Bone Mesenchymal Stromal Cells Research Articles

Retraction Note: miR-381 modulates human bone mesenchymal stromal cells (BMSCs) osteogenesis via suppressing Wnt signaling pathway during atrophic nonunion development

Retraction Note: miR-381 modulates human bone mesenchymal stromal cells (BMSCs) osteogenesis via suppressing Wnt signaling pathway during atrophic nonunion development

Identification of the metaphyseal skeletal stem cell building trabecular bone.

Skeletal stem cells (SSCs) that are capable of self-renewal and multipotent differentiation contribute to bone development and homeostasis. Several populations of SSCs at different skeletal sites have been reported. Here, we identify a metaphyseal SSC (mpSSC) population whose transcriptional landscape is distinct from other bone mesenchymal stromal cells (BMSCs). These mpSSCs are marked by Sstr2 or Pdgfrb+Kitl-, located just underneath the growth plate, and exclusively derived from hypertrophic chondrocytes (HCs). These HC-derived mpSSCs have properties of self-renewal and multipotency in vitro and in vivo, producing most HC offspring postnatally. HC-specific deletion of Hgs, a component of the endosomal sorting complex required for transport, impairs the HC-to-mpSSC conversion and compromises trabecular bone formation. Thus, mpSSC is the major source of BMSCs and osteoblasts in bone marrow, supporting the postnatal trabecular bone formation.

YBX1 promotes type H vessel-dependent bone formation in an m5C-dependent manner.

RNA-binding proteins (RBPs) interact with RNA and ubiquitously regulate RNA transcripts during their life cycle, playing a fundamental role in the progression of angiogenesis-related diseases. In the skeletal system, endothelium-dependent angiogenesis is indispensable for bone formation. However, the role of RBPs in endothelium-dependent bone formation is unclear. Here, we show that RBP-Y-box-binding protein 1 (YBX1) was strongly reduced in the bone vasculature of ovariectomy (OVX) mice. Endothelial cell-specific deletion of Ybx1 impaired CD31-high, endomucin-high (CD31hiEMCNhi) endothelium morphology and resulted in low bone mass whereas Ybx1 overexpression promoted angiogenesis-dependent osteogenesis and ameliorated bone loss. Mechanistically, YBX1 deletion disrupted CD31, EMCN, and bone morphogenetic protein 4 (BMP4) stability in an m5C-dependent manner and blocked endothelium-derived BMP4 release, thereby inhibiting osteogenic differentiation of bone mesenchymal stromal cells. Administration of recombinant BMP4 protein restored impaired bone formation in Ybx1 deletion mice. Tail vein injection of CD31-modified polyethylene glycol-poly (lactic-co-glycolic acid) carrying sciadopitysin, a natural YBX1 agonist, pharmacologically partially reversed CD31hiEMCNhi vessels' decline and improved bone mass in both OVX and aging animals. These findings demonstrated the role of RBP-YBX1 in angiogenesis-dependent bone formation and provided a therapeutic approach for ameliorating osteoporosis.

Exosomes derived from mesenchymal stromal cells exert a therapeutic effect on hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway

