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Delphinidin (Delp), a natural antioxidant, has shown promise in treating age-related ailments such as osteoarthritis (OA). This study investigates the impact of delphinidin on intervertebral disc degeneration (IVDD) using human nucleus pulposus cells (hNPCs) subjected to hydrogen peroxide. Various molecular and cellular assays were employed to assess senescence, extracellular matrix (ECM) degradation markers, and the activation of AMPK and autophagy pathways. Initially, oxidative stress (OS)-induced hNPCs exhibited notably elevated levels of senescence markers like p53 and p21, which were mitigated by Delp treatment. Additionally, Delp attenuated IVDD characteristics including apoptosis and ECM degradation markers in OS-induced senescence (OSIS) hNPCs by downregulating MMP-13 and ADAMTS-5 while upregulating COL2A1 and aggrecans. Furthermore, Delp reversed the increased ROS production and reduced autophagy activation observed in OSIS hNPCs. Interestingly, the ability of Delp to regulate cellular senescence and ECM balance in OSIS hNPCs was hindered by autophagy inhibition using CQ. Remarkably, Delp upregulated SIRT1 and phosphorylated AMPK expression while downregulating mTOR phosphorylation in the presence of AICAR (AMPK activator), and this effect was reversed by Compound C, AMPK inhibitor. In summary, our findings suggest that Delp can safeguard hNPCs from oxidative stress by promoting autophagy through the SIRT1/AMPK/mTOR pathway.

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Although three-dimensional (3D) printing techniques are used to mimic macro- and micro-structures as well as multi-structural human tissues in tissue engineering, efficient target tissue regeneration requires bioactive 3D printing scaffolds. In this study, we developed a bone morphogenetic protein-2 (BMP-2)-immobilized polycaprolactone (PCL) 3D printing scaffold with leaf-stacked structure (LSS) (3D-PLSS-BMP) as a bioactive patient-tailored bone graft. The unique LSS was introduced on the strand surface of the scaffold via heating/cooling in tetraglycol without significant deterioration in physical properties. The BMP-2 adsorbed on3D-PLSS-BMPwas continuously released from LSS over a period of 32 d. The LSS can be a microtopographical cue for improved focal cell adhesion, proliferation, and osteogenic differentiation.In vitrocell culture andin vivoanimal studies demonstrated the biological (bioactive BMP-2) and physical (microrough structure) mechanisms of3D-PLSS-BMPfor accelerated bone regeneration. Thus, bioactive molecule-immobilized 3D printing scaffold with LSS represents a promising physically and biologically activated bone graft as well as an advanced tool for widespread application in clinical and research fields.

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AIMS: As the process of bone regeneration is preceded by an inflammatory response, the immune system has long been considered important for fracture healing. Despite many studies on the contribution of immune cells to bone-related diseases, the role of immune cells in the regeneration therapy of lost bone is not well understood. In addition, various types of cells are involved in the clinical bone regeneration environment, but most of the osteo-biology studies are conducted in an osteoblast-only environment. MATERIALS AND METHODS: Here, we investigated the effects of macrophages and dendritic cells on osteogenic differentiation in a co-culture environment involving human periosteal cell-derived osteoblasts, human monocyte-derived osteoclasts, and myeloid-derived cells. In addition, the cluster of myeloid immune cells involved in the clinical bone regeneration process was analyzed through bone defect rat modeling. KEY FINDINGS: We found that specific types of myeloid cells and related cytokines increased osteogenic differentiation. These results were confirmed in experiments using myeloid cells originating from human primitive peripheral blood mononuclear cells and by measuring the colonization of macrophages and dendritic cells in an in vivo bone defect environment. In addition, Next generation sequencing (NGS) analysis was performed through RNA sequencing for osteogenesis caused by macrophages and dendritic cells in vitro, which implemented a clinical bone regeneration environment. The results of these experiments suggest that the role of M2 macrophages or dendritic cells is markedly increased during osteogenic differentiation. Therefore, we propose that the exchange of bioactive factors between macrophages and dendritic cells during the bone formation metabolic process is a crucial step of tissue regeneration rather than limited to the initial inflammatory response. SIGNIFICANCE: This study indicates that M2 macrophages, among myeloid cells, can be mediators that play a vital role in the effective bone regeneration process and shows the potential as a useful next-generation advanced cell therapy for bone regeneration treatment.

