Nerve-derived factors have attracted attention in bone regeneration therapy due to their ability to promote bone regeneration and nerve innervation. Mesenchymal stem cells transported to target sites promote osteogenesis. However, there are few reports on the effects of neural stem cells on bone regeneration. Therefore, the aim of this study was to investigate the role of neural stem cells in osteogenesis. Here, embryoid bodies (EB) or primary neurospheres (1NS) were generated using mouse induced pluripotent stem cells (iPS cells), which were then seeded onto gelatin (Gel) sponges. The seeded Gel sponges were then transplanted into mouse calvarial bone defects. We noted that 1NS-seeded Gel promoted bone regeneration and the presence of TRAP-positive cells, whereas the EB-seeded Gel did not. RNA-sequencing of the 1NS-seeded and EB seeded Gels showed an upregulation of the TGF-β signaling pathway in the 1NS-seeded Gel group. Immunostaining confirmed the presence of Id3 positive cells in mice with bone defects treated with the 1NS-seeded Gel. These findings suggest that the transplantation of neural stem cells may contribute to the promotion of bone regeneration. STATEMENT OF SIGNIFICANCE: This study aimed to investigate whether neural stem cells, when seeded in gelatin sponges, promoted bone regeneration. It has been well documented that bone is tightly linked with the nervous systems. Bioscaffolds comprising factors that promote innervation and bone regeneration have been investigated for use in bone therapy. However, there is limited research on the use of neural stem cells for promoting bone formation. To assess this relationship, we conducted both in vivo and in vitro assays to determine whether neural stem cells promoted bone formation. We noted that primary neurospheres-seeded gelatin sponges promoted bone formation significantly in mice with calvarial defects after four weeks. This study provides a novel approach of neural stem cells for bone therapy.

UNASSIGNED: Mammals have limited limb regeneration compared to amphibians. The role of Wnt signaling pathways in limb regeneration has rarely been studied. So, this study aimed to investigate the effect of Wnt-signaling using chemicals CHIR99021 and IWP-2 on amputated mice digit tips regeneration in an in vitro organ culture system.
UNASSIGNED: The distal phalanx of paws from C57BL/6J mouse fetuses at E14.5, E16.5, and E18.5 was amputated. Then, the hands were cultured for 7 days. Subsequently, paws were treated with 1-50 µg/ml concentration of CHIR99021 and 5-10 µg/ml concentration of IWP-2. Finally, the new tissue regrowth was assessed by histological analysis, immunohistochemistry for BC, TCF1, CAN, K14, and P63 genes, and beta-catenin and Tcf1 genes were evaluated with RT-qPCR.
UNASSIGNED: The paws of E14.5 and E16.5 days were shrinkaged and compressed after 7 days, so the paws of 18.5E that were alive were selected. As a result, newly-grown masses at digit tips were observed in 25 and 30 µl/ml concentrations of the CHR99021 group but not in the IWP2 treatment (*P<0.05; **P<0.01). qRT-PCR analysis confirmed the significant up-regulation of beta-catenin and Tcf1 genes in CHIR99021 group in comparison to the IWP-2 group (P<0.05). Moreover, Alcian-blue staining demonstrated the presence of cartilage-like tissue at regenerated mass in the CHIR group. In immunohistochemistry analysis beta-catenin, ACN, Keratin-14, and P63 protein expression were observed in digit tips in the CHIR-treated group.
UNASSIGNED: By activating the Wnt signaling pathway, cartilage-like tissue formed in the blastema-like mass in the mouse\'s amputated digit tips.

BACKGROUND: The mechanism of cortical bone adaptation to static forces is not well understood. This is an important process because static forces are applied to the cortical bone in response to the growth of soft tissues and during Orthodontic and Orthopedic corrections. The aim of this study was to investigate the cortical bone response to expanding forces applied to the maxilla.
METHODS: Overall, 375 adult Sprague-Dawley rats were divided into three groups: 1) static force group, 2) static force plus stimulation group, and 3) sham group. In addition to static force across the maxilla, some animals were exposed to anti-inflammatory medication. Samples were collected at different time points and evaluated by micro-computed tomography, fluorescence microscopy, immunohistochemistry, and gene and protein analyses.
RESULTS: The application of expansion forces to the maxilla increased inflammation in the periosteum and activated osteoclasts on the surface of the cortical plate. This activation was independent of the magnitude of tooth movement but followed the pattern of skeletal displacement. Bone formation on the surface of the cortical plate occurred at a later stage and resulted in the relocation of the cortical boundary of the maxilla and cortical drifting.
CONCLUSIONS: This study demonstrates that cortical bone adaptation to static forces originates from the periosteum, and it is an inflammatory-based phenomenon that can be manipulated by the clinician. Our findings support a new theory for cortical adaptation to static forces and an innovative clinical approach to promote cortical drifting through periosteal stimulation. Being able to control cortical drift can have a significant impact on clinical orthodontic and dentofacial orthopedics by allowing corrections of severe deformities without the need for maxillofacial surgery.

