Bone Differentiation Research Articles

Magnetic Field Boosts the Transmembrane Transport Efficiency of Magnesium Ions from PLLA Bone Scaffold.

In the system of magnesium-loaded scaffolds, the effect of magnesium ions (Mg2+ ) on the osteogenesis induction is restricted due to the low transmembrane transport efficiency of Mg2+ into the cell, which limits the application for bone defect repair. Inspired by the fact that magnetic field can regulate ion channel proteins on the cell membrane, magnetite nanoparticleis introduced into the poly (l-lactic acid) /magnesium oxide composite in this study, and a magnetic magnesium-loaded bone scaffold is prepared via selective laser sintering . Notably, the activities of the Mg2+ channel protein (MAGT1) on the membrane of bone marrow mesenchymal stem cells (rBMSCs) are enhanced via magnetic torque effect (via integrin αV β3/actin), under the action of static magnetic field (SMF), which promoted rBMSCs to capture Mg2+ in the microenvironment and induced osteogenesis. In vitro experiments showed that the magnetic magnesium-loaded scaffold, under the action of SMF, can accelerate the inflow of Mg2+ from surrounding microenvironment, which improved cellular activities, osteogenesis-related gene expression (ALP, Runx2, OCN, and OPN), and mineralization. Besides, in vivo skull defect repair experiments showed that the scaffolds possessed good ability to promote bone differentiation and new bone regeneration.

Interleukin-17A enhances osteogenic differentiation by activating ERK/MAPK in stem cells derived from human exfoliated deciduous teeth

Introduction: Human exfoliated deciduous teeth–derived stem cells (SHEDs) have been shown as an excellent source of bone regeneration. Interleukin-17A (IL-17A) facilitates bone differentiation in various cell types, including SHEDs. In this study, we have demonstrated IL-17A’s stimulating effect on SHEDs in osteogenic differentiation and further evaluated the role of the ERK/MAPK signaling pathway in this process. Methods: The function of IL-17A in osteogenic differentiation, proliferative activity, and MAPK cascade activation in SHEDs were investigated. Results: IL-17A significantly enhanced proliferative and alkaline phosphatase activities in SHEDs. Furthermore, the expression levels of different osteogenic proteins including COL1A1, ALP, OPN, RUNX, and OCN were significantly elevated in IL-17A-treated SHEDs. Moreover, IL-17A triggered MAPK signaling in SHEDs, as evidenced by significant upregulation of both downstream ERK targets, P38 and JNK pathways, and upstream activators. In addition, ERK/MAPK activation time-dependently established the participation of MAPK signaling in SHED osteogenic differentiation. Conclusion: These findings suggest that IL-17A-induced ERK/MAPK signaling pathway activation is necessary for SHEDs to differentiate into osteoblasts. This reiterates the significance of this particular intracellular signaling pathway in controlling SHED osteogenic differentiation, which is a promising source of bone tissue regeneration.

Biomechanical and Biological Characterization of XGel, a Human-Derived Hydrogel for Stem Cell Expansion and Tissue Engineering.

Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delivery of cells and drugs. Existing hydrogel classifications include synthetic, recombinant, chemically defined, plant- or animal-based, and tissue-derived matrices. There is a need for materials that can support both human tissue modeling and clinically relevant applications requiring stiffness tunability. Human-derived hydrogels are not only clinically relevant, but they also minimize the use of animal models for pre-clinical studies. This study aims to characterize XGel, a new human-derived hydrogel as an alternative to current murine-derived and synthetic recombinant hydrogels that features unique physiochemical, biochemical, and biological properties that support adipocyte and bone differentiation. Rheology studies determine the viscosity, stiffness, and gelation features of XGel. Quantitative studies for quality control support consistency in the protein content between lots. Proteomics studies reveal that XGel is predominantly composed of extracellular matrix proteins, including fibrillin, collagens I-VI, and fibronectin. Electron microscopy of the hydrogel provides phenotypic characteristics in terms of porosity and fiber size. The hydrogel demonstrates biocompatibility as a coating material and as a 3D scaffold for the growth of multiple cell types. The results provide insight into the biological compatibility of this human-derived hydrogel for tissue engineering.