ObjectiveHypoxic pulmonary hypertension (HPH) is a progressive and life-threatening disease characterized by perivascular inflammation, pulmonary vascular remodeling, and occlusion. Mesenchymal stromal cell-derived exosomes (MSC-exo) have emerged as potential therapeutic agents due to their role in cell communication and the transportation of bioactive molecules. In this study, we aimed to investigate the therapeutic effects of MSC-exo against HPH and elucidate the underlying molecular mechanism. MethodsExosomes were isolated from conditioned media of human bone mesenchymal stromal cells using ultracentrifugation and characterized through western blotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). An HPH animal model was established in male SD rats, and MSC-exo or phosphate-buffered saline (PBS) were administered via the tail vein for three weeks. Subsequently, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. Lung tissues from HPH rats and normal rats underwent high-throughput sequencing and transcriptomic analysis. Gene Ontology (GO) analysis was employed to identify upregulated differentially expressed genes. Additionally, rat pulmonary artery smooth muscle cells (PASMC) exposed to platelet-derived growth factor-BB (PDGF-BB) were used to simulate HPH-related pathological behavior. In vitro cellular models were established to examine the molecular mechanism of MSC-exo in HPH. ResultsMSC-exo administration protected rats from hypoxia-induced increases in RVSP, RVHI, and pulmonary vascular remodeling. Additionally, MSC-exo alleviated PDGF-BB-induced proliferation and migration of PASMC. Transcriptomic analysis revealed 267 upregulated genes in lung tissues of HPH rats compared to control rats. Gene Ontology analysis indicated significant differences in pathways associated with Yes Associated Protein 1 (YAP1), a key regulator of cell proliferation and organ size. RT-qPCR and western blot analysis confirmed significantly increased expression of YAP1 in HPH lung tissues and PASMC, which was inhibited by MSC-exo treatment. Furthermore, analysis of datasets demonstrated that Secreted Phosphoprotein 1 (SPP1), also known as Osteopontin (OPN), is a downstream binding protein of YAP1 and can be upregulated by PDGF-BB. MSC-exo treatment reduced the expression of both YAP1 and SPP1. Lentivirus-mediated knockdown of YAP1 inhibited PDGF-BB-induced PASMC proliferation, migration, and SPP1 protein levels. ConclusionOur findings demonstrate that MSC-exo exert a therapeutic effect against hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway. The inhibition of YAP1 and downstream SPP1 expression by MSC-exo may contribute to the attenuation of pulmonary vascular remodeling and PASMC proliferation and migration. These results suggest that MSC-exo could serve as a potential therapeutic strategy for the treatment of HPH. Further investigations are warranted to explore the clinical applicability of MSC-exo-based therapies in HPH patients.

Cartilage fragments combined with BMSCs-Derived exosomes can promote tendon-bone healing after ACL reconstruction

Anterior cruciate ligament reconstruction (ACLR) often fails due to the inability of tendon-bone integration to regenerate normal tissues and formation of fibrous scar tissues in the tendon-bone interface. Cartilage fragments and exosomes derived from bone mesenchymal stromal cells (BMSCs-Exos) can enhance enthesis healing. Nevertheless, the effects on the tendon-bone healing of ACLR remain unknown. This study found that BMSCs-Exos can promote the proliferation of chondrocytes in cartilage fragments, and activated the expression of chondro-related genes SOX9 and Aggrecan. The optimal effect concentration was 1012 events/uL. Besides, BMSCs-Exos could significantly upregulated the expression of BMP7 and Smad5 in cartilage fragments, and further enhanced the expression of chondrogenic genes. Moreover, this study established a rat model of ACLR and implanted the BMSCs-Exos/cartilage fragment complex into the femoral bone tunnel. Results demonstrated that the mean diameters of the femoral bone tunnels were significantly smaller in the BE-CF group than those in the CF group (p = 0.038) and control group (p = 0.007) at 8 weeks after surgery. Besides, more new bone formation was observed in the femoral tunnels in the BE-CF group, as demonstrated by a larger BV/TV ratio based on the reconstructed CT scans. Histological results also revealed the regeneration of tendon-bone structures, especially fibrocartilage. Thus, these findings provide a promising result that BMSCs-Exos/cartilage fragment complex can prevent the enlargement of bone tunnel and promote tendon-bone healing after ACLR, which may have resulted from the regulation of the BMP7/Smad5 signaling axis.

