Osteogenic Precursor Cells Research Articles

P178 CXCL12/CXCR4-mediated osteogenic precursor cells recruitment: a key mechanism in ankylosing spondylitis-associated pathological bone formation

Abstract Background/Aims Ankylosing spondylitis (AS) is a debilitating condition characterised by severe spinal deformities, inflammation, and excessive bone formation, imposing significant socioeconomic burdens. Current treatments alleviate symptoms but fail to address bone structural damage. Pathological bone formation often occurs at entheses, where tendons and ligaments connect to bone. Osteogenic precursor cells (OPCs) play a crucial role, but their migration to new bone sites is poorly understood. C-X-C motif chemokine ligand 12 (CXCL12) is implicated in cell migration and bone-related processes, but its role in AS remains unclear. This study explores CXCL12/CXCR4 signalling in OPC recruitment and new bone formation, providing insights into AS pathogenesis and potential therapeutic targets. Methods We established mouse models, including the DBA/1 model, lipopolysaccharide-induced spondyloarthritis model, and Achilles tendon semi-transection model to confirm the role of CXCL12-Induced OPCs migration in pathological new bone formation. Additionally, genetically edited mouse models were generated to confirm the underlying mechanism of OPCs migration and their spatiotemporal phenotypes of differentiation, including DBA1;CXCL12 col2 transgenic mice, CXCL12hi/hi;Col2a1-creERT mice, and CXCR4fl/fl;Prx1-creERT mice. Results In this study, we have identified CXCL12 as a crucial contributor to the initiation of pathological new bone formation by recruiting OPCs. CXCL12 was highly expressed in regions predisposed to pathological bone development, attracting OPCs to these upregulated areas. Inhibiting the CXCL12/CXCR4 axis with AMD3100 or the conditional knockout of CXCR4 effectively reduced OPC migration and subsequent pathological bone formation in animal models of AS. Overexpression of CXCL12 in genetically engineered animals with a DBA/1 background demonstrated a stable joint ankylosis phenotype, providing a valuable model for the study of AS. Furthermore, our research revealed the indispensable role of Rac1 in OPC migration and the ensuing pathological bone formation. Conclusion This study elucidates the novel role of CXCL12 in AS and offers potential strategies for targeting the CXCL12/CXCR4-Rac1 axis to prevent the progression of axial skeletal ankylosis. Disclosure H. Cui: None. H. Liu: None.

CYLD alleviates NLRP3 inflammasome-mediated pyroptosis in osteoporosis by deubiquitinating WNK1

BackgroundOsteoporosis (OP) is the result of bone mass reduction and bone structure disorder. Bone marrow mesenchymal stem cells (BMSCs) are the main source of osteogenic precursor cells involved in adult bone remodeling. The involvement of the deubiquitinating enzyme CYLD in OP has recently been discovered. However, the detailed role and mechanism of CYLD remain unknown.MethodsThe OP mouse model was established by performing ovariectomy (OVX) on mice. Hematoxylin and eosin staining, Masson and Immunohistochemical staining were used to assess pathologic changes. Real-time quantitative PCR, Western blot, and immunofluorescence were employed to assess the expression levels of CYLD, WNK1, NLRP3 and osteogenesis-related molecules. The binding relationship between CYLD and WNK1 was validated through a co-immunoprecipitation assay. The osteogenic capacity of BMSCs was determined using Alkaline phosphatase (ALP) and alizarin red staining (ARS). Protein ubiquitination was evaluated by a ubiquitination assay.ResultsThe levels of both CYLD and WNK1 were decreased in bone tissues and BMSCs of OVX mice. Overexpression of CYLD or WNK1 induced osteogenic differentiation in BMSCs. Additionally, NLRP3 inflammation was activated in OVX mice, but its activation was attenuated upon overexpression of CYLD or WNK1. CYLD was observed to reduce the ubiquitination of WNK1, thereby enhancing its protein stability and leading to the inactivation of NLRP3 inflammation. However, the protective effects of CYLD on osteogenic differentiation and NLRP3 inflammation inactivation were diminished upon silencing of WNK1.ConclusionCYLD mitigates NLRP3 inflammasome-triggered pyroptosis in osteoporosis through its deubiquitination of WNK1.