Evaluation of near-infrared Raman spectroscopy in the differentiation of cortical bone, trabecular bone, and Bio-Oss bone graft: an ex-vivo study

We evaluated the ability of near-infrared Raman spectroscopy (near-IR RS) to differentiate among cortical bone, trabecular bone, and Bio-Oss, a bovinebone-based graft material. We obtained a thinly sliced section of the mandible to collect cortical and trabecular bone samples and placed compacted Bio-Oss bone graft into a partially edentulous mandible in a dry human skull to obtain a comparable Bio-Oss sample. We performed near-IR RS of the 3 samples and evaluated the resultant Raman spectra to evaluate their differences. We identified 3 sets of spectroscopic markers that differentiated Bio-Oss from human bone. The first consisted of significant shifts in the location of the 960 cm-1 phosphate (PO43-) peak and a reduction in its width, suggesting that Bio-Oss is more crystalline than bone. The second was the reduced carbonate content of Bio-Oss compared to bone, as determined from the 1070 cm-1/960 cm-1 peak area ratio. The final marker was the lack of collagen-associated peaks in Bio-Oss compared to cortical and trabecular bone. Near-IR RS can reliably differentiate human cortical and trabecular bone from Bio-Oss via 3 sets of spectral markers associated with mineral crystallinity, carbonate content, and collagen content that differ significantly between them. Integrating this modality into dental practice may assist in implant treatment planning.

Tauroursodeoxycholic Acid Enhances Osteogenic Differentiation through EGFR/p-Akt/CREB1 Pathway in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are pluripotent stromal cells that are among the most appealing candidates for regenerative medicine and may aid in the repair and regeneration of skeletal disorders through multiple mechanisms, including angiogenesis, differentiation, and response to inflammatory conditions. Tauroursodeoxycholic acid (TUDCA) has recently been used in various cell types as one of these drugs. The mechanism of osteogenic differentiation by TUDCA in hMSCs remains unknown. Cell proliferation was performed by the WST-1 method, and alkaline phosphatase activity and alizarin red-sulfate staining were used to confirm the osteogenic differentiation indicator. Expression of genes related to bone differentiation and specific genes related to signaling pathways was confirmed by quantitative real-time polymerase chain reaction. We found that cell proliferation was higher as the concentration increased, and showed that the induction of osteogenic differentiation was significantly enhanced. We also show that osteogenic differentiation genes were upregulated, with the expression of the epidermal growth factor receptor (EGFR) and cAMP responsive element binding protein 1 (CREB1) being specifically high. To confirm the participation of the EGFR signaling pathway, the osteogenic differentiation index and expression of osteogenic differentiation genes were determined after using an EGFR inhibitor. As a result, EGFR expression was remarkably low, and that of CREB1, cyclin D1, and cyclin E1 was also significantly low. Therefore, we suggest that TUDCA-induced osteogenic differentiation of human MSCs is enhanced through the EGFR/p-Akt/CREB1 pathway.

Exosomes derived from mesenchymal stromal cells promote bone regeneration by delivering miR-182–5p-inhibitor

Exosomes, small extracellular vesicles that function as a key regulator of cell-to-cell communication, are emerging as a promising candidate for bone regeneration. Here, we aimed to investigate the effect of exosomes from pre-differentiated human alveolar bone-derived bone marrow mesenchymal stromal cells (AB-BMSCs) carrying specific microRNAs on bone regeneration. Exosomes secreted from AB-BMSCs pre-differentiated for 0 and 7 days were cocultured with BMSCs in vitro to investigate their effect on the differentiation of the BMSCs. MiRNAs from AB-BMSCs at different stages of osteogenic differentiation were analyzed. BMSCs seeded on poly-L-lactic acid(PLLA) scaffolds were treated with miRNA antagonist-decorated exosomes to verify their effect on new bone regeneration. Exosomes pre-differentiated for 7 days effectively promoted the differentiation of BMSCs. Bioinformatic analysis revealed that miRNAs within the exosomes were differentially expressed, including the upregulation of osteogenic miRNAs (miR-3182, miR-1468) and downregulation of anti-osteogenic miRNAs (miR-182–5p, miR-335–3p, miR-382–5p), causing activation of the PI3K/Akt signaling pathway. The treatment of BMSC-seeded scaffolds with anti-miR-182–5p decorated exosomes demonstrated enhanced osteogenic differentiation and efficient formation of new bone. In conclusion, Osteogenic exosomes secreted from pre-differentiated AB-BMSCs were identified and the gene modification of exosomes provides great potential as a bone regeneration strategy. Data Availability StatementData generated or analyzed in this paper partly are available in the GEO public data repository(http://www.ncbi.nlm.nih.gov/geo).