The polarization balance of M1/M2 macrophages with different functions is important in osteogenesis and bone repair processes. In a previous study, we succeeded in developing honeycomb tricalcium phosphate (TCP), which is a cylindrical scaffold with a honeycomb arrangement of straight pores, and we demonstrated that TCP with 300 and 500 μm pore diameters (300TCP and 500TCP) induced bone formation within the pores. However, the details of the influence of macrophage polarization on bone formation using engineered biomaterials, especially with respect to the geometric structure of the artificial biomaterials, are unknown. In this study, we examined whether differences in bone tissue formation due to differences in TCP geometry were due to the polarity of the assembling macrophages. Immunohistochemistry for IBA-1, iNOS, and CD163 single staining was performed. The 300TCP showed a marked infiltration of iNOS-positive cells, which are thought to be M1 macrophages, during the osteogenesis process, while no involvement of CD163-positive cells, which are thought to be M2 macrophages, was observed in the TCP pores. In addition, 500TCP showed a clustering of iNOS-positive cells and CD163-positive cells at 2 weeks, suggesting the involvement of M2 macrophages in the formation of bone tissue in the TCP pores. In conclusion, we demonstrated for the first time that the geometrical structure of the artificial biomaterial, i.e., the pore size of honeycomb TCP, affects the polarization of M1/2 macrophages and bone tissue formation in TCP pores.

BACKGROUND: Bone loss of residual alveolar ridges is a great challenge in the field of dental implantology. Deproteinized bovine bone mineral (DBBM) is commonly used for bone regeneration, however, it is loose and difficult to handle in clinical practice. Hyaluronic acid (HA) shows viscoelasticity, permeability and excellent biocompatibility. The aim of this study is to evaluate whether high-molecular-weight (MW) HA combined with DBBM could promote new bone formation in rat calvarial critical size defects (CSDs).
METHODS: Rat calvarial CSDs (5 mm in diameter) were created. Rats (n = 45) were randomly divided into 3 groups: HA-DBBM compound grafting group, DBBM particles only grafting group and no graft group. Defect healing was assessed by hematoxylin-eosin staining and histomorphometry 2, 4 and 8 weeks postop, followed by Micro-CT scanning 8 weeks postop. Statistical analyses were performed by ANOVA followed by Tukey\'s post hoc test with P < 0.05 indicating statistical significance.
RESULTS: All rats survived after surgery. Histomorphometric evaluation revealed that at 2, 4 and 8 weeks postop, the percentage of newly formed bone was significantly greater in HA-DBBM compound grafting group than in the other two groups. Consistently, Micro-CT assessment revealed significantly more trabecular bone (BV/TV and Tb.N) in HA-DBBM compound group than in the other two groups, respectively (P < 0.05). Moreover, the trabecular bone was significantly more continuous (Tb.Pf) in HA-DBBM compound group than in the other two groups, respectively (P < 0.05).
CONCLUSIONS: HA not only significantly promoted new bone formation in rats calvarial CSDs but also improved the handling ability of DBBM.

Intermittent and continuous mechanical loads are known to influence osteogenic activity. The present study examines the effects of matched intermittent and continuous load in vitro on bone formation markers. MC3T3 (mouse pre-osteoblasts) were cultured and placed in a bioreactor to undergo continuous, intermittent, or unloading for 1, 3 and 12 days. Loading conditions were matched for magnitude, duration and frequency. Each time point was analysed for alkaline phosphatase (ALP) activity, procollagen 1 N-terminal propeptide (PINP) and alizarin red staining (ARS). Intermittent load caused an increase in ALP activity across all time points compared to continuous loading (↑30%-59%) and unloaded conditions (↑70%-90%). PINP concentrations from intermittent load were lower than continuous load (↓112%) on day 3. However, no differences were observed in PINP concentrations between loading conditions at other time points. No differences were observed for ARS between loading conditions. Intermittent load caused an increase in bone formation marker ALP, but not PINP, when compared to continuous loading and unloaded conditions. These findings further our knowledge in bone formation response and provide additional tools for the analysis of osteogenesis in vitro.

