Reprogramming macrophages with R848-loaded artificial protocells to modulate skin and skeletal wound healing.

After tissue injury, inflammatory cells are rapidly recruited to the wound where they clear microbes and other debris, and coordinate the behaviour of other cell lineages at the repair site in both positive and negative ways. In this study, we take advantage of the translucency and genetic tractability of zebrafish to evaluate the feasibility of reprogramming innate immune cells in vivo with cargo-loaded protocells and investigate how this alters the inflammatory response in the context of skin and skeletal repair. Using live imaging, we show that protocells loaded with R848 cargo (which targets TLR7 and TLR8 signalling), are engulfed by macrophages resulting in their switching to a pro-inflammatory phenotype and altering their regulation of angiogenesis, collagen deposition and re-epithelialization during skin wound healing, as well as dampening osteoblast and osteoclast recruitment and bone mineralization during fracture repair. For infected skin wounds, R848-reprogrammed macrophages exhibited enhanced bactericidal activities leading to improved healing. We replicated our zebrafish studies in cultured human macrophages, and showed that R848-loaded protocells similarly reprogramme human cells, indicating how this strategy might be used to modulate wound inflammation in the clinic.

Initial Upper Palaeolithic Homo sapiens from Bacho Kiro Cave, Bulgaria.

The Middle to Upper Palaeolithic transition in Europe witnessed the replacement and partial absorption of local Neanderthal populations by Homo sapiens populations of African origin1. However, this process probably varied across regions and its details remain largely unknown. In particular, the duration of chronological overlap between the two groups is much debated, as are the implications of this overlap for the nature of the biological and cultural interactions between Neanderthals and H. sapiens. Here we report the discovery and direct dating of human remains found in association with Initial Upper Palaeolithic artefacts2, from excavations at Bacho Kiro Cave (Bulgaria). Morphological analysis of a tooth and mitochondrial DNA from several hominin bone fragments, identified through proteomic screening, assign these finds to H. sapiens and link the expansion of Initial Upper Palaeolithic technologies with the spread of H. sapiens into the mid-latitudes of Eurasia before 45 thousand years ago3. The excavations yielded a wealth of bone artefacts, including pendants manufactured from cave bear teeth that are reminiscent of those later produced by the last Neanderthals of western Europe4-6. These finds are consistent with models based on the arrival of multiple waves of H. sapiens into Europe coming into contact with declining Neanderthal populations7,8.

Guidelines derived from biomineralized tissues for design and construction of high-performance biomimetic materials: from weak to strong.

Living organisms in nature have undergone continuous evolution over billions of years, resulting in the formation of high-performance fracture-resistant biomineralized tissues such as bones and teeth to fulfill mechanical and biological functions, despite the fact that most inorganic biominerals that constitute biomineralized tissues are weak and brittle. During the long-period evolution process, nature has evolved a number of highly effective and smart strategies to design chemical compositions and structures of biomineralized tissues to enable superior properties and to adapt to surrounding environments. Most biomineralized tissues have hierarchically ordered structures consisting of very small building blocks on the nanometer scale (nanoparticles, nanofibers or nanoflakes) to reduce the inherent weaknesses and brittleness of corresponding inorganic biominerals, to prevent crack initiation and propagation, and to allow high defect tolerance. The bioinspired principles derived from biomineralized tissues are indispensable for designing and constructing high-performance biomimetic materials. In recent years, a large number of high-performance biomimetic materials have been prepared based on these bioinspired principles with a large volume of literature covering this topic. Therefore, a timely and comprehensive review on this hot topic is highly important and contributes to the future development of this rapidly evolving research field. This review article aims to be comprehensive, authoritative, and critical with wide general interest to the science community, summarizing recent advances in revealing the formation processes, composition, and structures of biomineralized tissues, providing in-depth insights into guidelines derived from biomineralized tissues for the design and construction of high-performance biomimetic materials, and discussing recent progress, current research trends, key problems, future main research directions and challenges, and future perspectives in this exciting and rapidly evolving research field.