Regulatory T cell-derived exosome mediated macrophages polarization for osteogenic differentiation in fracture repair

Refractory fracture presents an intractable challenge in trauma treatment. Selective polarization of macrophages as well as the recruitment of osteogenic precursor cells play key roles in osteogenic differentiation during fracture healing. Here we constructed regulatory T cell (Treg)-derived exosomes (Treg-Exo) for the treatment of fracture. The obtained exosomes displayed a spheroid shape with a hydrated particle size of approximately 130 nm. With further purification using CD39 and CD73 antibody-modified microfluidic chips, CD39 and CD73 specifically expressing exosomes were obtained. This kind of Treg-Exo utilized the ectonucleotidases of CD39 and CD73 to catalyze the high level of ATP in the fracture area into adenosine. The generated adenosine further promoted the selective polarization of macrophages. When interacting with mesenchymal stem cells (MSCs, osteogenic precursor cells), both Treg-Exo and Treg-Exo primed macrophages facilitated the proliferation and differentiation of MSCs. After administration in vivo, Treg-Exo effectively promoted fracture healing compared with conventional T cell-derived exosome. To further improve the delivery efficacy of exosomes and integrate multiple biological processes of fracture healing, an injectable hydrogel was fabricated to co-deliver Treg-Exo and stromal cell-derived factor 1 alpha (SDF-1α). With the dual effect of Treg-Exo for macrophage polarization and SDF-1α for MSC recruitment, the multifunctional hydrogel exerted a synergistic effect on fracture repair acceleration. This study provided a promising therapeutic candidate and synergistic strategy for the clinical treatment of fracture.

A distinct "repair" role of regulatory T cells in fracture healing.

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.

Preparation and properties of a 3D printed nHA/PLA bone tissue engineering scaffold loaded with a β-CD-CHX combined dECM hydrogel.

Jaw defects, which can result from a multitude of causes, significantly affect the physical well-being and psychological health of patients. The repair of these infected defects presents a formidable challenge in the clinical and research fields, owing to their intricate and diverse nature. This study aims to develop a personalized bone tissue engineering scaffold that synergistically offers antibacterial and osteogenic properties for treating infected maxillary defects. This study engineered a novel temperature-sensitive, sustained-release hydrogel by amalgamating β-cyclodextrin (β-CD) with chlorhexidine (CHX) and a decellularized extracellular matrix (dECM). This hydrogel was further integrated with a polylactic acid (PLA)-nano hydroxyapatite (nHA) scaffold, fabricated through 3D printing, to form a multifaceted composite scaffold (nHA/PLA/dECM/β-CD-CHX). Drug release assays revealed that this composite scaffold ensures prolonged and sustained release. Bacteriological studies confirmed that the β-CD-CHX loaded scaffold exhibits persistent antibacterial efficacy, thus effectively inhibiting bacterial growth. Moreover, the scaffold demonstrated robust mechanical strength. Cellular assays validated its superior biocompatibility, attributed to dECM and nHA components, significantly enhancing the proliferation, adhesion, and osteogenic differentiation of osteogenic precursor cells (MC3T3-E1). Consequently, the nHA/PLA/dECM/β-CD-CHX composite scaffold, synthesized via 3D printing technology, shows promise in inducing bone regeneration, preventing infection, and facilitating the repair of jaw defects, positioning itself as a potential breakthrough in bone tissue engineering.

Conformationally regulated “nanozyme-like” cerium oxide with multiple free radical scavenging activities for osteoimmunology modulation and vascularized osseointegration

Given post-operative aseptic loosening in orthopedic disease treatment, osteointegration occurs at the bone-implant interface as a holistic process, including immunoregulation (e.g., macrophage polarization), angiogenesis and osteogenesis in sequence. In order to achieve early rapid and satisfactory osseointegration, different nano-shaped (nanocone, nanopolyhedron and nanoflower abbr. NC, NP & NF) cerium oxide (CeO2-x) coatings, endowed with “nanozyme-like” activities for multiple free radical elimination and osteoimmunology regulation, were hydrothermally synthesized on titanium alloy (TC4). In vitro cell experiments showed that nano-CeO2-x coated TC4 not only induced polarization of RAW264.7 cells toward M2 phenotype, but also promoted angiogenesis and vascularization of endothelial cells along with differentiation and mineralization of osteogenic precursor cells. Improvements in M2-polarized macrophage, angiogenesis, and bone regeneration were further confirmed in a rat femoral condyle model. Among the above three nano-morphologies, NF exhibited the best osseoinetegration. RNA sequencing and mechanism exploration suggested that the inhibition of PI3K-AKT signaling pathway was essential for immunomodulatory capacity of NF. In conclusion, it provided promising insights into the immunomodulatory exploitation of orthopedic implants.