OBJECTIVE: Collagen membranes are extensively used for guided bone regeneration procedures, primarily for horizontal bone augmentation. More recently, it has been demonstrated that collagen membranes promote bone regeneration. Present study aimed at assessing if structural modifications of collagen membranes may enhance their osteoconductive capacity.
METHODS: Twenty-four adult Wistar rats were used. Bilateral calvaria defects with a diameter of 5 mm were prepared and covered with prototypes of collagen membranes (P1 or P2). The P1 membrane (positive control) presented a lower onset temperature of protein denaturation and a higher solubility than the P2 membrane (test). The contralateral defects were left uncovered (NC). After 1 and 4 weeks, the animals were euthanized. A microcomputed tomography analysis of the harvested samples was performed within and above the bony defect. Undecalcified ground sections were subjected to light microscopy and morphometric analysis.
RESULTS: Bone formation was observed starting from the circumferential borders of the defects in all groups at 1-week of healing. The foci of ossification were observed at the periosteal and dura mater sites, with signs of collagen membrane mineralization. However, there was no statistically significant difference between the groups. At 4 weeks, remnants of the collagen fibers were embedded in the newly formed bone. In the P2 group, significantly more bone volume, more new bone, and marrow spaces compared to the NC group were observed. Furthermore, the P2 group showed more bone volume ectocranially then the P1 group.
CONCLUSIONS: Bone formation subjacent to a P2 membrane was superior than subjacent to the P1 membrane and significantly better compared to the control. Modifications of the physico-chemical properties may enhance the osteoconductive competence of collagen membranes, supporting bone formation outside the bony defects.

Recent scientific investigations have revealed the intricate mechanisms underlying bone formation, emphasizing the essential role of long non-coding RNAs (lncRNAs) as critical regulators. This process, essential for skeletal strength and functionality, involves the transformation of mesenchymal stem cells into osteoblasts and subsequent deposition of bone matrix. lncRNAs, including HOX transcript antisense RNA (HOTAIR), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), differentiation antagonizing non-coding RNA (DANCR), and maternally expressed gene 3 (MEG3), have emerged as prominent players in this regulatory network. HOTAIR modulates osteoblast differentiation by interacting with chromatin-modifying enzymes, while MALAT1 regulates osteogenic differentiation through microRNA interactions. DANCR collaborates with Runx2 to fine-tune osteoblast differentiation, and MEG3 orchestrates multiple signaling pathways crucial for bone formation. Moreover, other lncRNAs such as H19, lncRNA for enhancing osteogenesis 3, rhabdomyosarcoma 2-associated transcript, urothelial cancer associated 1, taurine up-regulated gene 1, and nuclear enriched abundant transcript 1 contribute to the complex regulatory network governing osteoblast activities. Understanding the precise roles of these lncRNAs offers promising avenues for developing innovative therapeutic strategies targeting bone-related disorders like osteoporosis. Overall, this review summarizes the pivotal role of lncRNAs in bone formation, highlighting their potential as targets for future research endeavors aimed at advancing therapeutic interventions in bone diseases.

Bioactive ceramics, primarily consisting of bioactive glasses, glass-ceramics, calcium orthophosphate ceramics, calcium silicate ceramics and calcium carbonate ceramics, have received great attention in the past decades given their biocompatible nature and excellent bioactivity in stimulating cell proliferation, differentiation and tissue regeneration. Recent studies have tried to combine bioactive ceramics with bioactive ions, polymers, bioactive proteins and other chemicals to improve their mechanical and biological properties, thus rendering them more valid in tissue engineering scaffolds. This review presents the beneficial properties and potential applications of bioactive ceramic-based materials in dentistry, particularly in the repair and regeneration of dental hard tissue, pulp-dentin complex, periodontal tissue and bone tissue. Moreover, greater insights into the mechanisms of bioactive ceramics and the development of ceramic-based materials are provided.
[Box: see text].

UNASSIGNED: This meta-analysis aims to examine differences in biochemical markers of bone metabolism between individuals with type 2 diabetes (T2DM) and non-T2DM control groups.
UNASSIGNED: Two independent evaluators searched five databases: PubMed, EMBASE, EBSCOhost, Web of Science, and the Cochrane Library. We aimed to identify observational studies investigating the impact of T2DM on biochemical markers of bone metabolism. Literature retrieval covered the period from the establishment of the databases up to November 2022. Studies were included if they assessed differences in biochemical markers of bone metabolism between T2DM patients and non-T2DM control groups using cross-sectional, cohort, or case-control study designs.
UNASSIGNED: Fourteen studies were included in the analysis, comprising 12 cross-sectional studies and 2 cohort studies. Compared to the non-T2DM control group, T2DM patients showed reduced levels of Osteocalcin and P1NP, which are markers of bone formation. Conversely, levels of Alkaline phosphatase and Bone-specific alkaline phosphatase, other bone formation markers, increased. The bone resorption marker CTX showed decreased levels, while TRACP showed no significant difference.
UNASSIGNED: In individuals with T2DM, most bone turnover markers indicated a reduced rate of bone turnover. This reduction can lead to increased bone fragility despite higher bone mineral density, potentially increasing the risk of osteoporosis.
Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php? identifier CRD42022366430.