Search Results (149)

Objective: Growth retardation in adolescents caused by nutritional deficiency requires effective intervention. A novel dietary supplement containing bamboo shoot extract, amino acids, and calcium citrate (Kidtal + Ca, KDTCa) was evaluated for its growth-promoting effects. Methods: After acclimatization, sixty-three 3-week-old male Sprague-Dawley (SD) rats were randomly divided into a normal control group and model groups. Growth retardation was induced in the modeling groups through calcium-deficient feeding, followed by administration of KDTCa, bamboo shoot extract and amino acids (Kidtal), or calcium citrate (CC). After 6 weeks of intragastric administration, the mechanical properties, microstructure, and growth plate development of bone were evaluated using three-point bending, micro-CT, and H&E staining, respectively. Bone calcium/phosphorus distribution and fecal calcium apparent absorption rate were measured by ICP-MS. Results: All inter-group differences were analyzed using one-way analysis of variance and checked using the Tuckey test. KDTCa treatment dose-dependently enhanced bone development in calcium-deficient rats. Compared to the model group, H-KDTCa significantly restored naso-anal length (p < 0.05) and body weight (p < 0.01). KDTCa supplementation significantly restored calcium and phosphorus levels in blood and bone. Three-point bending experiments showed that the stiffness and bending energy were increased by 142.58% and 384.7%. In bone microarchitecture, both bone mineral density (BMD) and microstructural parameters were significantly improved. These findings were consistent with the increased long bone length (p < 0.05) and decreased serum BALP/TRACP levels (p < 0.001). Dose-dependent IGF-1 elevation (p < 0.01) potentially mediated growth plate elongation by 35.34%. Notably, KDTCa increased calcium apparent absorption by 6.1% versus calcium-only supplementation at equal intake. Conclusions: KDTCa improves bone microstructure and strength, restores bone metabolism, and enhances growth plate height via promoting IGF-1 secretion to facilitate bone development. Further studies are needed to determine whether the components and calcium in Kidtal have a synergistic effect. Full article

The Arhgap29 gene encodes Rho-GTPase-activating protein 29 (Arhgap29), which plays a crucial role in embryonic tissue development. Mutations in the Arhgap29 gene are significantly associated with non-syndromic cleft lip and palate (NSCL/P). Our study demonstrated that the deletion of Arhgap29 leads to syndromic cleft lip and palate (SCL/P) characteristics in mice, where, in addition to cleft palate, the mice exhibit craniofacial and systemic skeletal abnormalities. However, the mechanisms underlying these skeletal abnormalities remain unclear. Through micro-CT imaging, histological analysis, and transcriptomic methods, we discovered that the knockout of Arhgap29 delays the fusion of Meckel’s cartilage, widens cranial sutures, reduces bone quality, and alters the expression of osteoblasts and osteoclasts in the mandible. Digit defects, including ectrodactyly and impaired endochondral ossification, were also observed. Immunohistochemical analysis demonstrated the expression of Arhgap29 in both osteoblasts and osteoclasts, indicating its dual role in maintaining matrix homeostasis and regulating bone resorption equilibrium. Transcriptomic analysis revealed disrupted calcium and MAPK signaling pathways, while in vitro studies demonstrated impaired osteogenesis in Arhgap29-deficient calvarial cells, mirroring the in vivo defects. Furthermore, spatial transcriptomics linked the loss of Arhgap29 to defective bone differentiation and protein synthesis. Our findings underscore the critical role of Arhgap29 in the development of the mandible and digits, suggesting its potential as a pathogenic gene associated with syndromic cleft lip and palate (SCL/P). Full article