Mass Spectrometry-Based Proteomics of Poly(methylmethacrylate)-Embedded Bone.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely employed technique in proteomics research for studying the proteome biology of various clinical samples. Hard tissues, such as bone and teeth, are routinely preserved using synthetic poly(methyl methacrylate) (PMMA) embedding resins that enable histological, immunohistochemical, and morphological examination. However, the suitability of PMMA-embedded hard tissues for large-scale proteomic analysis remained unexplored. This study is the first to report on the feasibility of PMMA-embedded bone samples for LC-MS/MS analysis. Conventional workflows yielded merely limited coverage of the bone proteome. Using advanced strategies of prefractionation by high-pH reversed-phase liquid chromatography in combination with isobaric tandem mass tag labeling resulted in proteome coverage exceeding 1000 protein identifications. The quantitative comparison with cryopreserved samples revealed that each sample preparation workflow had a distinct impact on the proteomic profile. However, workflow replicates exhibited a high reproducibility for PMMA-embedded samples. Our findings further demonstrate that decalcification prior to protein extraction, along with the analysis of solubilization fractions, is not preferred for PMMA-embedded bone. The biological applicability of the proposed workflow was demonstrated using samples of human PMMA-embedded alveolar bone and the iliac crest, which revealed anatomical site-specific proteomic profiles. Overall, these results establish a crucial foundation for large-scale proteomics studies contributing to our knowledge of bone biology.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton.

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

Bone Endosteal Mimics Regulates Breast Cancer Development and Phenotype.

Bone is a frequent site for metastatic development in various cancer types, including breast cancer, with a grim prognosis due to the distinct bone environment. Despite considerable advances, our understanding of the underlying processes leading to bone metastasis progression remains elusive. Here, we applied a bioactive three-dimensional (3D) model capable of mimicking the endosteal bone microenvironment. MDA-MB-231 and MCF7 breast cancer cells were cultured on the scaffolds, and their behaviors and the effects of the biomaterial on the cells were examined over time. We demonstrated that close interactions between the cells and the biomaterial affect their proliferation rates and the expression of c-Myc, cyclin D, and KI67, leading to cell cycle arrest. Moreover, invasion assays revealed increased invasiveness within this microenvironment. Our findings suggest a dual role for endosteal mimicking signals, influencing cell fate and potentially acting as a double-edged sword, shuttling between cell cycle arrest and more active, aggressive states.

3D-Printed Silk Proteins for Bone Tissue Regeneration and Associated Immunomodulation.

Current bone repair methods have limitations, prompting the exploration of innovative approaches. Tissue engineering emerges as a promising solution, leveraging biomaterials to craft scaffolds replicating the natural bone environment, facilitating cell growth and differentiation. Among fabrication techniques, three-dimensional (3D) printing stands out for its ability to tailor intricate scaffolds. Silk proteins (SPs), known for their mechanical strength and biocompatibility, are an excellent choice for engineering 3D-printed bone tissue engineering (BTE) scaffolds. This article comprehensively reviews bone biology, 3D printing, and the unique attributes of SPs, specifically detailing criteria for scaffold fabrication such as composition, structure, mechanics, and cellular responses. It examines the structural, mechanical, and biological attributes of SPs, emphasizing their suitability for BTE. Recent studies on diverse 3D printing approaches using SPs-based for BTE are highlighted, alongside advancements in their 3D and four-dimensional (4D) printing and their role in osteo-immunomodulation. Future directions in the use of SPs for 3D printing in BTE are outlined.

Employing novel biocompatible composite scaffolds with bioglass 58S and poly L-lactic acid for effective bone defect treatment.

BACKGROUND: Bioglass materials have gained significant attention in the field of tissue engineering due to their osteoinductive and biocompatible properties that promote bone cell differentiation. In this study, a novel composite scaffold was developed using a sol-gel technique to combine bioglass (BG) 58 S with a poly L-lactic acid (PLLA). METHODS AND RESULTS: The physiochemical properties, morphology, and osteoinductive potential of the scaffolds were investigated by X-ray diffraction analysis, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The results showed that the SiO2-CaO-P2O5 system was successfully synthesized by the sol-gel method. The PLLA scaffolds containing BG was found to be osteoinductive and promoted mineralization, as demonstrated by calcium deposition assay, upregulation of alkaline phosphatase enzyme activity, and Alizarin red staining data. CONCLUSIONS: These in vitro studies suggest that composite scaffolds incorporating hBMSCs are a promising substitute material to be implemented in bone tissue engineering. The PLLA/BG scaffolds promote osteogenesis and support the differentiation of bone cells, such as osteoblasts, due to their osteoinductive properties.