Activation of Wnt/β‑Catenin‑p130/E2F4 promotes the differentiation of bone marrow‑derived mesenchymal stem cells into typeII alveolar epithelial cells through cell cycle arrest.

The results of our previous study demonstrated that activation of the Wnt/β-catenin pathway increased the differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells; however, the specific mechanisms remain unclear. The present study aimed to evaluate the role of Wnt/β-catenin-p130/E2F transcription factor 4 (E2F4) in regulating the differentiation of mouse MSCs (mMSCs) into AT II cells, and to determine the specific mechanisms. mMSCs with p130 or E2F4 overexpression were constructed using lentiviral vectors. Differentiation of mMSCs into AT II cells was promoted using a modified coculture system with murine lung epithelial-12 cells incubated in small airway growth medium for 7-14 days. The differentiation efficiency was detected using immunofluorescence, western blot analysis and transmission electron microscopy. To detect the association between the canonical Wnt pathway and p130/E2F4, 4 mmol/l lithium chloride (LiCl) or 200 ng/ml Dickkopf-related protein 1 (DKK-1) was also added to the coculture system. Following differentiation, the cell cycle of mMSCs was evaluated using flow cytometry. The results of the present study demonstrated that surfactant protein C (SP-C) protein expression was higher in the p130 overexpression (MSC-p130) and E2F4 overexpression (MSC-E2F4) groups compared with the normal control mMSCs group following differentiation into AT II cells. Similar results for SP-C protein expression and lamellar body-like structures were also observed using immunofluorescence analysis and electron microscopy. Following the addition of LiCl into the coculture system for activation of the Wnt/β-catenin signaling pathway, phosphorylated (p)-p130/p130 was slightly decreased at 7 days and E2F4 was increased both at 7 and 14 days in mMSCs. Furthermore, the p-p130/p130 ratio was significantly increased at 14 days and E2F4 was decreased both at 7 and 14 days following DKK-1-mediated inhibition of the Wnt pathway. The results of the present study demonstrated that the numbers of cells in G1 and S phases were increased following activation of the Wnt pathway and decreased following Wnt pathway inhibition. However, the number of cells in G1 phase was increased following the differentiation of mMSCs overexpressing p130 or E2F4. Therefore, the results of the present study revealed that the canonical Wnt signaling pathway may affect the differentiation of MSCs into AT II cells via regulation of downstream p130/E2F4. The specific mechanisms may be associated with G1 phase extension in the cell cycle of MSCs.

In Vitro Bone Differentiation of 3D Microsphere from Dental Pulp-Mesenchymal Stem Cells.

