Mineralized Bone Matrix Research Articles

Energetic Calcium Phosphate Nanominerals for Osteoporosis Treatment

AbstractEnergy metabolism disorders leading to tissue destruction are major causes of osteoporosis. While efficacious, bone repair strategies that modulate energy metabolism pose considerable challenges. Herein, an energetic calcium phosphate nanominerals (ECPN) is developed using polyphosphate as an energy source for osteoporosis treatment. ECPN promotes adenosine triphosphate (ATP) production in the physiological environment, providing energy to attain metabolic homeostasis. It significantly enhances rBMSCs’ autophagy capacity by activating the AMPK‐related pathway, promoting osteogenic differentiation, and rebuilding the bone regeneration microenvironment. ECPN's unique nanostructure can fully mineralize collagen fibers, enhancing the bone matrix's mechanical properties. In vivo, ECPN rapidly infiltrates osteoporotic bones, fills defects, mineralizes the matrix, and promotes new‐bone formation. The repaired bone exhibits mechanical properties comparable to those of normal bones. ECPN balances the time‐sensitive need for immediate bone matrix mineralization and long‐term construction of an osteogenic microenvironment during osteoporosis treatment. The potential of this metabolic fuel for generating functional nanomaterials for tissue engineering has been underestimated in the past. The concept of an energetic nanomineral for tissue regeneration may elicit new trends in tissue engineering.

Biology of bone mineralization and ectopic calcifications: the same actors for different plays

Bone has several crucial functions. It is essential for locomotion and allows our body to stand erect against gravity. A mismatch between the mechanical stresses applied to it and its mechanical resistance leads to fractures. Bone also has numerous endocrine functions. It acts as a reservoir for minerals such as calcium and phosphorus, making it the target of calciotropic hormones that mobilize these minerals, particularly calcium, according to the body's needs. Additionally, bone secretes hormones, notably fibroblast growth factor 23 (FGF23), which regulates urinary excretion of phosphate and the bioavailability of active vitamin D. Bone mineralization is the process that facilitates the organized deposition of minerals in the bone matrix, providing rigidity and appropriate mechanical resistance. This process is compromised in genetically related bone mineralization disorders, such as those causing hypophosphatemia or hypophosphatasia. Conversely, calcification can be pathological, affecting soft tissues like the blood vessels, as seen in generalized arterial calcification of infancy (GACI) or arterial calcification due to CD73 deficiency (ACDC). The aim of this article is to first present the composition and structure of the mineralized bone matrix, to review the current understanding of the molecular mechanisms of mineralization, and finally to discuss the conditions associated with ectopic calcification and the underlying mechanisms.

Converting ocean nacre into bone mineral matrix composite for bone regeneration- in vitro and in vivo studies

Nacre of Pinctada maxima is a natural biomineralized matrix, appeared 7 million years before hominins. In this study, we converted nacre into a self-setting particle bound with multiple calcium orthophosphates that reassemble mammals’ bone mineral matrices for induction of bone regeneration. The nacre-based calcium orthophosphates composite (NCOC) exhibited a compression strength of 10 MPa, which is superior to human trabecular bone. In vitro bioactivity tests revealed the formation of apatite with nano-porous flake-like crystals on the composite surface that mimic HA structure of a human bone matrix. NCOC demonstrated efficient attachment and proliferation of osteoblast cells, promoting osteogenic differentiation by increasing expressions of RUNX2 and OPN. In vivo studies using rabbit back fascia demonstrated that NCOC displays better bone healing and biocompatibility than conventional bone substitute apatite in critical bone defect models. The degradation of calcium carbonate crystal in vivo does not compromise structural integrity of NCOC. Overall, our data showed that NCOC produced through self-setting reactions, presents advantages such as accelerated biodegradation and osteostimulative properties, making it a promising bone substitute for effective bone regeneration.