New Insights into the Pro-Inflammatory and Osteoclastogenic Profile of Circulating Monocytes in Osteoarthritis Patients.

Osteoarthritis (OA) is a degenerative condition of the articular cartilage with chronic low-grade inflammation. Monocytes have a fundamental role in the progression of OA, given their implication in inflammatory responses and their capacity to differentiate into bone-resorbing osteoclasts (OCLs). This observational-experimental study attempted to better understand the molecular pathogenesis of OA through the examination of osteoclast progenitor (OCP) cells from both OA patients and healthy individuals (25 OA patients and healthy samples). The expression of osteoclastogenic and inflammatory genes was analyzed using RT-PCR. The OA monocytes expressed significantly higher levels of CD16, CD115, TLR2, Mincle, Dentin-1, and CCR2 mRNAs. Moreover, a flow cytometry analysis showed a significantly higher surface expression of the CD16 and CD115 receptors in OA vs. healthy monocytes, as well as a difference in the distribution of monocyte subsets. Additionally, the OA monocytes showed a greater osteoclast differentiation capacity and an enhanced response to an inflammatory stimulus. The results of this study demonstrate the existence of significant differences between the OCPs of OA patients and those of healthy subjects. These differences could contribute to a greater understanding of the molecular pathogenesis of OA and to the identification of new biomarkers and potential drug targets for OA.

Recent Advancements in Bone Tissue Engineering: Integrating Smart Scaffold Technologies and Bio-Responsive Systems for Enhanced Regeneration.

In exploring the challenges of bone repair and regeneration, this review evaluates the potential of bone tissue engineering (BTE) as a viable alternative to traditional methods, such as autografts and allografts. Key developments in biomaterials and scaffold fabrication techniques, such as additive manufacturing and cell and bioactive molecule-laden scaffolds, are discussed, along with the integration of bio-responsive scaffolds, which can respond to physical and chemical stimuli. These advancements collectively aim to mimic the natural microenvironment of bone, thereby enhancing osteogenesis and facilitating the formation of new tissue. Through a comprehensive combination of in vitro and in vivo studies, we scrutinize the biocompatibility, osteoinductivity, and osteoconductivity of these engineered scaffolds, as well as their interactions with critical cellular players in bone healing processes. Findings from scaffold fabrication techniques and bio-responsive scaffolds indicate that incorporating nanostructured materials and bioactive compounds is particularly effective in promoting the recruitment and differentiation of osteoprogenitor cells. The therapeutic potential of these advanced biomaterials in clinical settings is widely recognized and the paper advocates continued research into multi-responsive scaffold systems.

Therapeutic Potential of Ginsenosides on Bone Metabolism: A Review of Osteoporosis, Periodontal Disease and Osteoarthritis.

Ginsenosides, bioactive compounds from the genus Panax, have potential therapeutic effects on diverse ailments, including diabetes. Emerging evidence suggests their involvement in bone metabolism. The present review summarizes the current understanding of the effects of ginsenosides on osteoporosis, periodontal disease, and osteoarthritis. Their mechanisms of action include effects on osteoblasts, osteoclasts, periodontal ligament fibroblasts (PDLFs), and chondrocytes, which are pivotal in maintaining bone, periodontal tissue, and cartilage homeostasis. Ginsenosides may exert their beneficial effects by enhancing PDLF and osteoblast activity, suppressing osteoclast function, augmenting chondrocyte synthesis in the cartilage matrix, and mitigating connective tissue degradation. Moreover, they possess antioxidant, anti-inflammatory, antimicrobial, and anti-pyroptotic properties. Their efficacy in increasing bone density, ameliorating periodontitis, and alleviating osteoarthritis symptoms has been demonstrated in preclinical studies using animal models. In terms of their mechanism of action, ginsenosides modulate cellular differentiation, activity, and key signaling pathway molecules, such as mitogen-activated protein kinases (MAPKs), while also regulating various mediators. Furthermore, the symptomatic relief observed in animal models lends further credence to their therapeutic utility. However, to translate these preclinical findings into clinical practice, rigorous animal and clinical investigations are imperative to ascertain the safety, efficacy, and optimal dosing regimens in human subjects.

Luminescent Alendronic Acid-Conjugated Micellar Nanostructures for Potential Application in the Bone-Targeted Delivery of Cholecalciferol.