ScRNA-Seq Reveals a Distinct Osteogenic Progenitor of Alveolar Bone

The metabolism and remodeling of alveolar bone are the most active among the whole skeletal system, which is related to the biological characteristics and heterogeneity of the bone mesenchymal stromal cells (MSCs). However, there is a lack of systematic description of the heterogeneity of MSC-derived osteoblastic lineage cells as well as their distinct osteogenic differentiation trajectory of alveolar bone. In this study, we constructed a single-cell atlas of the mouse alveolar bone cells through single-cell RNA sequencing (scRNA-seq). Remarkably, by comparing the cell compositions between the alveolar bone and long bone, we uncovered a previously undescribed cell population that exhibits a high expression of protocadherin Fat4 (Fat4+ cells) and is specifically enriched around alveolar bone marrow cavities. ScRNA-seq analysis indicated that Fat4+ cells may initiate a distinct osteogenic differentiation trajectory in the alveolar bone. By isolating and cultivating Fat4+ cells in vitro, we demonstrated that they possess colony-forming, osteogenic, and adipogenic capabilities. Moreover, FAT4 knockdown could significantly inhibit the osteogenic differentiation of alveolar bone MSCs. Furthermore, we revealed that the Fat4+ cells exhibit a core transcriptional signature consisting of several key transcription factors, such as SOX6, which are involved in osteogenesis, and further demonstrated that SOX6 is required for the efficient osteogenic differentiation of the Fat4+ cells. Collectively, our high-resolution single-cell atlas of the alveolar bone reveals a distinct osteogenic progenitor that may contribute to the unique physiological characteristics of alveolar bone.

Pancreatic Lineage Cell Differentiation of Bone Marrow Mesenchymal Stromal Cells on Acellular Pancreatic Bioscaffold.

We evaluated the potential differentiation ability of bone mesenchymal stromal cells (BMSCs) into pancreatic lineage cells on a rat acellular pancreatic bioscaffold (APB) and the effect of differentiated BMSCs in vivo. The BMSCs were dynamically or statically cultured with or without growth factor in both culture systems. We assessed the cytological behavior and differentiation. We also evaluated the pancreatic fibrosis and pathological scores. The proliferation rates of BMSCs were significantly higher in the APB groups. The APB induced BMSCs to express mRNA markers at higher levels. All tested pancreatic functional proteins were also expressed at higher levels in the APB group. The secretion of metabolic enzymes was higher in the APB system. The ultrastructure of BMSCs in the APB group further revealed the morphological characteristics of pancreatic-like cells. For the in vivo study, the pancreatic fibrosis and pathological scores were significantly lower in the differentiated BMSCs group. In addition, in both the in vitro and the in vivo study, growth factor significantly improved proliferation, differentiation, and pancreatic cell therapy. The APB can promote BMSC differentiation toward pancreatic lineage and pancreatic-like phenotypes, giving it the potential for use in pancreatic cell therapies and tissue engineering.

Targeting macrophage endocytosis via platelet membrane coating for advanced osteoimmunomodulation

SummaryThe identification, uptake, and clearance of nanoparticles (NPs) by phagocytes are critical in NP-based therapeutics. The cell membrane coating technique has recently emerged as an ideal surface modification approach to help NP bypass phagocytosis. CD47, a regulatory protein for phagocytosis, is a cell surface glycoprotein expressed on all cell types, including platelets. Herein, we enclosed bioactive glass (BG) with a platelet membrane to bestow BG with unique cell surface functions for immune evasion and immunomodulation. Compared with the uncoated particles, platelet membrane-coated BG shows reduced cellular uptake and can generate an immune environment favorable for osteogenesis. This is evidenced by the triggering of robust osteogenic differentiation in bone mesenchymal stromal cells, suggesting the synergistic effect of platelet membrane and BG in bone regeneration. These collectively indicate that cell membrane coating is a promising approach to enhance the therapeutic efficacy of biomaterials and thus provide new insight into biomaterial-mediated bone regeneration.

Identification and validation of ferroptosis key genes in bone mesenchymal stromal cells of primary osteoporosis based on bioinformatics analysis.