Bone defects lead to the structural loss of normal architecture, and those in the field of bone tissue engineering are searching for new alternatives to aid bone regeneration. Dental pulp-mesenchymal stem cells (DP-MSC) could provide a promising alternative to repair bone defects, principally due to their multipotency and capacity to fabricate three-dimensional (3D) spheroids. The present study aimed to characterize the 3D DP-MSC microsphere and the osteogenic differentiation capacity potential cultured by a magnetic levitation system. To achieve this, the 3D DP-MSC microsphere was grown for 7, 14, and 21 days in an osteoinductive medium and compared to 3D human fetal osteoblast (hFOB) microspheres by examining the morphology, proliferation, osteogenesis, and colonization onto PLA fiber spun membrane. Our results showed good cell viability for both 3D microspheres with an average diameter of 350 μm. The osteogenesis examination of the 3D DP-MSC microsphere revealed the lineage commitment, such as the hFOB microsphere, as evidenced by ALP activity, the calcium content, and the expression of osteoblastic markers. Finally, the evaluation of the surface colonization exhibited similar patterns of cell-spreading over the fibrillar membrane. Our study demonstrated the feasibility of forming a 3D DP-MSC microsphere structure and the cell-behavior response as a strategy for the applications of bone tissue guiding.

Long term in vivo Dlk1 gene transfer protects against myocardial fibrosis-induced cardiac dysfunction

Introduction: Cardiac fibrosis can independently contribute to the impairment of cardiac function and development of heart failure. Early activation of pro-fibrotic genetic pathways has been suggested to occur well before adverse ventricular remodeling occurs in fibrosis as it progresses to heart failure. Delta-like homolog 1 (Dlk1), a paternally imprinted gene that encodes a transmembrane protein that contains multiple epidermal growth factor repeats, has been shown to play a critical role as a regulator of cell differentiation, neuronal differentiation, bone differentiation, and hematopoiesis. We hypothesize that Dlk-1 could regulate myocardial fibrosis via inhibition of fibroblast-myofibroblast conversion and therefore restrain cardiac fibrosis progression. Methods: Wildtype (WT) and Dlk1 knockout (Dlk1-/-) mice were subjected to myocardial infarction (MI) by permanent ligation of the left anterior descending artery to induce heart failure. A recombinant adeno-associated virus 9 (AAV9) was used to deliver Dlk1 into the myocardium in vivo. Cardiac function and hemodynamic changes were measured by echocardiography and left ventricular catheterization. Immunohistochemistry and confocal microscopy were used to determine the expression of pro-fibrotic markers. Total RNA was prepared from left ventricular (LV) tissue or cardiomyocytes and fibroblasts from Dlk1-/- and WT hearts. Masson trichrome staining was used to determine abnormal collagen deposition in the heart. Result: Myocardial fibrosis was demonstrated by increased expression of alpha-smooth muscle actin (αSMA), collagen, fibronectin, connective tissue growth factor, along with a substantial increase in the deposition of collagen as evident from Masson's trichrome staining. We observed downregulated Dlk-1 mRNA and protein expression in ischemic tissues from human patients and infarcted pigs’ hearts. The reduced levels of Dlk1 were associated with increased mRNA expression of pro-fibrotic molecules, collagen1a, LOX and αSMA in the scar area. Echocardiographic analysis revealed substantial LV chamber dilatation and reduced fractional shortening (FS %) in Dlk1 null mice as compared to WT, whereas the mice treated with AAV9-Dlk1 for 8-10 weeks showed a significant improvement in cardiac function. Consistently, Dlk1-deficient mice exhibited impairment in heart function, cardiac fibrosis and myocyte hypertrophy and increased expression of atrial natriuretic peptide, and B-type natriuretic peptide, while AAV9-Dlk1-treated mice significantly reduced cardiac remodeling and myocardial fibrosis. Conclusion: Restoration of Dlk1 in the heart following MI-induced heart failure improved cardiac function and attenuated myocardial fibrosis. The findings from the current study suggest a critical role for Dlk1 in myocardial fibrosis-induced cardiac remodeling, indicating that Dlk1 gene therapy may be a viable therapeutic approach for heart failure. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of p53 in promoting BMP9‑induced osteogenic differentiation of mesenchymal stem cells through TGF‑β1.