Cartilage is a crucial component of the vertebrate skeletal system, providing structural integrity, flexibility, and adaptive functions across species. In teleost fish, cartilage exhibits significant morphological and functional diversity, providing specialized biomechanical properties essential for aquatic life. This study presents a detailed histological, immunohistochemical, and ultrastructural investigation of cartilage in molly fish (Poecilia sphenops), identifying five distinct types of cartilage: hyaline-cell, scleral, cell-rich hyaline, elastic cell-rich, and matrix-rich hyaline cartilage. Histological staining techniques revealed notable differences in cellular architecture and composition of the extracellular matrix among the cartilage types. Immunohistochemical analysis demonstrated the expression of S100 protein and acetylcholinesterase (Ach), suggesting their involvement in cartilage regulation and maintenance. Endochondral ossification was observed in the head and gill arches. Electron microscopy provided detailed insights into chondrocyte morphology, interactions between cartilage and the perichondrium, and interactions between telocytes and fibroblasts. The findings enhance our understanding of skeletal adaptations in teleost fish, emphasizing the functional diversity of cartilage in aquatic environments. This study contributes to evolutionary biology and may have implications for regenerative medicine and biomaterials research. Full article

Although the roles of proteoglycans (PGs) have been well documented in the development and homeostasis of the temporomandibular joint (TMJ), how the glycosaminoglycan (GAG) chains of PGs contribute to TMJ chondrogenesis and osteogenesis still requires explication. In this study, we found that FAM20B, a hexokinase essential for attaching GAG chains to the core proteins of PGs, was robustly activated in the condylar mesenchyme during TMJ development. The inactivation of Fam20b in craniofacial neural crest cells (CNCCs) dramatically reduced the synthesis and accumulation of GAG chains rather than core proteins in the condylar cartilage, which resulted in a hypoplastic condylar cartilage by severely promoting chondrocyte hypertrophy and perichondral ossification. In the condyles of Wnt1-Cre;Fam20b^f/f mouse embryos, enlarged Ihh- and COL10-expressing domains indicated premature hypertrophy resulting from an attenuated IHH-PTHRP negative feedback in condylar chondrocytes, while increased osteogenic markers, canonical Wnt activity, and type-H angiogenesis verified the enhanced osteogenesis in the perichondrium. Further ex vivo investigations revealed that the loss of Fam20b decreased the domain area but increased the activity of HH signaling in the embryonic condylar mesenchyme. Moreover, the abrogation of GAG chains in heparan sulfate and chondroitin sulfate proteoglycans led to a rapid up- and then downregulation of HH signaling in condylar chondrocytes, implicating a “slow-release” manner of growth factors controlled by GAG chains. Overall, this study revealed a comprehensive role of the FAM20B-catalyzed GAG chain synthesis in the chondrogenic and osteogenic differentiation of the embryonic TMJ condyle. Full article

The temporomandibular joint (TMJ) is unique in both developmental origin and functional maintenance. The role of bone morphogenic protein (BMP) signaling in endochondral ossification has been widely investigated but not in the context of the TMJ. We employed a histomorphometric analysis approach to understand how augmented BMP signaling in the cranial neural crest affects the postnatal development of the TMJ. Our analysis showed that cartilage length in the mandibular condyle was reduced in Wnt1 Cre;caBmpr1a mice before the weaning stage (P17). However, following weaning, the mandibular condylar cartilage showed recovered length (P28 and P42). Furthermore, the changes in cartilage length coincide with alterations in cell death in the superficial region of the mandibular condyle. These results suggest that BMP signaling influences chondrocyte cell death and TMJ development in a timepoint-specific manner. Full article

Background/Objectives: In contrast to endochondral bone healing, the process of intramembranous bone regeneration is poorly understood. This limits our ability to repair and regenerate the craniofacial skeleton to either correct deformity or optimally heal tissues following injury. While there are several preclinical models of intramembranous regeneration within the craniofacial skeleton, some are not load bearing and others are technically challenging. The goal of this pilot study is therefore to describe a simple method for induction of cortical defects within the mandible that does not involve compounding injury to the surrounding tissues. Methods: Single cortex defects were generated in the mandible body of 8-week-old male and female mice. The extent of bone regeneration within the defect was characterized at days 0, 3, 14, and 28 following defect generation via micro-computed tomography and histology. Conclusions: Observed healing was predictable and reproducible and resulted in intramembranous bone formation. This model will help aid the understanding of intramembranous bone healing in load bearing bones (e.g., mandible) within the craniofacial skeleton Full article