Primary osteoporosis has long been underdiagnosed and undertreated. Currently, ferroptosis may be a promising research direction in the prevention and treatment of primary osteoporosis. However, the specific mechanism of ferroptosis in primary osteoporosis remains a mystery. Differentially expressed genes (DEGs) were identified in bone mesenchymal stromal cells (BMSCs) of primary osteoporosis and heathy patients from the GEO databases with the help of bioinformatics analysis. Then, we intersected these DEGs with the ferroptosis dataset and obtained 80 Ferr-DEGs. Several bioinformatics algorithms (PCA, RLE, Limma, BC, MCC, etc.) were adopted to integrate the results. Additionally, we explored the potential functional roles of the Ferr-DEGs via GO and KEGG. Protein–protein interactions (PPI) were used to predict potential interactive networks. Finally, 80 Ferr-DEGs and 5 key Ferr-DEGs were calculated. The 5 key Ferr-DEGs were further verified in the OVX mouse model. In conclusion, through a variety of bioinformatics methods, our research successfully identified 5 key Ferr-DEGs associated with primary osteoporosis and ferroptosis, namely, sirtuin 1(SIRT1), heat shock protein family A (Hsp70) member 5 (HSPA5), mechanistic target of rapamycin kinase (MTOR), hypoxia inducible factor 1 subunit alpha (HIF1A) and beclin 1 (BECN1), which were verified in an animal model.

MiR-100-5p inhibits osteogenic differentiation of human bone mesenchymal stromal cells by targeting TMEM135.

Osteoporosis is a disorder characterized by reduced bone mass, disruption of bone microarchitecture, and a propensity to fracture. The osteogenic differentiation of human bone mesenchymal stromal cells (hBMSCs) exerts a critical effect on preventing bone loss during osteoporosis. Herein, the study recognized miR-100-5p as a deregulated miRNA during osteoporosis (upregulated) and BMSC osteogenic differentiation (downregulated). miR-100-5p was upregulated in osteoporosis patients-isolated BMSCs compared to non-osteoporosis trauma patients-isolated BMSCs. hBMSCs, overexpression inhibited hBMSC proliferation and osteogenic differentiation, whereas miR-100-5p inhibition exerted opposite effects. TMEM135 was downregulated in osteoporosis and upregulated in differentiated osteoblasts, as well as downregulated upon the overexpression of miR-100-5p. MiR-100-5p directly targeted and inhibited TMEM135. In hBMSCs, TMEM135 silencing also inhibited hBMSC osteogenic differentiation. When co-transfected to hBMSCs, antagomir-100-5p promoted, whereas TMEM135 silencing inhibited hBMSC osteogenic differentiation; TMEM135 knockdown dramatically attenuated the effects of miR-100-5p inhibition. Taken together, miR-100-5p forms a regulatory axis with TMEM135 by direct binding. The miR-100-5p/TMEM135 axis modulates hBMSC differentiation into osteoblast. Considering the critical effect of BMSC osteogenesis on osteoporosis, this axis might play a role in osteoporosis, and further in vivo and clinical investigations are required.

Effect of Combined Intraosseous and Intraarticular Infiltrations of Autologous Platelet-Rich Plasma on Subchondral Bone Marrow Mesenchymal Stromal Cells from Patients with Hip Osteoarthritis.

Osteoarthritis (OA) is a debilitating condition that significantly impacts its patients and is closely associated with advancing age and senescence. Treatment with autologous platelet rich plasma (PRP) is a novel approach that is increasingly being researched for its effects. Subchondral bone mesenchymal stromal cells (MSCs) are key progenitors that form bone and cartilage lineages that are affected in OA. This study investigated the changes in subchondral bone MSCs before and after combined intraosseous (IO) and intraarticular (IA) PRP infiltration. Patient bone marrow aspirates were collected from 12 patients (four male, eight female) aged 40–86 years old (median 59.5). MSCs were expanded in standard media containing human serum to passage 1 and analysed for their colony-forming potential, senescence status, and gene expression. Hip dysfunction and Osteoarthritis Outcome Score (HOOS) at baseline and 6 months post second infiltration were used to assess the clinical outcomes; seven patients were considered responders and five non-responders. The number of colony-forming MSCs did not increase in the post treatment group, however, they demonstrated significantly higher colony areas (14.5% higher compared to Pre) indicative of enhanced proliferative capacity, especially in older donors (28.2% higher). Senescence assays also suggest that older patients and responders had a higher resistance to senescent cell accumulation. Responder and non-responder MSCs tended to differ in the expression of genes associated with bone formation and cartilage turnover including osteoblast markers, matrix metalloproteinases, and their inhibitors. Taken together, our data show that in hip OA patients, combined IO and IA PRP infiltrations enhanced subchondral MSC proliferative and stress-resistance capacities, particularly in older patients. Future investigation of the potential anti-ageing effect of PRP infiltrations and the use of next-generation sequencing would contribute towards better understanding of the molecular mechanisms associated with OA in MSCs.