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Bone morphogenetic proteins (BMPs) have tremendous therapeutic potential regarding the treatment of bone and musculoskeletal disorders due to their osteo-inductive ability. More than twenty BMPs have been identified in the human body with various functions, such as embryonic development, skeleton genesis, hematopoiesis, and neurogenesis. BMPs can induce the differentiation of MSCs into the osteoblast lineage and promote the proliferation of osteoblasts and chondrocytes. BMP signaling is also involved in tissue remodeling and regeneration processes to maintain homeostasis in adults. In particular, growth factors, such as BMP-2 and BMP-7, have already been approved and are being used as treatments, but it is unclear as to whether they are the most potent BMPs that induce bone formation. According to recent studies, BMP-9 is known to be the most potent inducer of the osteogenic differentiation of mesenchymal stem cells, both in vitro and in vivo. However, its exact role in the skeletal system is still unclear. In addition, research results suggest that the molecular mechanism of BMP-9-mediated bone formation is also different from the previously known BMP family, suggesting that research on signaling pathways related to BMP-9-mediated bone formation is actively being conducted. In this study, we performed a phosphorylation array to investigate the signaling mechanism of BMP-9 compared with BMP-2, another influential bone-forming growth factor, and we compared the downstream signaling system. We present a mechanism for the signal transduction of BMP-9, focusing on the previously known pathway and the p53 factor, which is relatively upregulated compared with BMP-2.

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Extrusion-based bioprinting technology is widely used for tissue regeneration and reconstruction. However, the method that uses only hydrogel as the bioink base material exhibits limited biofunctional properties and needs improvement to achieve the desired tissue regeneration. In this study, we present a three-dimensionally printed bioactive microparticle-loaded scaffold for use in bone regeneration applications. The unique structure of the microparticles provided sustained release of growth factor for > 4 weeks without the use of toxic or harmful substances. Before and after printing, the optimal particle ratio in the bioink for cell viability demonstrated a survival rate of ≥ 85% over 7 days. Notably, osteogenic differentiation and mineralization-mediated by human periosteum-derived cells in scaffolds with bioactive microparticles-increased over a 2-week interval. Here, we present an alternative bioprinting strategy that uses the sustained release of bioactive microparticles to improve biofunctional properties in a manner that is acceptable for clinical bone regeneration applications.

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PURPOSE: The purpose of this study was to determine the significance of hinge position through comparison between open-wedge and closed-wedge high tibial osteotomy (HTO) and to determine the ideal hinge position to minimize the effect of HTO on the posterior tibial slope (PTS) and medial proximal tibial angle (MPTA). METHODS: Procedures were performed on 32 cadaveric knees using open-wedge HTO with the standard hinge position or a low hinge position or closed-wedge HTO with the standard hinge position or a low hinge position. To define the standard hinge position in open wedge HTO, we drew a line 3-cm inferior to the medial tibial plateau toward the fibular head and located the intersection of this line with a longitudinal line 1-cm medial to fibular shaft. The low hinge position was then defined as the point 1-cm inferior to the standard position. For the standard hinge position for closed-wedge HTO, we drew a line parallel with joint line from 2-cm inferior to the lateral tibial plateau. The low hinge position was then defined as the point 1-cm inferior to the standard position. RESULTS: For the open-wedge procedure, osteotomy through the low hinge position resulted in a significantly greater PTS compared to osteotomy through the standard hinge position. MPTA was also significantly greater for the low hinge position compared to standard hinge position. In the closed-wedge HTO, neither the PTS nor MPTA was significantly different for the low and standard hinge positions. CONCLUSIONS: Hinge position significantly affects changes in the PTS and MPTA following open-wedge but not closed-wedge HTO. Understanding how to hinge position affects the PTS and MPTA is critical for surgeons performing open-wedge HTO procedures. Adopting an accurate hinge position is crucial for preventing complications, especially in open-wedge osteotomy, due to postoperative changes in the PTS and MPTA.