Metabolically healthy obesity in adults with X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is characterized by increased concentrations of circulating fibroblast growth factor 23 (FGF-23) resulting in phosphate wasting, hypophosphatemia, atypical growth plate and bone matrix mineralization. Epidemiologic studies suggest a relationship between FGF-23, obesity, and metabolic dysfunction. The prevalence of overweight and obesity is high in children with XLH. We aimed to evaluate the prevalence of obesity and metabolic complications in adults with XLH. We conducted a prospective cohort study in adult XLH patients from a single tertiary referral center. The proportion of patients with a BMI >25 kg/m2 was the main outcome measure. Body fat mass percentage (FM%) and adipose tissue surfaces were secondary outcome measures. Glucose homeostasis (plasma glucose and insulin concentrations after fasting and 2 hours after an oral glucose tolerance test) was explored in a subgroup of patients and compared with age-, sex-, and BMI-matched healthy controls. Among 113 evaluated patients, 85 (75%) were female and 110 (97%) carried a PHEX mutation. Sixty-three (56%) patients were overweight or obese, with a median BMI of 25.3 [IQR, 22.7; 29.2] kg/m2. BMI was correlated with FM%, abdominal and thigh subcutaneous and intra-abdominal adipose tissue surfaces. The prevalence of impaired fasting glucose, impaired glucose tolerance, and diabetes was not different between XLH patients and matched controls. The prevalence of overweight and obesity is high among XLH patients and is associated with excess fat mass. However, the prevalence of glucose homeostasis abnormalities is not increased in patients compared to healthy controls, suggesting that metabolically healthy overweight or obesity predominates.

Micro-Nanostructured Polymeric Scaffolds for Bone Tissue Engineering

While bone tissue allograft and autograft are commonly used in bone healing, their application is limited by factors such as availability, donor site morbidity, and immune response to the grafted tissue. Tissue-engineered implants, such as acellular or cellular polymeric structures, offer a promising alternative, and are a current trend in tissue engineering. Leveraging recent advancements in bone tissue engineering (BTE), we utilize 3D printing to develop biodegradable scaffolds that combine mechanical strength and bioactivity to facilitate bone repair and regeneration. This study focuses on the design and fabrication of mechanically competent 3D printed poly (L-lactic acid) (PLLA) micro-structured scaffolds. These scaffolds are enhanced with collagen type I nanofibrils to create bioactive scaffolds that promote tissue regeneration. The performance of these mechanically competent, micro-nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in PLLA and PLLA-collagen scaffolds. The resulting micro-nanostructured PLLA-Collagen scaffolds mimic trabecular bone architecture, mechanical strength, and the extracellular matrix environment found in native bone tissue. The composite PLLA-collagen scaffolds exhibit mechanical properties in the mid-range of human trabecular bone. Both PLLA and PLLA-Collagen scaffolds support human BMSCs adhesion, proliferation, and osteogenic differentiation. A significantly higher number of implanted host cells are distributed in the PLLA-Collagen scaffolds with greater bone density, more uniform cell distribution, and attachment compared to the PLLA microstructure. Additionally, the biomimetic collagen nanostructure potently induces osteogenic transcription evidenced by increased alkaline phosphatase activity and upregulation of bone markers such as sialoprotein and collagen type I, ultimately guiding stem cell-mediated formation of a mature, mineralized bone matrix throughout the interconnected scaffold pores. This study underscores the benefits of micro-nanostructured scaffolds in successfully generating the inductive microenvironment of native bone extracellular matrix, triggering the cascade of cellular events required for functional bone regeneration, repairing critical-sized bone defects, and ultimately serving as an alternative material platform for bone regeneration, thereby instilling confidence in the potential of our research.

Cuttlefish Bone-Derived Calcium Phosphate Bioceramics Have Enhanced Osteogenic Properties.

Cuttlefish bones are byproducts of cuttlefish processing and are readily available in the marine food industry. In this study, calcium phosphate bioceramics were prepared from cuttlefish bones using a two-stage hydrothermal calcination process. The results indicated that all bioceramics derived from cuttlefish bones had a higher degradation capacity, better bone-like apatite formation ability, and higher degree of osteogenic differentiation than commercially available hydroxyapatite. Notably, β-tricalcium phosphate, which had the highest degree of Ca2+ and Sr2+ dissolution among the bioceramics extracted, can significantly upregulate osteogenic markers (alkaline phosphatase, osteocalcin) and stimulate bone matrix mineralization. Thus, it is a promising bioceramic material for applications in bone regeneration.

Biopsies from patients with sacral insufficiency fracture are characterized by low bone matrix mineralization and high turnover.