Cytostatic Effects of Polyethyleneimine Surfaces on the Mesenchymal Stromal Cell Cycle.

Polyelectrolytes assembled layer-by-layer (PEMs) are commonly used as functional coatings to build-up biological interfaces, particularly suitable as compatible layers for the interaction with a biological medium, providing suitable conditions to promote or prevent cell seeding while maintaining the phenotype. The proper assessment of the biocompatibility of PEMs and the elucidation of the related mechanisms are therefore of paramount importance. In this study, we report in detail the effect of two different PEM endings, polystyrene sulfonate (PSS) and polyethylenimine (PEI), respectively, on the cell adhesion, growth, and viability of human bone mesenchymal stromal cells (MSCs). The results have shown that PSS-ended substrates appear to be the most suitable to drive the cell adhesion and phenotype maintenance of MSCs, showing good biocompatibility. On the contrary, while the cells seem to adhere more quickly and strongly on the PEI-ended surfaces, the interaction with PEI significantly affects the growth and viability, reducing the cell spreading capability, by sequestering the adhesion molecules already in the very early steps of cell–substrate contact. These results point to the promotion of a cytostatic effect of PEI, rather than the often-claimed cytotoxicity.

A heterogeneous TiO2/SrTiO3 coating on titanium alloy with excellent photocatalytic antibacterial, osteogenesis and tribocorrosion properties

In order to meet the clinical demand for titanium implants, the heterogeneous structure of TiO2/SrTiO3 coating was in situ fabricated on the surface of Ti6Al4V alloy by a facile way. The well-designed Ti6Al4V/TiO2/SrTiO3 composite exhibits a strong photocatalytic antibacterial efficiency against Escherichia coli (E. coli). The suitable energy band structure, improved light absorption and enhanced carrier separation of TiO2/SrTiO3 heterojunction facilitate the generation of bactericidal reactive oxygen species (ROS) under the illumination of UV light. The biocompatible SrTiO3 component effectively favors the adhesion, proliferation and osteogenic differentiation of human bone mesenchymal stromal cells (hBMSCs) in vitro. It is worth noting that the dense oxide coating provides a reliable barrier against tribocorrosion for the titanium alloy substrate, and the wear volume loss of the Ti6Al4V/TiO2/SrTiO3 reduces to 1/8 of that of the bare Ti6Al4V alloy in simulated body fluid. In this work, a multifunctional coating system was proposed to provide inspiration for the development of novel artificial bone implant materials.

CCL2 is a critical mechano-responsive mediator in crosstalk between osteoblasts and bone mesenchymal stromal cells.

It has been known that moderate mechanical loading, like that caused by exercise, promotes bone formation. However, its underlying mechanisms remain elusive. Here we showed that moderate running dramatically improved trabecular bone in mice tibias with an increase in bone volume fraction and trabecular number and a decrease in trabecular pattern factor. Results of immunohistochemical and histochemical staining revealed that moderate running mainly increased the number of osteoblasts but had no effect on osteoclasts. In addition, we observed a dramatic increase in the number of colony forming unit-fibroblast in endosteal bone marrow and the percentage of CD45- Leptin receptor+ (CD45- LepR+ ) endosteal mesenchymal progenitors. Bioinformatics analysis of the transcriptional data from gene expression omnibus (GEO) database identified chemokine c-c-motif ligands (CCL2) as a critical candidate induced by mechanical loading. Interestingly, we found that CCL2 was up-regulated mainly in osteoblastic cells in the tibia of mice after moderate running. Further, we found that mechanical loading up-regulated the expression of CCL2 by activating ERK1/2 pathway, thereby stimulating migration of endosteal progenitors. Finally, neutralizing CCL2 abolished the recruitment of endosteal progenitors and the increased bone formation in mice after 4weeks running. These results therefore uncover an unknown connection between osteoblasts and endosteal progenitors recruited in the increased bone formation induced by mechanical loading.