The surface topography and chemistry of titanium–aluminum–vanadium (Ti6Al4V) implants play critical roles in the osteoblast differentiation of human bone marrow stromal cells (MSCs) and the creation of an osteogenic microenvironment. To assess the effects of a microscale/nanoscale (MN) topography, this study compared the effects of MN-modified, anodized, and smooth Ti6Al4V surfaces on MSC response, and for the first time, directly contrasted MN-induced osteoblast differentiation with culture on tissue culture polystyrene (TCPS) in osteogenic medium (OM). Surface characterization revealed distinct differences in microroughness, composition, and topography among the Ti6Al4V substrates. MSCs on MN surfaces exhibited enhanced osteoblastic differentiation, evidenced by increased expression of RUNX2, SP7, BGLAP, BMP2, and BMPR1A (fold increases: 3.2, 1.8, 1.4, 1.3, and 1.2). The MN surface also induced a pro-healing inflammasome with upregulation of anti-inflammatory mediators (170–200% increase) and downregulation of pro-inflammatory factors (40–82% reduction). Integrin expression shifted towards osteoblast-associated integrins on MN surfaces. RNA-seq analysis revealed distinct gene expression profiles between MSCs on MN surfaces and those in OM, with only 199 shared genes out of over 1000 differentially expressed genes. Pathway analysis showed that MN surfaces promoted bone formation, maturation, and remodeling through non-canonical Wnt signaling, while OM stimulated endochondral bone development and mineralization via canonical Wnt3a signaling. These findings highlight the importance of Ti6Al4V surface properties in directing MSC differentiation and indicate that MN-modified surfaces act via signaling pathways that differ from OM culture methods, more accurately mimicking peri-implant osteogenesis in vivo. Full article

Ex vivo regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold, and then implanted into a bone defect to exert a therapeutic effect. Compared to recombinant human BMP-2 (rhBMP-2), which is approved for clinical use, regional gene therapy may have unique benefits related to the addition of MSCs and the sustained release of BMP-2. However, the cellular and transcriptional mechanisms regulating the response to these two strategies for BMP-2 mediated bone regeneration are largely unknown. Here, for the first time, we performed single-cell RNA sequencing (10x Genomics) of hematoma tissue in six rats with critical-sized femoral defects that were treated with either regional gene therapy or rhBMP-2. Our unbiased bioinformatic analysis of 2393 filtered cells in each group revealed treatment-specific differences in their cellular composition, transcriptional profiles, and cellular communication patterns. Gene therapy treatment induced a more robust chondrogenic response, as well as a decrease in the proportion of fibroblasts and the expression of profibrotic pathways. Additionally, gene therapy was associated with an anti-inflammatory microenvironment; macrophages expressing canonical anti-inflammatory markers were more common in the gene therapy group. In contrast, pro-inflammatory markers were more highly expressed in the rhBMP-2 group. Collectively, the results of our study may offer insights into the unique pathways through which ex vivo regional gene therapy can augment bone regeneration compared to rhBMP-2. Furthermore, an improved understanding of the cellular pathways involved in segmental bone defect healing may allow for the further optimization of regional gene therapy or other bone repair strategies. Full article

In the context of bone fractures, the influence of the mechanical environment on the healing outcome is widely accepted, while its influence at the cellular level is still poorly understood. This study explores the influence of mechanical load on naïve mesenchymal stem cell (MSC) differentiation, focusing on hypertrophic chondrocyte differentiation. Unlike primary bone healing, which involves the direct differentiation of MSCs into bone-forming cells, endochondral ossification uses an intermediate cartilage template that remodels into bone. A high-throughput uniaxial bioreactor system (StrainBot) was used to apply varying percentages of strain on naïve MSCs encapsulated in GelMa hydrogels. This research shows that cyclic uniaxial compression alone directs naïve MSCs towards a hypertrophic chondrocyte phenotype. This was demonstrated by increased cell volumes and reduced glycosaminoglycan (GAG) production, along with an elevated expression of hypertrophic markers such as MMP13 and Type X collagen. In contrast, Type II collagen, typically associated with resting chondrocytes, was poorly detected under mechanical loading alone conditions. The addition of chondrogenic factor TGFβ1 in the culture medium altered these outcomes. TGFβ1 induced chondrogenic differentiation, as indicated by higher GAG/DNA production and Type II collagen expression, overshadowing the effect of mechanical loading. This suggests that, under mechanical strain, hypertrophic differentiation is hindered by TGFβ1, while chondrogenesis is promoted. Biochemical analyses further confirmed these findings. Mechanical deformation alone led to a larger cell size and a more rounded cell morphology characteristic of hypertrophic chondrocytes, while lower GAG and proteoglycan production was observed. Immunohistology staining corroborated the gene expression data, showing increased Type X collagen with mechanical strain. Overall, this study indicates that mechanical loading alone drives naïve MSCs towards a hypertrophic chondrocyte differentiation path. These insights underscore the critical role of mechanical forces in MSC differentiation and have significant implications for bone healing, regenerative medicine strategies and rehabilitation protocols. Full article