Sacral insufficiency fractures are known to occur primarily in older women without adequate trauma. While an association with low bone mineral density (ie, osteoporosis) has been reported, more detailed information on local bone quality properties in affected patients is not available. In the present study, core biopsies were obtained from the S1 sacral ala in patients with a bilateral sacral insufficiency fracture (type IV according to the fragility fractures of the pelvis classification) who required surgical stabilization. Dual energy X-ray absorptiometry (DXA) and laboratory bone metabolism analyses were performed. For comparison, control biopsies were acquired from skeletally intact age- and sex-matched donors during autopsy. A total of 31 biopsies (fracture: n = 19; control: n = 12) were evaluated by micro-computed tomography, histomorphometry on undecalcified sections, and quantitative backscattered electron imaging (qBEI). DXA measurements showed mean T-scores in the range of osteoporosis in the fracture cohort (T-scoremin -2.6 ± 0.8). Biochemical analysis of bone metabolism parameters revealed high serum alkaline phosphatase and urinary deoxypyridinoline/creatinine levels. In the biopsies, a loss of trabecular microstructure along with increased osteoid values were detected in the fracture patients compared with controls (osteoid volume per bone volume 5.9 ± 3.5 vs. 0.9 ± 0.5%, p <.001). We also found evidence of microfractures with chronic healing processes (ie, microcallus) as well as pronounced hypomineralization in the biopsies of the fracture cohort compared with the controls as evidenced by lower CaMean measured by qBEI (22.5 ± 1.6 vs. 24.2 ± 0.5wt%, p =.003). In conclusion, this high-resolution biopsy study provides evidence of local hypomineralization in patients with sacral insufficiency fractures, pointing to reduced fracture resistance but also a distinct phenotype other than the predominant loss of trabeculae as in postmenopausal osteoporosis. Our data highlight the importance of therapies that promote bone mineralization to optimally treat and prevent sacral insufficiency fractures.

3D printed O2-generating scaffolds enhance osteoprogenitor- and type H vessel recruitment during bone healing

Oxygen (O2)-delivering tissue substitutes have shown tremendous potential for enhancing tissue regeneration, maturation, and healing. As O2 is both a metabolite and powerful signaling molecule, providing controlled delivery is crucial for optimizing its beneficial effects in the treatment of critical-sized injuries. Here, we report the design and fabrication of 3D-printed, biodegradable, O2-generating bone scaffold comprising calcium peroxide (CPO) that once hydrolytically activated, provides long-term generation of oxygen at a controlled, concentration-dependent manner, and polycaprolactone (PCL), a hydrophobic polymer that regulate the interaction of CPO with water, preventing burst release of O2 at early time points. When anoxic conditions were simulated in vitro, CPO-PCL scaffolds maintained the survival and proliferation of human adipose-derived stem/stromal cells (hASCs) relative to PCL-only controls. We assessed the in vivo osteogenic efficacy of hASC-seeded CPO-PCL scaffolds implanted in a non-healing critical-sized 4-mm calvarial defects in nude mice for 8 weeks. Even without exogenous osteoinductive factors, CPO-PCL scaffolds demonstrated increased new bone volume compared to PCL-only scaffolds as verified by both microcomputed tomography analysis and histological assessments. Lastly, we employed a quantitative 3D lightsheet microscopy platform to determine that O2-generating scaffolds had similar vascular volumes with slightly higher presence of CD31hiEmcnhi pro-osteogenic, type H vessels and increased number of Osterix+ skeletal progenitor cells relative to PCL-only scaffolds. In summary, 3D-printed O2 generating CPO-PCL scaffolds with tunable O2 release rates provide a facile, customizable strategy for effectively treating, craniofacial bone defects. Statement of significanceOxygen(O2)-delivering bone substitutes show promise in defect repair applications by supplying O2 to the cells within or around the graft, improving cell survivability and enhancing bone matrix mineralization. A novel O2-generating bone scaffold has been 3D printed for the first-time which ensures patient and defect specificity. 3D printed calcium peroxide-polycaprolactone (CPO-PCL) bone scaffold provides uninterrupted O2 supply for 22 days allowing cell survival in deprived O2 and nutrient conditions. For the first time, O2-driven bone regenerative environment in mice calvaria has been captured by light-sheet imaging which illuminates abundance of Osterix+ cells, angiogenesis at a single cell resolution indicating active site of bone remodeling and growth in the presence of O2.