Regulation of an Antimicrobial Peptide GL13K-Modified Titanium Surface on Osteogenesis, Osteoclastogenesis, and Angiogenesis Base on Osteoimmunology.

Creating a pro-regenerative immune microenvironment around implant biomaterial surfaces is significant to osseointegration. Immune cells, especially macrophages that participate in the osseointegration, including osteogenesis, osteoclastogenesis, and angiogenesis, should be considered when testing biomaterials. In this study, we immobilized an antimicrobial peptide GL13K with immunomodulatory properties onto a titanium surface via silanization. The modified surfaces show good biocompatibility with bone mesenchymal stromal cells (BMSCs), human umbilical vein endothelial cells (HUVECs), and RAW264.7. By co-culturing BMSCs with RAW264.7, we found that the GL13K-coated titanium surfaces could promote late-stage osteogenesis as demonstrated by the upregulated expression of recombinant collagen type I alpha 1 (COL-1α1) and more extracellular matrix mineralization, while the early phase remained unchanged. The surfaces inhibited the osteoclastogenic differentiation of RAW264.7 cells by restraining nuclear factor-activated T cells, cytoplasmic 1 (NFATc1), the main factor of the receptor activator of nuclear factor-κ B, and the receptor activator of the nuclear factor-κ B ligand signaling pathway, from entering the nucleus and further reduced the expression of the activating osteoclastogenic tartrate-resistant acid phosphatase gene. Moreover, the GL13K-coated titanium surface demonstrated significant promotion of angiogenesis differentiation of HUVECs as indicated by the upregulated expression of essential angiogenesis function genes, including hypoxia-inducible factor-1α, endothelial nitric oxide synthase, kinase insert domain receptor, and vascular endothelial growth factor A (HIF-1α, eNOS, KDR, and VEGF-A). Taken together, these results demonstrated that the GL13K coating had properties of osteogenesis, angiogenesis, and anti-osteoclastogenesis via its immunomodulatory potential.

Bioengineered Fluorescent Nanoprobe Conjugates for Tracking Human Bone Cells: In Vitro Biocompatibility Analysis.

Herein, we validated novel functionalized hybrid semiconductor bioconjugates made of fluorescent quantum dots (QD) with the surface capped by chitosan (polysaccharide) and chemically modified with O-phospho-L-serine (OPS) that are biocompatible with different human cell sources. The conjugation with a directing signaling molecule (OPS) allows preferential accumulation in human bone mesenchymal stromal cells (HBMSC). The chitosan (Chi) shell with the fluorescent CdS core was characterized by spectroscopical (UV spectrophotometry and photoluminescence), by morphological techniques (Transmission Electron Microscopy (TEM)) and showed small size (ø 2.3 nm) and a stable photoluminescence emission band. The in vitro biocompatibility results were not dependent on the polysaccharide chain length (Chi with higher and lower molecular weight) but were remarkably affected by the surface modification (Chi or Chi-OPS). In addition, the efficiency of nanoparticles uptake by the cells was dependent on cells nature (human primary cells or cell lines) and tissue source (bone or skin) in the presence or absence of the OPS modification. The complex cellular uptake pathways involved in the cell labeling with the nanoparticles do not interfere on the normal cellular biology (adhesion and proliferation), osteogenic differentiation, and gene expression. The bone cells particles uptake evaluation showed a possible pathway by Caveolin-1 that regulates cell transduction in the membrane’s Caveolae. Caveolae mediates non-specific endocytosis, and it is upregulated in HBMSC. The OPS-modified nanoparticles promoted an intense intracellular trafficking by the HBMSCs that showed late-osteoblast phenotype with an increase of extracellular matrix (ECM) mineralization (Alizarin red and Von Kossa staining for calcium phosphate crystals). In this work, the OPS modified bioconjugated QD proved to be a reliable and stable fluorescent bioprobe for cell imaging and targeting research that could also help in clarifying some cellular mechanisms of particles intracellular traffic through the cytoplasmic membrane and osteogenic differentiation induction. The in vitro HBMSC’s biocompatibility responses indicated that the OPS-modified chitosan QDs have a prospective future in laboratory and pre-clinical applications such as bioimaging analysis and for ex-vivo cellular evaluation of biomedical implants.