While the flat bones of the face, most of the cranial bones, and the clavicles are formed directly from sheets of undifferentiated mesenchymal cells, most bones in the human body are first formed as cartilage templates. Cartilage is subsequently replaced by bone via a very tightly regulated process termed endochondral ossification, which is led by chondrocytes of the growth plate (GP). This process requires continuous communication between chondrocytes and invading cell populations, including osteoblasts, osteoclasts, and vascular cells. A deeper understanding of these signaling pathways is crucial not only for normal skeletal growth and maturation but also for their potential relevance to pathophysiological processes in bones and joints. Due to limited information on the communication between chondrocytes and other cell types in developing bones, this review examines the current knowledge of how interactions between chondrocytes and bone-forming cells modulate bone growth. Full article

Endochondral ossification is the process by which cartilage is mineralized into bone, and is essential for the development of long bones. Osteocalcin (OCN), a protein abundant in bone matrix, also exhibits high expression in chondrocytes, especially hypertrophic chondrocytes, while its role in endochondral ossification remains unclear. Utilizing a new CRISPR/Cas9-mediated bglap–bglap2 deficiency (OCN^em) mouse model generated in our laboratory, we provide the first evidence of OCN’s regulatory function in chondrocyte differentiation and endochondral ossification. The OCN^em mice exhibited significant delays in primary and secondary ossification centers compared to wild-type mice, along with increased cartilage length in growth plates and hypertrophic zones during neonatal and adolescent stages. These anomalies indicated that OCN deficiency disturbed endochondral ossification during embryonic and postnatal periods. Mechanism wise, OCN deficiency was found to increase chondrocyte differentiation and postpone vascularization process. Furthermore, bone marrow mesenchymal stromal cells (BMSCs) from OCN^em mice demonstrated an increased capacity for chondrogenic differentiation. Transcriptional network analysis implicated that BMP and TGF-β signaling pathways were highly affected in OCN^em BMSCs, which is closely associated with cartilage development and maintenance. This elucidation of OCN’s function in chondrocyte differentiation and endochondral ossification contributes to a more comprehensive understanding of its impact on skeletal development and homeostasis. Full article

Degenerative scoliosis (DS), encompassing conditions like spondylolisthesis and spinal stenosis, is a common type of spinal deformity. Lumbar interbody fusion (LIF) stands as a conventional surgical intervention for this ailment, aiming at decompression, restoration of intervertebral height, and stabilization of motion segments. Despite its widespread use, the precise mechanism underlying spinal fusion remains elusive. In this review, our focus lies on endochondral ossification for spinal fusion, a process involving vertebral development and bone healing. Endochondral ossification is the key step for the successful vertebral fusion. Endochondral ossification can persist in hypoxic conditions and promote the parallel development of angiogenesis and osteogenesis, which corresponds to the fusion process of new bone formation in the hypoxic region between the vertebrae. The ideal material for interbody fusion cages should have the following characteristics: (1) Good biocompatibility; (2) Stable chemical properties; (3) Biomechanical properties similar to bone tissue; (4) Promotion of bone fusion; (5) Favorable for imaging observation; (6) Biodegradability. Utilizing cartilage-derived bone-like constructs holds promise in promoting bony fusion post-operation, thus warranting exploration in the context of spinal fusion procedures. Full article