Vitamin D, an essential micronutrient crucial for skeletal integrity and various non-skeletal physiological functions, exhibits limited bioavailability and stability in vivo. This study is focused on the development of polyethylene glycol (PEG)-grafted phospholipid micellar nanostructures co-encapsulating vitamin D3 and conjugated with alendronic acid, aimed at active bone targeting. Furthermore, these nanostructures are rendered optically traceable in the UV-visible region of the electromagnetic spectrum via the simultaneous encapsulation of vitamin D3 with carbon dots, a newly emerging class of fluorescents, biocompatible nanoparticles characterized by their resistance to photobleaching and environmental friendliness, which hold promise for future in vitro bioimaging studies. A systematic investigation is conducted to optimize experimental parameters for the preparation of micellar nanostructures with an average hydrodynamic diameter below 200 nm, ensuring colloidal stability in physiological media while preserving the optical luminescent properties of the encapsulated carbon dots. Comprehensive chemical-physical characterization of these micellar nanostructures is performed employing optical and morphological techniques. Furthermore, their binding affinity for the principal inorganic constituent of bone tissue is assessed through a binding assay with hydroxyapatite nanoparticles, indicating significant potential for active bone-targeting. These formulated nanostructures hold promise for novel therapeutic interventions to address skeletal-related complications in cancer affected patients in the future.

[Research Progress in the Molecular Regulatory Mechanisms of Alveolar Bone Restoration]. / ç‰™æ§½éª¨ä¿®å¤é‡å»ºåˆ†å­è°ƒæŽ§æœºåˆ¶çš„ç ”ç©¶æ–°è¿›å±•.

Alveolar bone, the protruding portion of the maxilla and the mandible that surrounds the roots of teeth, plays an important role in tooth development, eruption, and masticatory performance. In oral inflammatory diseases, including apical periodontitis, periodontitis, and peri-implantitis, alveolar bone defects cause the loosening or loss of teeth, impair the masticatory function, and endanger the physical and mental health of patients. However, alveolar bone restoration is confronted with great clinical challenges due to the the complicated effect of the biological, mechanical, and chemical factors in the oral microenvironment. An in-depth understanding of the underlying molecular regulatory mechanisms will contribute to the exploration of new targets for alveolar bone restoration. Recent studies have shown that Notch, Wnt, Toll-like receptor (TLR), and nuclear factor-κB (NF-κB) signaling pathways regulate the proliferation, differentiation, apoptosis, and autophagy of osteoclasts, osteoblasts, osteocytes, periodontal ligament cells, macrophages, and adaptive immune cells, modulate the expression of inflammatory mediators, affect the balance of the receptor activator for nuclear factor-κB ligand/receptor activator for nuclear factor-κB/osteoprotegerin (RANKL/RANK/OPG) system, and ultimately participate in alveolar bone restoration. Additionally, alveolar bone restoration involves AMP-activated protein kinase (AMPK), phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT), Hippo/YAP, Janus kinase/signal transducer and activator of transcription (JAK/STAT), and transforming growth factor ß (TGF-ß) signaling pathways. However, current studies have failed to construct mature molecular regulatory networks for alveolar bone restoration. There is an urgent need for further research on the molecular regulatory mechanisms of alveolar bone restoration by using new technologies such as single-cell transcriptome sequencing and spatial transcriptome sequencing.

[New Advances in the Application of Bacterial Cellulose Composite Materials in the Field of Bone Tissue Engineering]. / ç»†èŒçº¤ç»´ç´ å¤åˆææ–™åœ¨éª¨ç»„ç»‡å·¥ç¨‹é¢†åŸŸçš„åº”ç”¨æ–°è¿›å±•.

Bacterial cellulose (BC) is a type of extracellular polymeric nanomaterial secreted by microorganisms over the course of their growth. It has gained significant attention in the field of bone tissue engineering due to its unique structure of three-dimensional fibrous network, excellent biocompatibility, biodegradability, and exceptional mechanical properties. Nevertheless, BC still has some weaknesses, including low osteogenic activity, a lack of antimicrobial properties, small pore size, issues with the degradation rate, and a mismatch in bone tissue regeneration, limiting its standalone use in the field of bone tissue engineering. Therefore, the modification of BC and the preparation of BC composite materials have become a recent research focus. Herein, we summarized the relationships between the production, modification, and bone repair applications of BC. We introduced the methods for the preparation and the modification of BC. Additionally, we elaborated on the new advances in the application of BC composite materials in the field of bone tissue engineering. We also highlighted the existing challenges and future prospects of BC composite materials.