Known as a tumour suppressor gene, p53 also plays a key role in controlling the differentiation of mesenchymal stem cells (MSCs). Bone morphogenetic protein 9 (BMP9) has been identified as a potent factor in inducing osteogenic differentiation of MSCs, but its relationship with p53 remains unclear. The present study revealed that TP53 was expressed at higher levels in MSCs from patients with osteoporosis and was associated with the top 10 core central genes found in the current osteoporosis genetic screen. p53 was expressed in C2C12, C3H10T1/2, 3T3-L1, MEFs, and MG-63 cell lines, and could be upregulated by BMP9, as measured by western blotting and reverse-transcription quantitative PCR (RT-qPCR). Furthermore, overexpression of p53 increased the mRNA and protein levels of osteogenic marker Runx2 and osteopontin, as evaluated by western blotting and RT-qPCR in BMP9-induced MSCs, whereas the p53 inhibitor pifithrin (PFT)-α attenuated these effects. The same trend was found in alkaline phosphatase activities and matrix mineralization, as measured by alkaline phosphatase staining and alizarin red S staining. Moreover, p53 overexpression reduced adipo-differentiation markers of PPARγ and lipid droplet formation, as measured by western blotting, RT-qPCR and oil red O staining, respectively, whereas PFT-α facilitated adipo-differentiation in MSCs. In addition, p53 promoted TGF-β1 expression and inhibition of TGF-β1 by LY364947 partially attenuated the effects of p53 on promoting BMP9-induced MSC osteo-differentiation and inhibiting adipo-differentiation. The inhibitory effect of PFT-α on osteogenic markers and the promoting effect on adipogenic markers can be reversed when combined with TGF-β1. TGF-β1 may enhance the promotion of osteo-differentiation of MSCs by p53 through inhibition of adipo-differentiation. Collectively, by promoting BMP9-induced MSCs bone differentiation and inhibiting adipose differentiation, p53 may be a novel therapeutic target for bone-related diseases.

Inhibition of GSK-3β Enhances Osteoblast Differentiation of Human Mesenchymal Stem Cells through Wnt Signalling Overexpressing Runx2.

Small-molecule-inhibitor-based bone differentiation has been recently exploited as a novel approach to regulating osteogenesis-related signaling pathways. In this study, we identified 1-Azakenpaullone, a highly selective inhibitor of glycogen synthase kinase-3β (GSK-3β), as a powerful inducer of osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). GSK-3β is a serine-threonine protein kinase that plays a major role in different disease development. GSK-3β is a key regulator of Runx2 activity in osteoblastic formation. We evaluated alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin Red staining to assess the mineralization of cultured human MSCs. Gene expression profiling was assessed using an Agilent microarray platform, and bioinformatics were performed using Ingenuity Pathway Analysis software. Human MSCs treated with 1-Azakenpaullone showed higher ALP activity, increased in vitro mineralized matrix formation, and the upregulation of osteoblast-specific marker gene expression. Global gene expression profiling of 1-Azakenpaullone-treated human MSCs identified 1750 upregulated and 2171 downregulated mRNA transcripts compared to control cells. It also suggested possible changes in various signaling pathways, including Wnt, TGFβ, and Hedgehog. Further bioinformatics analysis employing Ingenuity Pathway Analysis recognized significant enrichment in the 1-Azakenpaullone-treated cells of genetic networks involved in CAMP, PI3K (Complex), P38 MAPK, and HIF1A signaling and functional categories associated with connective tissue development. Our results suggest that 1-Azakenpaullone significantly induced the osteoblastic differentiation and mineralization of human MSCs mediated by the activation of Wnt signaling and the nuclear accumulation of β-catenin, leading to the upregulation of Runx2, a key transcription factor that ultimately promotes the expression of osteoblast-specific genes. Thus, 1-Azakenpaullone could be used as an osteo-promotor factor in bone tissue engineering.