Before calcification begins, the early embryonic and fetal skeletal development of both mammalian Homo sapiens and the chondrichthyan fish Raja asterias consists exclusively of cartilage. This cartilage is formed and shaped through processes involving tissue segmentation and the frequency, distribution, and orientation of chondrocyte mitoses. In the subsequent developmental phase, mineral deposition in the cartilage matrix conditions the development further. The stiffness and structural layout of the mineralized cartilage have a significant impact on the shape of the anlagen (early formative structure of a tissue, a scaffold on which the new bone is formed) and the mechanical properties of the skeletal segments. The fundamental difference between the two studied species lies in how calcified cartilage serves as a scaffold for osteoblasts to deposit bone matrix, which is then remodeled. In contrast, chondrichthyans retain the calcified cartilage as the definitive skeletal structure. This study documents the distinct mineral deposition pattern in the cartilage of the chondrichthyan R. asterias, in which calcification progresses with the formation of focal calcification nuclei or “tesserae”. These are arranged on the flat surface of the endo-skeleton (crustal pattern) or aligned in columns (catenated pattern) in the radials of the appendicular skeleton. This anatomical structure is well adapted to meet the mechanical requirements of locomotion in the water column. Conversely, in terrestrial mammals, endochondral ossification (associated with the remodeling of the calcified matrix) provides limb bones with the necessary stiffness to withstand the strong bending and twisting stresses of terrestrial locomotion. In this study, radiographs of marine mammals (reproduced from previously published studies) document how the endochondral ossification in dolphin flippers adapts to the mechanical demands of aquatic locomotion. This adaptation includes the reduction in the length of the stylopodium and zeugopodium and an increase in the number of elements in the autopodium’s central rays. Full article

The objective of this work was to analyze the in vitro and in vivo tests of a novel Mg-based biodegradable alloy—Mg-0.5%Ca—with various amounts of Zn (0.5, 1, 1.5, 2.0, and 3.0 wt.%). In terms of in vitro biocompatibility, MTT and Calcein-AM cell viability assays, performed on the MG-63 cell line through the extract method, revealed that all five alloy extracts are non-cytotoxic at an extraction ratio of 0.025 g alloy per mL of cell culture medium. In the in vivo histological analysis, Mg-0.5Ca-1.5Zn demonstrated exceptional potential for stimulating bone remodeling and showed excellent biocompatibility. It was observed that Mg-0.5Ca-0.5Zn, Mg-0.5Ca-1.5Zn, and Mg-0.5Ca-3Zn displayed good biocompatibility. Furthermore, the histological examination highlighted the differentiation of periosteal cells into chondrocytes and subsequent bone tissue replacement through endochondral ossification. This process highlighted the importance of the initial implant’s integrity and the role of the periosteum. In summary, Mg-0.5Ca-1.5Zn stands out as a promising candidate for bone regeneration and osseointegration, supported by both in vitro and in vivo findings. Full article

As a result of screening a panel of marine organisms to identify lead molecules for the stimulation of endochondral bone formation, the calcareous sponge Pericharax heteroraphis was identified to exhibit significant activity during endochondral differentiation. On further molecular networking analysis, dereplication and chemical fractionation yielded the known clathridine A-related metabolites 3–6 and the homodimeric complex (clathridine A)₂ Zn²⁺ (9), together with the new unstable heterodimeric complex (clathridine A–clathridimine)Zn²⁺ (10). With the presence of the zinc complexes annotated through the LC-MS analysis of the crude extract changing due to the instability of some metabolites and complexes constituting the mixture, we combined the isolation of the predicted molecules with their synthesis in order to confirm their structure and to understand their reactivity. Interestingly, we also found a large quantity of the contaminant benzotriazoles BTZ (7) and its semi-dimer (BTZ)₂CH₂ (8), which are known to form complexes with transition metals and are used for preventing corrosion in water. All isolated 2-aminoimidazole derivatives and complexes were synthesized not only for structural confirmation and chemical understanding but to further study their bioactivity during endochondral differentiation, particularly the positively screened imidazolone derivatives. Compounds leucettamine B, clathridine A and clathridimine were found to increase type X collagen transcription and stimulate endochondral ossification in the ATDC5 micromass model. Full article