ASPN Synergizes with HAPLN1 to Inhibit the Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells and Extracellular Matrix Mineralization of Osteoblasts.

Bone marrow mesenchymal stromal cells (BMSCs) are major sources of osteogenic precursor cells in bone remodeling, which directly participate in osteoporosis (OP) progression. However, the involved specific mechanisms of BMSCs in OP warrant mass investigations. Initially, our bioinformatics analysis uncovered the prominent up-regulation of Asporin (ASPN) and proteoglycan link protein 1 (HAPLN1) in osteoblasts (OBs) of OP patients and their possible protein interaction. Hence, this study aimed to explore the effects of ASPN and HAPLN1 on osteogenic differentiation of BMSCs, extracellular matrix (ECM) mineralization of OBs, and osteoclastogenesis, hoping to offer research basis for OP treatment. GSE156508 dataset was used for analysis and screening to acquire the differentially expressed genes in OBs of OP patients, followed by the predicative analysis via STRING. OP mouse models were induced by ovariectomy (OVX), and ASPN and HAPLN1 expression was determined. BMSCs and bone marrow macrophages (BMMs) were isolated from OVX mice and induced for osteogenic differentiation and osteoclastogenesis, respectively. After knockdown experiments, we assessed adipogenic differentiation and osteogenic differentiation in BMSCs. Osteogenic (OPN, OCN, and COL1A1) and osteoclast (Nfatc1 and c-Fos) marker protein expression was determined. The binding of ASPN to HAPLN1 was analyzed. High expression of ASPN and HAPLN1 and their protein interaction were observed in OBs of OP patients via bioinformatics and in bone tissues of OVX mice. ASPN interacted with HAPLN1 in BMSCs of OVX mice. ASPN/HAPLN1 knockdown increased ALP, OPN, OCN, and COL1A1 protein expression and ECM mineralization in BMSCs while decreasing Nfatc1 and c-Fos expression in BMMs. These effects were aggravated by the simultaneous knockdown of ASPN and HAPLN1. Our results indicate that ASPN synergises with HAPLN1 to suppress the osteogenic differentiation of BMSCs and ECM mineralization of OBs and promote the osteoclastogenesis in OP.

Genesis of osteoclasts on calcium phosphate ceramics and their role in material-induced bone formation.

Innate immune responses play important roles in material-induced bone formation and such roles were further explored in the current study with an emphasis on M2 macrophages and osteoclastogenesis. With the presence of M-CSF and RANKL, M0 macrophages from FVB mouse bone marrow-derived monocytes (BMMs) fused to osteoclasts with both M2 marker and osteoclast marker at day 5, and such osteoclast formation at day 5 was enhanced when the cells were treated with IL-4 at day 3. With IL-4 treatment alone for 24 h, M0 polarized into M2 macrophages. Conditioned medium of M2 macrophages enhanced osteogenic differentiation of MC3T3-E1 (pre-osteoblasts) while osteoclast conditioned medium enhanced osteogenic differentiation of CRL-12424 (osteogenic precursors). TCPs (a typical osteoinductive material) supported M2 macrophage polarization at day 4 and osteoclast formation at day 5, while TCPb (a typical non-osteoinductive material) was less effective. Moreover, osteoclasts formed on TCPs produced osteogenic factors including S1P, Wnt10B and BMP-6, resulting osteogenic differentiation of CRL-12424 cells. Similar to in vitro testing, TCPs favored M2 macrophage polarization followed by the formation of osteoclasts in vivo, as compared to TCPb. The overall data provided evidence of a coupling between M2 macrophages, osteoclasts and material-induced bone formation: osteoclasts formed from M2 macrophages secrete osteogenic cytokines to induce osteogenic differentiation of osteogenic precursor cells to finally form bone. The current findings outlined a biological mechanism of material-induced bone formation and further rationalized the use of osteoinductive materials for bone regeneration. STATEMENT OF SIGNIFICANCE: This paper provides evidence for finding out the relationship between M2 macrophages, osteoclasts and osteogenesis in material-induced bone formation. It suggested that osteoinductive materials enhanced macrophage polarization to M2 macrophages which fuses to osteoclasts, osteoclasts subsequently secret osteogenic cytokines to differentiate finally osteogenic precursors to form bone in osteoinductive materials. The data supports scientifically the superiority of osteoinductive materials for bone regeneration in clinics.

