 and bone marrow mesenchymal stem cells (BMSC). Finally, we used RNA-seq to explore the regulation of osteogenesis by the target gene. Results: We identified PPP2R2A, RRBP1, HSPB6, SLC22A15, ADAMTS4, ATP8B1, CTNNB1, ROBO1, and EFR3B, which may contribute to osteoporosis. ROBO1 was the most significant regulator of osteogenesis in both human and mouse osteoblast. The inhibitory effect of Robo1 knockdown on osteogenic differentiation may be related to the activation of inflammatory signaling pathways. Conclusion: Our study provides several novel molecular mechanisms involved in the pathogenesis of osteoporosis. ROBO1 is a potential target for osteoporosis intervention.

The Biomimetic Electrical Stimulation System Inducing Osteogenic Differentiations of BMSCs.

Electrical stimulation has been used clinically as an adjunct therapy to accelerate the healing of bone defects, and its mechanism requires further investigations. The complexity of the physiological microenvironment makes it challenging to study the effect of electrical signal on cells alone. Therefore, an artificial system mimicking cell microenvironment in vitro was developed to address this issue. In this work, a novel electrical stimulation system was constructed based on polypyrrole nanowires (ppyNWs) with a high aspect ratio. Synthesized ppyNWs formed a conductive network in the composited hydrogel which contained modified gelatin with methacrylate, providing a conductive cell culture matrix for bone marrow mesenchymal stem cells. The dual-network conductive hydrogel had improved mechanical, electrical, and hydrophilic properties. It was able to imitate the three-dimensional structure of the cell microenvironment and allowed adjustable electrical stimulations in the following system. This hydrogel was integrated with cell culture plates, platinum electrodes, copper wires, and external power sources to construct the artificial electrical stimulation system. The optimum voltage of the electrical stimulation system was determined to be 2 V, which exhibited remarkable biocompatibility. Moreover, this system had significant promotion in cell spreading, osteogenic makers, and bone-related gene expression of stem cells. RNA-seq analysis revealed that osteogenesis was correlated to Notch, BMP/Smad, and calcium signal pathways. It was proven that this biomimetic system could regulate the osteogenesis procedure, and it provided further information about how the electrical signal regulates osteogenic differentiations.

N6-methyladenosine-modified circCDK14 promotes ossification of the ligamentum flavum via epigenetic modulation by targeting AFF4.

BACKGROUND: The ligamentum flavum (LF) is an important anatomical structure of the spine. Ossification of the LF (OLF) has become the leading cause of thoracic spinal stenosis. Circular RNAs (circRNAs) and N6-methyladenosine (m6A) modification are reported to be associated with several human diseases. However, the role of circRNAs and m6A modification in the pathogenesis of OLF has not been fully investigated. Here, we aimed to explore the vital function of circRNAs and m6A modification in OLF. MATERIALS AND METHODS: We analysed the circRNA expression of 4 OLF tissues and 4 normal LF tissues using bioinformatic analysis and identified circCDK14 for further analysis. We investigated the effects of circCDK14 on the osteogenic differentiation of LF cells. We observed that circCDK14 regulated its target genes by binding to miRNAs as a miRNA sponge. Moreover, the circRNA pull-down assay indicated that RNA-binding proteins might regulate the expression of circCDK14 via m6A modification. RESULTS: CircCDK14 was significantly upregulated in OLF tissues compared to normal LF tissues. Overexpression of circCDK14 promoted the osteogenic differentiation of LF cells. Mechanistically, CircCDK14 promoted the expression of ALF transcription elongation Factor 4 (AFF4) by serving as a sponge for miR-93-5p. Moreover, Wilms tumour 1-associated protein (WTAP) increased the stability of circCDK14 via N6-methyladenosine modification. CONCLUSION: The m6A-modified CircCDK14 binding to miR-93-5p played an important role in the osteogenesis of LF cells by targeting AFF4, providing a promising therapeutic target for OLF.

The accordion technique did not improve bone healing in a mouse model of distraction osteogenesis.