Matrix first, minerals later: fine-tuned dietary phosphate increases bone formation in zebrafish.

Bone matrix formation and mineralization are two closely related, yet separated processes. Matrix formation occurs first, mineralization is a second step strictly dependent on the dietary intake of calcium and phosphorus (P). However, mineralization is commonly used as diagnostic parameter for bone-related diseases. In this context, bone loss, often characterized as a condition with reduced bone mineral density, represents a major burden for human health, for which increased dietary mineral intake is generally recommended. Using a counterintuitive approach, we use a low-P diet followed by a sufficient-P intake to increase bone volume. We show in zebrafish by histology, qPCR, micro-CT, and enzyme histochemistry that a two-months period of reduced dietary P intake stimulates extensive formation of new bone matrix, associated with the upregulation of key genes required for both bone matrix formation and mineralization. The return to a P-sufficient diet initiates the mineralization of the abundant matrix previously deposited, thus resulting in a striking increase of the mineralized bone volume as proven at the level of the vertebral column, including vertebral bodies and arches. In summary, bone matrix formation is first stimulated with a low-P diet, and its mineralization is later triggered by a sufficient-P dietary intake. In zebrafish, the uncoupling of bone formation and mineralization by alternating low and sufficient dietary P intake significantly increases the bone volume without causing skeletal malformations or ectopic mineralization. A modification of this approach to stimulate bone formation, optimized for mammalian models, can possibly open opportunities to support treatments in patients that suffer from low bone mass.

A Gelatin-Based Biomimetic Scaffold Promoting Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells.

In bone tissue engineering segment, numerous approaches have been investigated to address critically sized bone defects via 3D scaffolds, as the amount of autologous bone grafts are limited, accompanied with complications on harvesting. Moreover, the use of bone-marrow-derived stem cells is also a limiting factor owing to the invasive procedures involved and the low yield of stem cells. Hence, research is ongoing on the search for an ideal bone graft system promoting bone growth and regeneration. This study aims to develop a unique platform for tissue development via stem cell differentiation towards an osteogenic phenotype providing optimum biological cues for cell adhesion, differentiation and proliferation using biomimetic gelatin-based scaffolds. The use of adipose-derived mesenchymal stem cells in this study also offers an ideal approach for the development of an autologous bone graft. A gelatin-vinyl acetate-based 3D scaffold system incorporating Bioglass was developed and the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs) on the highly porous freeze-dried gelatin-vinyl acetate/ Bioglass scaffold (GB) systemwas analyzed. The physicochemical properties, cell proliferation and viability were investigated by seeding rat adipose tissue-derived mesenchymal stem cells (ADSCs) onto the scaffolds. The osteogenic differentiation potential of the ADMSC seeded GeVAc/bioglass system was assessed using calcium deposition assay and bone-related protein and genes and comparing with the 3D Gelatin vinyl acetate coppolymer (GeVAc) constructs. According to the findings, the 3D porous GeVAc/bioglass scaffold can be considered as a promising matrix for bone tissue regeneration and the 3D architecture supports the differentiation of the ADMSCs into osteoblast cells and enhances the production of mineralized bone matrix.

Induction of mineralized matrix production by recombinant human BMP-2 Immobilized in TEMPO-Oxidized Cellulose Hydrogel: a novel target for tissue repair

Bone morphogenetic proteins (BMPs) are potent promoters of osteogenesis, especially BMP-2, which has been highlighted for acting as a growth and differentiation factor that promotes new bone formation. There are several biomaterials that can be used to release bioactive substances, such as natural polymers. Cellulose has stood out for the possibility of its chemical modification using the reagent 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) to obtain a cellulose derivative (TEMPO oxidized cellulose nanofibers - ToCNF), which is shown to be a promising material for biological application. The objective of this work was to evaluate TEMPO cellulose immobilized with rhBMP-2 against the activity of inducing bone cell proliferation and differentiation in vitro, evaluating the ability to form bone matrix in pre-osteoblastic cell lineage of rats - MC3T3. Cell viability assays using resazurin were performed and for detection of mineralized matrix, Alizarin Red solution was used. The results reveal the good capacity of TEMPO cellulose functionalized with rhBM-2 in inducing the synthesis of mineralized bone matrix.