Advances in osseointegration of biomimetic mineralized collagen and inorganic metal elements of natural bone for bone repair.

At this stage, bone defects caused by trauma, infection, tumor, or congenital diseases are generally filled with autologous bone or allogeneic bone transplantation, but this treatment method has limited sources, potential disease transmission and other problems. Ideal bone-graft materials remain continuously explored, and bone defect reconstruction remains a significant challenge. Mineralized collagen prepared by bionic mineralization combining organic polymer collagen with inorganic mineral calcium phosphate can effectively imitate the composition and hierarchical structure of natural bone and has good application value in bone repair materials. Magnesium, strontium, zinc and other inorganic components not only can activate relevant signaling pathways to induce differentiation of osteogenic precursor cells but also stimulate other core biological processes of bone tissue growth and play an important role in natural bone growth, and bone repair and reconstruction. This study reviewed the advances in hydroxyapatite/collagen composite scaffolds and osseointegration with natural bone inorganic components, such as magnesium, strontium and zinc.

Creating morphological diversity in reptilian temporal skull region: A review of potential developmental mechanisms.

Reptilian skull morphology is highly diverse and broadly categorized into three categories based on the number and position of the temporal fenestrations: anapsid, synapsid, and diapsid. According to recent phylogenetic analysis, temporal fenestrations evolved twice independently in amniotes, once in Synapsida and once in Diapsida. Although functional aspects underlying the evolution of tetrapod temporal fenestrations have been well investigated, few studies have investigated the developmental mechanisms responsible for differences in the pattern of temporal skull region. To determine what these mechanisms might be, we first examined how the five temporal bones develop by comparing embryonic cranial osteogenesis between representative extant reptilian species. The pattern of temporal skull region may depend on differences in temporal bone growth rate and growth direction during ontogeny. Next, we compared the histogenesis patterns and the expression of two key osteogenic genes, Runx2 and Msx2, in the temporal region of the representative reptilian embryos. Our comparative analyses suggest that the embryonic histological condition of the domain where temporal fenestrations would form predicts temporal skull morphology in adults and regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating morphological diversity in the temporal skull region of reptiles.

Study on complications of osteoporosis based on network pharmacology.

Osteoporosis is a serious threat to human life. Guben Zenggu Granule is an empirical prescription for clinical treatment of osteoporosis. MC3T3-E1 cells are mouse osteogenic precursor cells with osteogenic differentiation, and are classic cells for studying bone metabolism and osteogenic mechanism, as well as mechanical stimulation sensitive cells. Therefore, it can be inferred that Guben Zenggu granule can repair MC3T3-E1 cells under continuous static pressure overload. This study aims to through the network of pharmacology and gene sequencing method, reveal thrift increase bone particles under the condition of continuous static pressure overload on osteogenesis mechanism of MC3T3-E1 cells. In the process of analysis, from a variety of 98 compounds was predicted in the database, a collection of 474 goals, a total of 29,164 difference between two groups of genes. Then, construction of composite targets between cells and predict targets and protein - protein interaction networks, and through the cluster analysis to further explore the relationship between the target. In addition, linkages between target proteins and cells were further identified using Gene Ontology (GO) and Pathways (KEGG Pathway). Finally, the repair effect of Guben Zenggu granule on MC3T3-E1 cells under continuous static pressure overload was verified through experiments, so as to accurately explain the pharmacodynamic mechanism of Traditional Chinese medicine.

Circulating Osteogenic Progenitor Cells Enhanced with Teriparatide or Denosumab Treatment.

Circulating osteogenic precursor (COP) cells are peripheral blood cells with a capacity for osteogenesis. The objective of our study was to ascertain the percentage of COPs as an early biomarker of osteoporosis and the effect of these cells in response to Denosumab (DmAb) (anti-resorptive) or to Teriparatide (TPDP) (anabolic) as very effective drugs in the treatment of the illness. A first study was conducted on healthy volunteers, with three age ranges, to determine the percentage of COPs and relate it to their anthropometric and biochemical characteristics, followed by a second longitudinal study on patients with osteoporosis, whereby one group of patients was treated with TPTD and another with DmAb. All were analyzed by cytometry for COP percentage in blood, bone turnover markers, and bone mass. Our findings show that COPs are influenced by age and become more prolific in the stages of growth and skeletal maturation. A higher percentage of COPs is found in osteoporotic disease, which could constitute a predictive marker thereof. We also show how treatment with TPTD or DmAb mobilizes circulating osteogenic precursors in the blood. Significant increases in % COPs were observed after 12 months of treatment with Dmb (21.9%) and TPTD (17%). These results can be related to an increase in osteogenesis and, consequently, a better and more efficient repair of bone tissue.