GSTT1 PROMOTES OSTEOGENIC DIFFERENTIATION OF ADIPOSE STEM CELLS

We found that adipose stem cells are poorly differentiated into bone and that their ability to differentiate into bone varies from cell line to cell line. The osteogenic differentiation ability of the adipose stem cell lines was distinguished through Alzarin Red Staining, and the cell lines that performed well and those that did not were subjected to RNA-seq analysis. The selected gene GSTT1 (glutathione S-transferase theta-1) gene is a member of a protein superfamily that catalyzes the conjugation of reduced glutathione to a variety of hydrophilic and hydrophobic compounds. The purpose of this study is to treat avascular necrosis and bone defect by improving bone regeneration with adipose stem cells introduced with a new GSTT1 gene related to osteogenic differentiation of adipose stem cells. In addition, the GSTT1 gene has the potential as a genetic marker that can select a specific cell line in the development of an adipose stem cell bone regeneration drug.Total RNA was extracted from each sample using the TRIzol reagent. Its concentration and purity were determined based on A260 and A260/A280, respectively, using a spectrophotometer. RNA sequencing library of each sample was prepared using a TruSeq RNA Library Prep Kit. RNA-seq experiments were performed for hADSCs. Cells were transfected with either GSTT1 at 100 nM or siControl (scramble control) by electroporation using a 1050 pulse voltage for 30 ms with 2 pulses using a 10 μl pipette tip.The purpose of this study is to discover genetic markers that can promote osteogenic differentiation of adipose stem cells (hADSCs) through mRNA-seq gene analysis. The selected GSTT1 gene was found to be associated with the enhancement of osteogenic differentiation of adipose stem cells. siRNA against GSTT1 reduced osteogenic differentiation of hADSCs, whereas GSTT1 overexpression enhanced osteogenic differentiation of hADSCs under osteogenic conditions.In this study, GSTT1 transgenic adipose stem cells could be used in regenerative medicine to improve bone differentiation. In addition, the GSTT1 gene has important significance as a marker for selecting adipose stem cells with potential for bone differentiation in the development of a therapeutic agent for bone regeneration cells.

Chronic adrenergic stress and generation of myeloid-derived suppressor cells: Implications for cancer immunotherapy in dogs.

Recent studies have highlighted a key role played by the sympathetic nervous system (SNS) and adrenergic stress in mediating immune suppression associated with chronic inflammation in cancer and other diseases. The connection between chronic SNS activation, adrenergic stress and immune suppression is linked in part to the ability of catecholamines to stimulate the bone marrow release and differentiation of myeloid-derived suppressor cells (MDSC). Rodent model studies have revealed an important role for β-adrenergic receptor signalling in suppression of cancer immunity in mice subjected to chronic stresses, including thermal stress. Importantly, therapeutic blockade of beta-adrenergic responses by drugs such as propranolol can partially reverse the generation and differentiation of MDSC, and partly restore tumour immunity. Clinical trials in both humans and dogs with cancer have demonstrated that propranolol blockade can improve responses to radiation therapy, cancer vaccines and immune checkpoint inhibitors. Thus, the SNS stress response has become an important new target to relieve immune suppression in cancer and other chronic inflammatory conditions.

Deproteinized Bone Tissue as a Matrix for Tissue-Engineered Construction: Experimental Study

Background. At present, for a number of reasons the complete bone defect replacement with autogenous bone is not always possible. Bone substitute materials are used as an alternative to autogenous bone tissue and can be of either biological or non-biological origin. One of the ways of development of reconstructive technologies is the use of tissue-engineered constructs that fully imitate autogenous bone tissue in the required volume.
 Aim of study to define in vivo the possibility of using deproteinized human cancellous bone tissue as a matrix for creating tissue-engineered constructs.
 Methods. An in vivo study was carried out on NZW rabbits. To create a construct, we used the fragments of deproteinized cancellous bone tissue of the human femoral head and stromal vascular fraction of rabbit adipose tissue as a matrix. Bone defect modeling with its subsequent replacement was performed to evaluate the efficacy of reparative osteogenesis during bone defects reconstruction. Study groups were defined: group 1 (control) surgical modeling of a bone defect of the femur without its reconstruction; group 2 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix; group 3 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix in combination with stromal vascular fraction of adipose tissue (according to ACP SVF technology).
 Results. Comparative analysis of reparative processes in case of applying tissue-engineered constructs based on deproteinized human cancellous bone matrix in combination with adipose tissue-derived stromal vascular fraction on in vivo experimental model revealed that the use of these bone substitute materials contributes not only to an early activation of reparative regeneration of main structural elements of the bone tissue in the area of the bone defect replacement, but also to its well-timed differentiation. This determines the restoration of structural and functional viability of the bone tissue at the damage site without developing discernible reactive inflammation. Moreover, the effect of the selected tissue-engineered construct with the combined influence of several factors (ACP SVF) in its composition turned out to be more effective in stimulating bone tissue repair and differentiation.
 Conclusion. Combination of SVF and deproteinized bone matrix for creating tissue-engineered constructs enables to engage several regeneration mechanisms and accelerate the process of bone defect replacement in comparison with isolated deproteinized bone matrix without bone defect reconstruction.