Distraction osteogenesis (DO) is a valuable surgical method for limb lengthening and bone defect correction, but its lengthy consolidation phase presents challenges. The accordion technique (AT), involving compression and distraction of bone segments, has shown potential for enhancing healing. This study aimed to investigate the effectiveness of the AT conducted at three different time points (distraction phase, early consolidation phase, or late consolidation phase) compared to conventional DO in a mouse osteotomy model. Healing was evaluated using in vivo microCT, histology, and computational modeling. Results showed that bridging frequency, BV, and callus tissue composition were similar between conventional DO and late consolidation AT. In contrast, distraction phase AT led to delayed healing at day 15 with a 72% reduction in BV compared to DO, but no significant differences by the endpoint. Early consolidation AT showed significantly impaired healing compared to DO, with only 29% of mice achieving bony bridging, and significantly reduced bone marrow area of the endpoint callus. In silico modeling was generally predictive of in vivo findings and suggested that application of the AT during early consolidation results in destruction of newly-formed vascular tissue. Overall, no benefit was observed for the AT compared to conventional DO with the parameters employed in this study.

Development of a standardized and reproducible murine femoral distraction osteogenesis model.

Objective: Distraction osteogenesis (DO) has been widely used to treat bone defects as its effectiveness in bone regeneration. Currently, distraction devices for establishing DO models are mainly developed for rats or large animals. However, a mouse DO model is in great need for in-depth mechanistic investigations using various transgenic mice. The current study reports the development of a reproducible murine DO model. Methods: A mini-titanium lengthener was designed and fabricated. The mini-lengthener was applied on the murine femur with four threaded pins using a designed clamp as the drilling and insertion guide. After transverse osteotomy using a Gigli saw, and after 5 days of latency, DO procedures started at 0.3 mm/day for 10 days, and the consolidation period was left for 28 days. The bone formation was monitored by radiography and histology. Potential effects on animal locomotion during DO were also measured by behavior tests. Results: Separated bone segments maintained good alignment during the entire DO phases. New bone formation was found as early as the end of the distraction phase. Active bone remodeling was found between the separated bone segments at late distraction and early consolidation phases. At the mature consolidation phase, bone remodeling was mainly observed in the contact cortical bone. Mice underwent DO procedure did not have significant impairment in their locomotion. Conclusion: We have successfully developed a murine femoral DO model, which may be used to study the biological processes of DO. We also developed the mini-lengthener and the guide clamp to ensure the standardization and reproducibility of the mouse DO model.The translational potential of this article: Current study reports the development of a murine femoral DO model. A well-established murine DO model will facilitate further investigations of the biological mechanisms of DO in various transgenic and normal mice.

Osteogenic effects and safety of human induced pluripotent stem cell-derived megakaryocytes and platelets produced on a clinical scale.

Introduction: Platelet-rich plasma obtained by centrifuging peripheral blood can promote osteogenesis owing to its abundant growth factors but has drawbacks, including rapid growth factor loss and inconsistent effects depending on donor factors. To overcome these issues, we were the first in the world to use freeze-dried human induced pluripotent stem cell-derived megakaryocytes and platelets (S-FD-iMPs) and found that they have osteogenesis-promoting effects. Since turbulence was found to activate platelet biogenesis and iPS cell-derived platelets can now be produced on a clinical scale by a device called VerMES, this study examined the osteogenesis-promoting effect and safety of clinical-scale FD-iMP (V-FD-iMPs) for future human clinical application. Method: We administered either S-FD-iMPs, V-FD-iMPs, or saline along with artificial bone to the lumbar spine of 8-week-old male Sprague-Dawley rats (n = 4 each) and evaluated bone formation by computed tomography (CT) and pathology. Next, we administered V-FD-iMPs or saline along with artificial bone to the lumber spines of 5-week-old male New Zealand White rabbits (n = 4 each) and evaluated the bone formation by CT and pathology. Rats (n = 10) and rabbits (n = 6) that received artificial bone and V-FD-iMPs in the lumbar spine were also observed for 6 months for adverse events, including infection, tumor formation, and death. Results: Both V-FD-iMPs and S-FD-iMPs significantly enhanced osteogenesis in the lumber spines of rats in comparison with the controls 8 weeks postoperatively, with no significant differences between them. Furthermore, V-FD-iMPs vigorously promoted osteogenesis in the lumber spines of rabbits 8 weeks postoperatively. In rats and rabbits, V-FD-iMPs showed no adverse effects, including infection, tumor formation, and death, over 6 months. Conclusion: These results suggest that V-FD-iMPs promote safe osteogenesis.