CCL2 is a Critical Mechano-Responsive Mediator in Crosstalk Between Osteoblasts and Bone Mesenchymal Stromal Cells

Background: It has known that moderate mechanical loading, like that caused by exercise, promotes bone formation. However its underlying mechanisms remain elusive. Methods: Mice were randomly maintained a sedentary lifestyle in cage or subjected to run on a rodent treadmill for 5 weeks. Bone metabolism of mice was evaluated with micro-CT and immunohistochemistry staining. Bioinformatics analysis was performed to identify the candidate chemokine for mechanical-induced bone formation. Flow cytometry and in vivo and in vitro functional assays were conducted to explore the mechanisms of chemokine promoting mesenchymal progenitors recruitment and bone remodeling. Findings: Moderate running dramatically improved trabecular bone remodeling and increased the number of osteoblasts but had no effect on osteoclasts. A dramatic increase in the number of colony forming unit-fibroblast (CFU-F) in endosteal bone marrow and the percentage of CD45- Leptin receptor+ endosteal mesenchymal progenitors were observed. Bioinformatics analysis identified chemokine c-c-motif ligands (CCL2) as a critical candidate induced by mechanical loading. Interestingly, we found that CCL2 was up-regulated mainly in osteoblastic cells in the tibia of mice after moderate running. Further, we found that mechanical loading up-regulates the expression of CCL2 by activating ERK1/2 pathway, thereby stimulating the migration of endosteal mesenchymal progenitors. Interpretation: These results uncover an unknown connection between osteoblasts and endosteal mesenchymal progenitors recruited in the increased bone formation induced by mechanical loading. Funding Statement: This study was supported by the National Natural Science Foundation of China (81830079), Science and Technology Key Planning Project of Guangdong Province (2019B020201013), Natural Science Foundation of Guangdong (2019A1515110818), and President Foundation of Nanfang Hospital, Southern Medical University (2019Z012). Declaration of Interests: The authors declare that there is no conflict of interests. Ethics Approval Statement: All animal protocols were approved by Institutional Animal Care and Use Committee (IACUC) at Nanfang Hospital, Southern Medical University before study and all experiments were performed in accordance with IACUC guidelines.

A multi-functionalized calcitriol sustainable delivery system for promoting osteoporotic bone regeneration both in vitro and in vivo

Among metabolic and systemic bone diseases, osteoporosis is known as one of the most progressive disease characterized by bone microstructural deterioration and bone mass reduction. Calcitriol (Cal) is widely accepted as a fat-soluble small molecule drug with reported anti-osteoporotic activity. However, delivery systems capable of local sustained release of small molecule drugs remain a significant challenge. Herein, based on poly(D, L-lactide)-poly(ethylene glycol)-poly(D, L-lactide) (PDLLA-PEG-PDLLA) hydrogels, we developed a thermosensitive and multifunctional sustainable delivery system by doubly integrating with polydopamine modified nano-hydroxyapatite (HA-D) and calcitriol-loaded amino-terminated poly(ethylene glycol)-block-poly(epsilon-caprolactone) (PCL-PEG-NH2) micelles to realize the long-term delivery of small molecule drugs. The sustained release of Cal from the system was associate with triple controlled release effects including the protective screen of the hydrogels over micelles, the reservoir effect of the HA nanoparticles for Cal, and the encapsulation of Cal in micelles. In vitro, results from the present study demonstrated that sustainable delivery system effectively activated osteogenesis, proliferation and migration in these bone mesenchymal stromal cells of ovariectomized rats (OVX-rBMSCs). In vivo, the composite Cal-loaded system was verified to promote bone regeneration in the critical-size femoral epicondyle bone defect model. Meanwhile, little cytotoxicity and appropriate degradation rate of the composite Cal delivery system were revealed. Thus, incorporating Cal into a multifunctional delivery system to perform a long-acting drug delivery in an injectable hydrogel form is a promising therapeutic to improve anti-osteoporotic ability and promote bone regeneration of osteoporosis.