Variables Influencing Bone Formation After Transcrestal Sinus Floor Elevation: Radiographic and Tomographic Evaluations.

To evaluate the influence of initial implant protrusion within the subantral space on hard tissue gain for implants placed simultaneously with transcrestal sinus floor elevation (TSFE) with a biomaterial. A total of 50 implants were placed after TSFE in 44 patients using either a human demineralized bone matrix or a deproteinized bone mineral matrix. Intraoral radiographs were obtained before and immediately after surgery. CBCT scans were obtained at the last follow-up (mean: 6.6 years). The initial bone crest height was 4.6 ± 1.4 mm, and the initial protrusion of the implants above the sinus floor was 3.5 ± 1.4 mm. At the follow-up assessments, the hard tissue mean gain was 2.5 ± 1.5 mm, resulting in a mean residual protrusion of 1.1 ± 1.3 mm. Only 10 implants did not protrude above the apical level of hard tissue. Positive correlations were found between hard tissue gain and initial protrusion (r = 0.55; 95% CI: 0.32 to 0.72; P = .0001), between the initial and final protrusions (r = 0.38; 95% CI: 0.10 to 0.60; P = .007), and between the follow-up period and final protrusion (r = 0.35; 95% CI: 0.07 to 0.58; P = .012). The higher the initial protrusion was, the higher were the hard tissue gain and the final protrusion of the implant above the apical level of the hard tissue.

Effect of collagen fibril orientation on the anisotropic properties of peri-implant bone.

In orthopedic and dental surgery, the implantation of biomaterials within the bone to restore the integrity of the treated organ has become a standard procedure. Their long-term stability relies on the osseointegration phenomena, where bone grows onto and around metallic implants, creating a bone-implant interface. Bone is a highly hierarchical material that evolves spatially and temporally during this healing phase. A deeper understanding of its biomechanical characteristics is needed, as they are determinants for surgical success. In this context, we propose a multiscale homogenization model to evaluate the effective elastic properties of bone as a function of the distance from the implant, based on the tissue's structure and composition at lower scales. The model considers three scales: hydroxyapatite foam (nanoscale), ultrastructure (microscale), and tissue (mesoscale). The elastic properties and the volume fraction of the elementary constituents of bone matrix (mineral, collagen, and water), the orientation of the collagen fibril relative to the implant surface, and the mesoscale porosity constitute the input data of the model. The effect of a spatiotemporal variation in the collagen fibrils' orientation on the bone anisotropic properties in the proximity of the implant was investigated. The findings revealed a strong variation of the components of the effective elasticity tensor of the bone as a function of the distance from the implant. The effective elasticity appears to be primarily sensitive to the porosity (mesoscale) rather than to the collagen fibrils' orientation (sub-micro scale). However, the orientation of the fibrils has a significant influence on the isotropy of the bone. When analyzing the symmetry properties of the effective elasticity tensor, the ratio between the isotropic and hexagonal components is determined by a combination of the porosity and the fibrils' orientation. A decrease in porosity leads to a decrease in bone isotropy and, in turn, an increase in the impact of the fibrils' orientation. These results demonstrate that the collagen fibril orientation should be taken into account to properly describe the effective elastic anisotropy of bone at the organ scale.

Ligand-Selective Targeting of Macrophage Hydrogel Elicits Bone Immune-Stem Cell Endogenous Self-Healing Program to Promote Bone Regeneration.

Targeting macrophages can facilitate the site-specific repair of critical bone defects. Herein, a composite hydrogel, gelatin-Bletilla striata polysaccharide-mesoporous bioactive glass hydrogel (GBMgel), is constructed via the self-assembly of mesoporous bioactive glass on polysaccharide structures, through the Schiff base reaction. GBMgel can efficiently capture macrophages and drive the recruitment of seed stem cells and vascular budding required for regeneration in the early stages of bone injury, and the observed sustained release of inorganic silicon ions further enhances bone matrix deposition, mineralization, and vascular maturation. Moreover, the use of macrophage-depleted rat calvarial defect models further confirms that GBMgel, with ligand-selective macrophage targeting, increases the bone regeneration area and the proportion of mature bone. Mechanistic studies reveal that GBMgel upregulates the TLR4/NF-κB and MAPK macrophage pathways in the early stages and the JAK/STAT3 pathway in the later stages; thus initiating macrophage polarization at different time points. In conclusion, this study is based on the endogenous self-healing properties of bone macrophages, which enhances stem cell homing, and provides a research and theoretical basis upon which bone tissue can be reshaped and regenerated using the body's immune power, providing a new strategy for the treatment of critical bone defects.