Comparison of perioperative serum osteocrin concentrations between surgical techniques in dogs with cranial cruciate ligament rupture

The cranial cruciate ligament (CCL) rupture is a common orthopedic disease in dogs that is usually managed with tibial plateau leveling osteotomy (TPLO) or extracapsular lateral suture (ECLS). Osteotomy is generally associated with some complications, including nonunion. The periosteum plays an important role in bone growth and remodeling. Osteocrin (OSTN), which was recently identified and is involved in bone formation and differentiation, is produced in the periosteum and osteoblasts. The aimed to investigate whether the concentrations of serum OSTN change before and after stifle surgery in dogs and compare the OSTN concentrations in the two surgical techniques (TPLO: n = 20 vs. ECLS: n = 36). The postoperative serum OSTN concentration in the TPLO group was significantly lower than the preoperative value (p < 0.05), while serum OSTN concentrations differed statistically between the preoperative and suture-removal periods. In contrast, no significant differences were observed in the ECLS group. In conclusion, osteotomy affects serum OSTN concentrations during the perioperative period in dogs, which may be related to periosteal injury.

Macromolecular crowding in animal component-free, xeno-free and foetal bovine serum media for human bone marrow mesenchymal stromal cell expansion and differentiation.

Background: Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of human bone marrow stromal cells (hBMSCs). Methods: Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain hBMSC phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and/or differentiation, can more tightly control hBMSC fate. Results: Cells expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBMSCs. Conclusion: Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant hBMSC medicines.

Yam-derived exosome-like nanovesicles stimulate osteoblast formation and prevent osteoporosis in mice.

Plants-releasing exosome-like nanovesicles (PENs) contain miRNA, bioactive lipids, mRNAs, and proteins to exert antioxidant, anti-inflammatory, and regenerative activity. Substances extracted from yams have been reported to promote osteoblast growth in bone regeneration, which prevent weak and brittle bones in osteoporosis. Herein, we describe the beneficial effects of yam-derived exosome-like nanovesicles (YNVs) on promoting differentiation and mineralization of osteoblasts for bone regeneration in ovariectomized (OVX)-induced osteoporotic mice. YNVs were successfully isolated and characterized. YNVs stimulate the proliferation, differentiation, and mineralization of osteoblasts with increased bone differentiation markers (OPN, ALP, and COLI). Interestingly, YNVs do not contain saponins including diosgenin and dioscin known to mainly exert osteogenic activity of yams. Instead, the osteogenic activity of YNVs was revealed to be resulted from activation of the BMP-2/p-p38-dependent Runx2 pathway. As a result, YNVs promote longitudinal bone growth and mineral density of the tibia in the OVX-induced osteoporotic mice in vivo, and these results positively correlate the significant increases in osteoblast-related parameters. In addition, the orally administered YNVs were transported through the GI tract and absorbed through the small intestine. These results showed an excellent systemic biosafety determined by histological analysis and liver/kidney toxicity tests. Taken together, YNVs can serve as a safe and orally effective agent in the treatment of osteoporosis.

Bone Differentiation Ability of CD146-Positive Stem Cells from Human Exfoliated Deciduous Teeth.