Epithelial-like transport of mineral distinguishes bone formation from other connective tissues.

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ-carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs.

NPP1 and TNAP hydrolyze ATP synergistically during biomineralization.

Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (Pi), a calcification promoter, and pyrophosphate (PPi), a calcification inhibitor. The Pi/PPi ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PPi as well as Pi from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi to generate Pi. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48 h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210 nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201 nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25 nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a Pi/PPi ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.

Osteoblastic Cell Behavior and Gene Expression Related to Bone Metabolism on Different Titanium Surfaces.

The surface topography of titanium dental implants has a great influence on osseointegration. In this work, we try to determine the osteoblastic behavior and gene expression of cells with different titanium surfaces and relate them to the physicochemical properties of the surface. For this purpose, we have used commercial titanium discs of grade 3: as-received corresponds to machined titanium without any surface treatment (MA), chemically acid etched (AE), treated via sand blasting with Al2O3 particles (SB) and a sand-blasting treatment with acid etching (SB+AE). The surfaces have been observed using scanning electron microscopy (SEM) and the roughness, wettability and surface energy with dispersive and polar components have been characterized. Osteoblastic cultures were performed with SaOS-2 osteoblastic cells determining cell viability as well as alkaline phosphatase levels for 3 and 21 days, and osteoblastic gene expression was determined. The roughness values of the MA discs was 0.02 µm, which increases to 0.3 µm with acid attack and becomes the maximum for the sand-blasted samples, reaching values of 1.2 µm for SB and SB+AE. The hydrophilic behavior of the MA and AE samples with contact angles of 63° and 65° is superior to that of the rougher samples, being 75° for SB and 82° for SB+AE. In all cases, they show good hydrophilicity. GB and GB+AE surfaces present a higher polar component in the surface energy values, 11.96 and 13.18 mJ/m2, respectively, than AE and MA, 6.64 and 9.79 mJ/m2, respectively. The osteoblastic cell viability values at three days do not show statistically significant differences between the four surfaces. However, the viability of the SB and SB+AE surfaces at 21 days is much higher than that of the AE and MA samples. From the alkaline phosphatase studies, higher values were observed for those treated with sand blasting with and without acid etching compared to the other two surfaces, indicating a greater activity in osteoblastic differentiation. In all cases except in the Osterix (Ostx) -osteoblast-specific transcription factor-a decrease in gene expression is observed in relation to the MA samples (control). The most important increase was observed for the SB+AE condition. A decrease in the gene expression of Osteoprotegerine (OPG), Runt-related transcription factor 2 (Runx2), Receptor Activator of NF-κB Ligand (RANKL) and Alkaline Phosphatase (Alp) genes was observed in the AE surface.

Molecular Mechanisms of Craniofacial and Dental Abnormalities in Osteopetrosis.

Osteopetrosis is a group of genetic bone disorders characterized by increased bone density and defective bone resorption. Osteopetrosis presents a series of clinical manifestations, including craniofacial deformities and dental problems. However, few previous reports have focused on the features of craniofacial and dental problems in osteopetrosis. In this review, we go through the clinical features, types, and related pathogenic genes of osteopetrosis. Then we summarize and describe the characteristics of craniofacial and dental abnormalities in osteopetrosis that have been published in PubMed from 1965 to the present. We found that all 13 types of osteopetrosis have craniomaxillofacial and dental phenotypes. The main pathogenic genes, such as chloride channel 7 gene (CLCN7), T cell immune regulator 1 (TCIRG1), osteopetrosis-associated transmembrane protein 1 (OSTM1), pleckstrin homology domain-containing protein family member 1 (PLEKHM1), and carbonic anhydrase II (CA2), and their molecular mechanisms involved in craniofacial and dental phenotypes, are discussed. We conclude that the telltale craniofacial and dental abnormalities are important for dentists and other clinicians in the diagnosis of osteopetrosis and other genetic bone diseases.

The role of Gdf5 in the development of the zebrafish fin endoskeleton.

BACKGROUND: The development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota. RESULTS: We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. CONCLUSIONS: Our study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.