Novel insights into osteocyte and inter-organ/tissue crosstalk.

Osteocyte, a cell type living within the mineralized bone matrix and connected to each other by means of numerous dendrites, appears to play a major role in body homeostasis. Benefiting from the maturation of osteocyte extraction and culture technique, many cross-sectional studies have been conducted as a subject of intense research in recent years, illustrating the osteocyte-organ/tissue communication not only mechanically but also biochemically. The present review comprehensively evaluates the new research work on the possible crosstalk between osteocyte and closely situated or remote vital organs/tissues. We aim to bring together recent key advances and discuss the mutual effect of osteocyte and brain, kidney, vascular calcification, muscle, liver, adipose tissue, and tumor metastasis and elucidate the therapeutic potential of osteocyte.

Direct Reprogramming of Mouse Fibroblasts to Osteoblast-like Cells Using Runx2/Dlx5 Factors on Engineered Stiff Hydrogels.

Direct reprogramming of somatic cells into functional cells still faces major limitations in terms of efficiency and achieving functional maturity of the reprogramed cells. While different approaches have been developed commonly based on exploiting biochemical signals, introducing appropriate mechanical cues that stimulate the reprogramming process is rarely reported. In this study, collagen-coated polyacrylamide (PAM) hydrogels with stiffness close to that of collagenous bone (40 kPa) were adopted to augment the direct reprogramming process of mouse fibroblasts to osteoblastic-like cells. The results suggested that culturing cells on a hydrogel substrate enhanced the overexpression of osteogenic transcription factors using nonviral vectors and improved the yield of osteoblast-like cells. Particularly, a synergistic effect on achieving osteogenic functionality has been observed for the mechanical cues and overexpression of transcriptional factors, leading to enhanced osteogenic transformation and production of bone mineral matrix. Animal experiments suggested that reprogramed cells generated on matrix hydrogels accelerated bone regeneration and stimulated ectopic osteogenesis. Mechanism analysis suggested the critical involvement of actomyosin contraction and mechanical signal-mediated pathways like the RhoA-ROCK pathway, leading to a synergistic effect on the key transcriptional processes, including chromatin remodeling, nuclear translocation, and epigenetic transition. This study provides insights into the mechanical cue-enhanced direct reprogramming and cell therapy.

Moderate static magnetic field promotes fracture healing and regulates iron metabolism in mice

BackgroundFractures are the most common orthopedic diseases. It is known that static magnetic fields (SMFs) can contribute to the maintenance of bone health. However, the effect and mechanism of SMFs on fracture is still unclear. This study is aim to investigate the effect of moderate static magnetic fields (MMFs) on bone structure and metabolism during fracture healing.MethodsEight-week-old male C57BL/6J mice were subjected to a unilateral open transverse tibial fracture, and following treatment under geomagnetic field (GMF) or MMF. The micro-computed tomography (Micro-CT) and three-point bending were employed to evaluate the microarchitecture and mechanical properties. Endochondral ossification and bone remodeling were evaluated by bone histomorphometric and serum biochemical assay. In addition, the atomic absorption spectroscopy and ELISA were utilized to examine the influence of MMF exposure on iron metabolism in mice.ResultsMMF exposure increased bone mineral density (BMD), bone volume per tissue volume (BV/TV), mechanical properties, and proportion of mineralized bone matrix of the callus during fracture healing. MMF exposure reduced the proportion of cartilage in the callus area during fracture healing. Meanwhile, MMF exposure increased the number of osteoblasts in callus on the 14th day, and reduced the number of osteoclasts on the 28th day of fracture healing. Furthermore, MMF exposure increased PINP and OCN levels, and reduced the TRAP-5b and β-CTX levels in serum. It was also observed that MMF exposure reduced the iron content in the liver and callus, as well as serum ferritin levels while elevating the serum hepcidin concentration.ConclusionsMMF exposure could accelerate fracture healing via promote the endochondral ossification and bone formation while regulating systemic iron metabolism during fracture healing. This study suggests that MMF may have the potential to become a form of physical therapy for fractures.