Regenerative therapy for tissues by mesenchymal stem cell (MSCs) transplantation has received much attention. The cluster of differentiation (CD)146 marker, a surface-antigen of stem cells, is crucial for angiogenic and osseous differentiation abilities. Bone regeneration is accelerated by the transplantation of CD146-positive deciduous dental pulp-derived mesenchymal stem cells contained in stem cells from human exfoliated deciduous teeth (SHED) into a living donor. However, the role of CD146 in SHED remains unclear. This study aimed to compare the effects of CD146 on cell proliferative and substrate metabolic abilities in a population of SHED. SHED was isolated from deciduous teeth, and flow cytometry was used to analyze the expression of MSCs markers. Cell sorting was performed to recover the CD146-positive cell population (CD146+) and CD146-negative cell population (CD146-). CD146 + SHED without cell sorting and CD146-SHED were examined and compared among three groups. To investigate the effect of CD146 on cell proliferation ability, an analysis of cell proliferation ability was performed using BrdU assay and MTS assay. The bone differentiation ability was evaluated using an alkaline phosphatase (ALP) stain after inducing bone differentiation, and the quality of ALP protein expressed was examined. We also performed Alizarin red staining and evaluated the calcified deposits. The gene expression of ALP, bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) was analyzed using a real-time polymerase chain reaction. There was no significant difference in cell proliferation among the three groups. The expression of ALP stain, Alizarin red stain, ALP, BMP-2, and OCN was the highest in the CD146+ group. CD146 + SHED had higher osteogenic differentiation potential compared with SHED and CD146-SHED. CD146 contained in SHED may be a valuable population of cells for bone regeneration therapy.

A Review on Manufacturing Processes of Biocomposites Based on Poly(α-Esters) and Bioactive Glass Fillers for Bone Regeneration.

The incorporation of bioactive and biocompatible fillers improve the bone cell adhesion, proliferation and differentiation, thus facilitating new bone tissue formation upon implantation. During these last 20 years, those biocomposites have been explored for making complex geometry devices likes screws or 3D porous scaffolds for the repair of bone defects. This review provides an overview of the current development of manufacturing process with synthetic biodegradable poly(α-ester)s reinforced with bioactive fillers for bone tissue engineering applications. Firstly, the properties of poly(α-ester), bioactive fillers, as well as their composites will be defined. Then, the different works based on these biocomposites will be classified according to their manufacturing process. New processing techniques, particularly additive manufacturing processes, open up a new range of possibilities. These techniques have shown the possibility to customize bone implants for each patient and even create scaffolds with a complex structure similar to bone. At the end of this manuscript, a contextualization exercise will be performed to identify the main issues of process/resorbable biocomposites combination identified in the literature and especially for resorbable load-bearing applications.

Bisphosphonate-Modified Functional Supramolecular Hydrogel Promotes Periodontal Bone Regeneration by Osteoclast Inhibition.

Regeneration of periodontal tissue remains a challenge. Under periodontitis, osteoclasts are overactivated and bone loss occurs. We incorporated sodium alendronate (Alen), a medication commonly used to treat osteoporosis, into a supramolecular hydrogel system in order to create a novel biomaterial that would promote periodontal bone regeneration by inhibiting osteoclast overactivation. The Nap-Gly-Phe-Phe (NapGFF) peptide chain was modified to synthesize the functional Nap-Alen gelator. Afterward, the Nap-Alen/HAP supramolecular hydrogel composite with a suitable hydroxyapatite (HAP) ratio was constructed, which has outstanding mechanical properties and 3D structure. In addition to its good biocompatibility, it can inhibit the proliferation of bone marrow-derived macrophages (BMDMs) and differentiation of osteoclasts. Due to the simultaneous introduction of porous HAP, the hydrogel with a nanofiber structure was formed into a 3D mesh-like sparse porous composite hydrogel. While enhancing the mechanical properties of the gel, the porous structure facilitated the attachment and migration of bone regeneration-related cells. Therefore, it can effectively promote the regeneration of periodontal bone. In the future, by modifying the biophysical properties and loading stem cells or cytokines, this supramolecular hydrogel composite constructed in this study may provide a new strategy for tissue regeneration engineering and provide a preliminary experimental basis for relevant clinical translational studies.