Urine-derived stem cells-extracellular vesicles ameliorate diabetic osteoporosis through HDAC4/HIF-1α/VEGFA axis by delivering microRNA-26a-5p.

Critical roles of stem cell-extracellular vesicles (EVs) in the management of osteoporosis have been documented. Here, this study was designed to enlarge the research of the specific effects and underlying mechanism of urine-derived stem cells-EVs (USCs-EVs) on osteoporosis in diabetes rats. Firstly, miR-26a-5p and histone deacetylase 4 (HDAC4) expression in USCs of rats after diabetic osteoporosis (DOP) modeling induced by streptozotocin injection was determined, followed by study of their interaction. Then, USCs-EVs were co-cultured with osteogenic precursor cells, the effects of miRNA-26a-5p (miR-26a-5p) on osteoblasts, osteoclasts, bone mineralization deposition rate were evaluated. Meanwhile, the effect of USCs-EVs carrying miR-26a-5p on DOP rats was assessed. Elevated miR-26a-5p was seen in USCs-EVs which limited HDAC4 expression. Moreover, USCs-EVs delivered miR-26a-5p to osteogenic precursor cells, thereby promoting their differentiation, enhancing the activity of osteoblasts, and inhibiting the activity of osteoclasts, thereby preventing DOP through the activation of hypoxia inducible factor 1 subunit alpha (HIF-1α)/vascular endothelial growth factor A (VEGFA) pathway by repressing HDAC4. In a word, USCs-EVs-miR-26a-5p is a promising therapy for DOP by activating HIF-1α/VEGFA pathway through HDAC4 inhibition. 1. USCs-EVs-miR-26a-5p targeted HDAC4 and limited HDAC4 expression. 2. miR-26a-5p was delivered by USCs-EVs into osteoblast precursor cells. 3. USCs-EVs-miR-26a-5p promoted the differentiation of osteoblast precursor cells into osteoblasts. 4. miR-26a-5p delivered by USCs-EVs could inhibit HDAC4. 5. USCs-EVs-miR-26a-5p could prevent the pathogenesis of DOP via HIF-1α/VEGFA aix.

CXCL12/CXCR4-Rac1-mediated migration of osteogenic precursor cells contributes to pathological new bone formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by inflammatory back pain and spinal ankylosis due to pathological new bone formation. Here, we identified CXCL12 as a critical contributor to pathological new bone formation through recruitment of osteogenic precursor cells (OPCs). CXCL12 was found highly expressed in the regions that would potentially develop pathological new bone. OPCs were recruited to the regions where CXCL12 was up-regulated. Inhibition of CXCL12/CXCR4 axis with AMD3100 or conditional knockout of CXCR4 attenuated OPCs migration and subsequent pathological new bone formation in animal models of AS. By contrast, a genetically engineered animal model with CXCL12 overexpression developed a joint ankylosis phenotype. Furthermore, Rac1 was found essential for OPCs migration and pathological new bone formation. These findings ravel the novel role of CXCL12 in AS and indicate a potential strategy for targeting the CXCL12/CXCR4-Rac1 axis to prevent progression of axial skeleton ankylosis.

Cerium Oxide Nanoparticles Promote Osteoplastic Precursor Differentiation by Activating the Wnt Pathway