Dynamic chromatin accessibility landscapes of osteoblast differentiation and mineralization

Bone acts as a self-healing organ, which undergoes continuous regeneration process that is tightly regulated by the cooperation of osteoclasts with the capability of bone resorption and osteoblasts with the capability of bone formation. Generally, bone marrow derived mesenchymal stem cells (BMSCs) differentiated to final osteoblasts have been considered as critical role in bone remodeling. In this regard, several transcription factors (TFs) whose binding sites are initially hidden deep within accessible chromatin that participate in modulating osteoblast differentiation and bone matrix mineralization. Then, it is necessary to explore further the dynamic changes about the epigenetic transcription machinery during osteoblastogenesis. Here, we performed the chromatin accessibility and transcriptomic landscape of osteoblast differentiation and mineralization by using transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-Seq). Our data found that global chromatin accessibility during osteoblastogenesis was extensively improved. Above this, it is shown that key target genes including Col6a3, Serpina3n, Ms4a4d, Lyz2, Phf11b and Grin3a were enriched in differential loci RNA-seq and ATAC-Seq peaks with continuous changed tendency during osteoblasts differentiation and mineralization. In addition, Analysis of Motif Enrichment (AME) was used to elucidate TFs which modulated these target genes. In this study, it was shown for the first time that these important TFs including MEF2A, PRRX1, Shox2 and HOXB13 could alter promoter accessibility of target genes during osteoblastogenesis. This helps us understand how TF binding motif accessibility influences osteoblast differentiation. In addition, it also suggests that modulating the chromatin accessibility of osteogenesis could be developed as the promising strategies to regulate bone regeneration.

Evaluation of nanoscale versus hybrid micro/nano surface topographies for endosseous implants

We examined the effect of a nanoscale titanium surface topography (D) versus two hybrid micro/nanoscale topographies (B and OS) on adherent mesenchymal stem cells (MSCs) and bone marrow derived macrophages (BMMs) function in cell culture and in vivo. In the in vitro study, compared to OS and B surfaces, D surface induced earlier and greater cell spreading, and earlier and profound mRNA expression of RUNX2, Osterix and BMP2 in MSCs. D surface induced earlier and higher expression of RUNX2 and BMP2 and lower expression of inflammatory genes in implant adherent cells in vivo. Measurement of osteogenesis at implant surfaces showed greater bone-to-implant contact at D versus OS surfaces after 21 days. We explored the cell population on the D and OS implant surfaces 24 h after placement using single-cell RNA sequencing and identified distinct cell clusters including macrophages, neutrophils and B cells. D surface induced lower expression and earlier reduction of inflammatory genes expression in BMMs in vitro. BMMs on D, B and OS surfaces demonstrated a marked increase of BMP2 expression after 1 and 3 days, and this increase was significantly higher on D surface at day 3. Our data implicates a dynamic process that may be influenced by nanotopography at multiple stages of osseointegration including initial immunomodulation, recruitment of MSCs and later osteoblastic differentiation leading to bone matrix production and mineralization. The results suggest that a nanoscale topography (D) favorably modulates adherent macrophage polarization toward anti-inflammatory and regenerative phenotypes and promotes the osteoinductive phenotype of adherent mesenchymal stem cells. Statement of significanceOur manuscript contains original data developed to define effects of a novel nanotopography on the process of osseointegration at the cell and tissue level. Few studies have compared the effects of a nanoscale surface versus the more typical hybrid micro/nano-scale surfaces used today. We have utilized single-cell RNA sequencing for the first time to identify earliest cell populations on implant surfaces in vivo. We provide data indicating that the nanoscale surface acts upon both osteoprogenitor and immune cell (macrophages) to alter the process of bone formation in a surface-specific manner. This work represents new observations regarding osseointegration and immunomodulation.

Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone.

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.