Grooved hydroxyapatite scaffold modulatesmitochondria homeostasis and thus promotes osteogenesis in bone mesenchymal stromal cells.

Hydroxyapatite scaffolds (HASs) are widely studied as suitable materials for bone replacement scaffolds due to their chemical similarities to organic materials. In our previous study, a novel HAS with a 25–30-µm groove structure (HAS-G) exhibited enhanced osteogenesis of bone mesenchymal stromal cells (BMSCs) compared with HAS, potentially by modulating the macrophage-induced immune microenvironment. However, the exact effects of different surface patterns on the physiological processes of attached cells is not known. The present study aimed to determine the effects of HAS-G on the osteogenesis and physiological processes in BMSCs. Cell counting kit-8 assays and propidium iodide staining followed by flow cytometry were performed, and the results demonstrated that both in normal medium and differentiating medium, HAS-G promoted cell proliferation by decreasing the proportion of G1/G0 cells and decreased reactive oxygen species (ROS) accumulation in BMSCs compared with HAS. Detection markers of osteogenesis revealed that compared with HAS, HAS-G increased runt-related transcription factor 2, osteocalcin and osteopontin protein levels and promoted osteogenesis, which was further confirmed by Alizarin Red S staining. Following JC-1 staining, it was observed that HAS-G maintained the mitochondrial membrane potential, similar to that achieved by N-acetylcysteine pretreatment. In addition, compared with those of HAS, HAS-G decreased mitochondrial ROS levels, which potentially contributed to the promotion of osteogenesis. The results also demonstrated that HAS-G inhibited mitophagy induced by ROS accumulation and ATP synthesis compared with HAS. In conclusion, HAS-G decreased ROS accumulation and mitophagy and thus promoted osteogenesis of BMSCs, indicating that ROS modulation of HAS-G may serve a key role in osteogenesis.

Strontium-substituted calcium sulfate hemihydrate/hydroxyapatite scaffold enhances bone regeneration by recruiting bone mesenchymal stromal cells.

Fabrication of osteoconductive scaffold with osteoinductive capability and appropriate resorption rate is of great significance for treating bone defects. To achieve this aim, strontium-substituted calcium sulfate hemihydrate (Sr-CSH) and hydroxyapatite (HA) were mixed to develop a novel composite. Sr-CSH containing 5% and 10% strontium was mixed with HA at the weight ratio of 6:4, respectively. Female Sprague-Dawley rats underwent bone defect surgery in left tibia were randomly assigned to three different treatment groups filled with CSH/HA, 5% and 10% Sr-CSH/HA. Micro-CT analysis showed increased new bone formation in 10% Sr-CSH/HA group compared to CSH/HA group. In addition, histological analysis showed large amounts of chondrocytes and osteoblasts within the pores of Sr-CSH/HA composites as a result of the CSH resorption. Further, CFU-F assay demonstrated the increased amount of bone marrow mesenchymal stromal cells (BMSCs) colonies in 10% Sr-CSH/HA group. In primary BMSCs, extraction from Sr-CSH/HA composite significantly increased the migration of cells, up-regulated the expression of osteoblastic marker genes, and increased the area of mineralized nodules. Together, Sr-CSH/HA may promote bone formation by recruiting and stimulating osteogenic differentiation of BMSCs. Therefore, this composite may be proposed as an ideal substitute to repair bone defects.