Osteoplastic precursors are critical for fracture repair and bone homeostasis maintenance. Cerium oxide nanoparticles (CeO2 NPs) can promote the osteogenic differentiation of mesenchymal stem cells and secrete vascular endothelial growth factors. However, little is known about its role in precursor osteoblasts; therefore, we further investigated the effect and mechanism of CeO2 NPs in precursor osteoblasts. Cell counting kit-8 analysis was utilized to detect the toxicity of CeO2 NPs on MC3T3-E1 mouse osteogenic precursor cells. Then, alizarin red S staining was employed to assess the degree of extracellular matrix mineralization, and quantitative real-time polymerase chain reaction analysis was performed to measure the levels of osteogenesis-related genes. To identify differentially expressed genes, mRNA-sequencing was performed. Subsequently, GO and KEGG analyses were deployed to identify the major downstream pathways, whereas Western blot was used for verification. CeO2 NPs significantly enhanced the ability of MC3T3-E1 precursor osteoblasts to enhance matrix mineralization and increased the expression of osteogenic genes such as runt-related transcription factor 2, collagen Iα1, and osteocalcin. Pathway analysis revealed that CeO2 NPs enhanced the nuclear translocation of β-catenin and activated the Wnt pathway by promoting family with sequence similarity 53 member B/simplet expression, while Western blot analysis indicated the same results. After using a Wnt pathway inhibitor (KYA1797K), the simulative effect of CeO2 NPs was abolished. This study revealed that CeO2 NPs promoted MC3T3-E1 precursor osteoblast differentiation by activating the Wnt pathway.

The Biology of COP Cells: Mesenchymal of Hematopoietic?

Circulating osteogenic precursor (COP) cells constitute a recently discovered population of circulating progenitor cells with the capacity to form not only bone but other mesenchymal tissues. A small but growing body of literature explores these cells, but with a great deal of disagreement and contradiction within it, mainly whether these cells are from mesenchymal or hematopoietic origin. This session will discuss the origins and biological characterization of these cells, including the identification strategies used to isolate these cells from the peripheral blood. It also examines the available knowledge on the in vitro and in vivo behaviour of these cells in plastic adherence, differentiation capacity, proliferation, and cellular homing. We will also review the profound and exciting implications for future use of COP cells in clinical practice, particularly in comparison with other types of stem cells.

Preparation of Antibiotic-Loaded Copper-Doped Hydroxyapatite Microspheres and Evaluation of Their Antibacterial and Osteogenic Effect

To explore the preparation method of copper (Cu)-doped hydroxyapatite (HA) microspheres loaded with vancomycin (Van), and evaluate their antibacterial and osteogenic effects in vitro. The Cu doped HA microspheres (Cu-HA) with molar doping ratios of 1%, 5%, 10%, and 20% were prepared by hydrothermal synthesis. The microscopic morphology changes were observe with scanning electron microscope. X-ray diffractometer (XRD) was used to study the phase composition and analyze the crystallinity of the sample. Cu-HA with a molar doping ratio of 10% was selected for analysis of the elemental composition of the sample with energy dispersive X-ray spectroscopy (EDS), and was then coated with polydopamine (PDA) as the medium to prepare Cu-HA-PDA. XRD and Fourier infrared spectrometer were used to examine the coating effect of the sample. Van was load on Cu-HA-PDA to prepare Cu-HA-PDA-Van. HA, Cu-HA, HA-PDA, and Cu-HA-PDA-Van were added to α medium at 10 mg/mL to prepare different groups of extract solutions.The main components of the extract solutions were examined, and the Van concentration was checked. We examined the toxic effect of material extract solutions on osteogenic precursor cells and the proliferation and differentiation of bone marrow mesenchymal stem cells, and checked the expression of osteocalcin ( OCN), runt-related transcription factor 2 ( RUNX-2), and alkaline phosphatase ( ALP), the osteogenic related genes. Sterilized HA, Cu-HA, HA-PDA, Cu-HA-PDA, Cu-HA-DPA-Van microsphere materials were prepared, and the colony counting method was used to evaluate the antibacterial effect of the materials for Staphylococcus aureus. Various types of Cu-doped HA (Cu-HA) were successfully synthesized. As the proportion of Cu increased, the morphology gradually changed from being strip or belt-shaped to a uniform spherical shape. Cu-HA of 10% molar doping ratio showed a clearly microspherical shape and a petal-like porous micro-nano morphology on the surface. EDS and XRD analyses showed that the main structure of the material was still made up of hydroxyapatite crystals and Cu was successfully doped with HA. The infrared spectrometer showed that the PDA was successfully coated on the surface of the material. Examination of the main components of the extract solution once again verified that the Cu element had successfully entered and replaced part of the Ca element in the HA. The 10 mg/mL Cu-HA-PDA-Van extract solution contained 0.27 mg/mL of Van. In vitro cell experiments and bone-formation-related gene testing showed that Cu-HA-Van had good biological activity and promoted bone differentiation. The minimum inhibitory concentration (MIC) of Cu-HA-PDA-Van microspheres was 16 μg/mL. Compared with Cu-HA, HA-PDA and pure HA, Cu-HA-Van microspheres had significant and long-lasting antibacterial effects. Cu element was used to control the microscopic morphology of HA, and the Cu-HA-PDA-Van microspheres prepared by successfully coating of PDA and loading of Van had good antibacterial properties and biological activity.

Development and validation of a new method to isolate, expand, and differentiate circulating osteogenic precursor (COP) cells

Circulating osteogenic precursor (COP) cells are a population of progenitor cells in the peripheral blood with the capacity to form bone in vitro and in vivo. They have characteristics of the mesenchymal stem and progenitor pool found in the bone marrow; however, more recently, a population of COP cells has been identified with markers of the hematopoietic lineage such as CD45 and CD34. While this population has been associated with several bone pathologies, a lack of cell culture models and inconsistent characterization has limited mechanistic research into their behavior and physiology. In this study, we describe a method for the isolation of CD45+/CD34+/alkaline phosphatase (ALP) + COP cells via fluorescence-activated cell sorting, as well as their expansion and differentiation in culture. Hematopoietic COP cells are a discreet population within the monocyte fraction of the peripheral blood mononuclear cells, which form proliferative, fibroblastoid colonies in culture. Their expression of hematopoietic markers decreases with time in culture, but they express markers of osteogenesis and deposit calcium with differentiation. It is hoped that this will provide a standard for their isolation, for consistency in future research efforts, to allow for the translation of COP cells into clinical settings.

PHOTOBIOMODULATORY EFFECT OF LOW-INTENSITY LASER RADIATION ON MULTICELLULAR SPHEROIDS

Photobiomodulatory effects of low-intensity laser radiation (LILR) in cells cultured in standard, two-dimensional conditions are well established. Conversely, the characteristics of this effect in three-dimensional (3D) cultures, which are currently recommended due to the greater similarity with cellular behavior in vivo, have not yet been widely investigated. The objective of this work was to analyze the biomodulator effect of LILR, on the wavelength (λ) of 685 nm, on the constitution process and on the viability of cells cultured as multicellular spheroid (MSs). For this, agarose molds containing microwells were seeded (2x105 cell/ml) with osteogenic precursor cells (OPCs - MC3T3-E1) and kept under ideal culture conditions. The molds were irradiated for five consecutive days with doses of 0.5, 1.0 and 1.5 J/cm², the first irradiation being performed immediately after sowing. The process of constitution of MSs was analyzed and the cultures were submitted to the cell viability test. The results demonstrated that the LILR at λ 685 nm exerted a dose-dependent biomodulatory effect on cell metabolism and on the process of constituting the MSs of OPCs. These results demonstrate the potential of photobiomodulation to contribute to the process of constituting MSs, which can be explored in the strategies of multicellular spheroids therapy used in regenerative medicine and bioprinting.

Electrospinning With Lyophilized Platelet-Rich Fibrin Has the Potential to Enhance the Proliferation and Osteogenesis of MC3T3-E1 Cells.

Platelet-rich fibrin (PRF) as a reservoir of various growth factors plays an essential role in wound healing and tissue engineering at present. Electrospinning technology is an efficient approach to acquire artificial scaffold which has large specific surface area and high porosity. The goal of this study was to investigate the potential of electrospinning on the proliferation and osteogenesis of osteogenic precursor cells in vitro, with lyophilized PRF added as a component for electrospinning preparation. The surface structure of lyophilized PRF and nanofibers were investigated, and the proliferation, osteogenesis of MEC3T3-E1 cells with lyophilized PRF or nanofibers extract were studied. The results showed that the diameters of the lyophilized PRF pores were 1.51 ± 0.75 μm, and lyophilized PRF medium promoted the proliferation and osteocalcin (OCN) and osteopontin (OPN) genes expression of MEC3T3-E1 cells. Furthermore, the diameters of the polyvinyl alcohol/sodium alginate/lyophilized PRF (PVA/SA/PRF) fibers were 201.14 ± 40.14 nm. Compared to PVA/SA nanofibers extract and control medium, PVA/SA/PRF nanofibers extract also enhanced the proliferation and mineralization activity of MEC3T3-E1 cells. These results might be instructive to future therapeutics with PVA/SA/PRF electrospinning for bone tissue engineering or other applications.