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Tiron is a potent antioxidant that counters the pathological effects of reactive oxygen species (ROS) production due to oxidative stress in various cell types. We examined the effects of tiron on mitochondrial function and osteoblastic differentiation in human periosteum-derived cells (hPDCs). Tiron increased mitochondrial activity and decreased senescence-associated ß-galactosidase activity in hPDCs; however, it had a detrimental effect on osteoblastic differentiation by reducing alkaline phosphatase (ALP) activity and alizarin red-positive mineralization, regardless of H2O2 treatment. Osteoblast-differentiating hPDCs displayed increased ROS production compared with non-differentiating hPDCs, and treatment with tiron reduced ROS production in the differentiating cells. Antioxidants decreased the rates of oxygen consumption and ATP production, which are increased in hPDCs during osteoblastic differentiation. In addition, treatment with tiron reduced the levels of most mitochondrial proteins, which are increased in hPDCs during culture in osteogenic induction medium. These results suggest that tiron exerts negative effects on the osteoblastic differentiation of hPDCs by causing mitochondrial dysfunction.

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Even though bony defects can be recovered to their original condition with full functionality, critical-sized bone injuries continue to be a challenge in clinical fields due to deficiencies in the scaffolding matrix and growth factors at the injury region. In this study, we prepared bone morphogenetic protein-2 (BMP-2)-loaded porous particles as a bioactive bone graft for accelerated bone regeneration. The porous particles with unique leaf-stacked morphology (LSS particles) were fabricated by a simple cooling procedure of hot polycaprolactone (PCL) solution. The unique leaf-stacked structure in the LSS particles provided a large surface area and complex release path for the sufficient immobilization of BMP-2 and sustained release of BMP-2 for 26 days. The LSS was also recognized as a topographical cue for cell adhesion and differentiation. In in vitro cell culture and in vivo animal study using a canine mandible defect model, BMP-2-immobilized LSS particles provided a favorable environment for osteogenic differentiation of stem cells and bone regeneration. In vitro study suggests a dual stimulus of bone mineral-like (leaf-stacked) structure (a physical cue) and continuously supplied BMP-2 (a biological cue) to be the cause of this improved healing outcome. Thus, LSS particles containing BMP-2 can be a promising bioactive grafting material for effective new bone formation.

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Human interleukin-15 (hIL-15) has attracted a considerable attention as a promising cancer immunotherapeutic due to its function to directly stimulate the proliferation and cytotoxic activity of NK and T cells. Nevertheless, a relatively short half-life of hIL-15 requires repeated administration and higher doses, causing serious side effects. Here, we demonstrate an enhanced blood half-life and biological activity of hIL-15 through genetic fusion of a human serum albumin-specific protein binder (rHSA). The fusion construct (rHSA-IL15) was observed to maintain respective binding activities for both hIL-15 receptor α and human serum albumin. The rHSA-IL15 led to a significant increase in the secretion of Granzyme B and INF-γ by immune cells compare to free hIL-15, expanding the population of activated T cell subset such as CD4 + T and CD8+ T cells. The terminal half-life of the rHSA-IL15 was prolonged by around a 40-fold in transgenic mice expressing human serum albumin, compared to free hIL-15. The rHSA-IL15 resulted in distinct anti-tumor activities in xenograft SCC (squamous cell carcinoma) mouse and allograft melanoma mouse models through activation of NK and CD8+ T cells. The rHSA-IL15 is expected to be used in cancer immunotherapy, assisting in the development of other cytokines as immunotherapeutic agents with greater efficacy.

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Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.

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It is accepted that biomimetic supply of signaling molecules during bone regeneration can provide an appropriate environment for accelerated new bone formation. In this study, we developed a growth factor delivery system based on porous particles and a thermosensitive hydrogel that allowed fast, continuous, and delayed/continuous release of growth factors to mimic their biological production during bone regeneration. It was observed that the Continuous group (continuous release of growth factors) provides a better environment for the osteogenic differentiation of hPDCs than the Biomimetic group (biomimetic release of growth factors), and thus is anticipated to promote bone regeneration. However, contrary to expectation, the Biomimetic group promoted significant new bone formation compared to the Continuous group. From the systematic cell culture experiments, the initial supply of VEGF was considered to have more favorable effects on the osteoclastogenesis than osteogenesis, which may hinder bone regeneration. Our results indicated that the continuous supply of VEGF (in particular, at early stage) from VEGF-loaded biomaterial might not be conducive to new bone formation. Therefore, we suggest that a biomimetic supply of growth factors is a more pivotal parameter for sufficient tissue regeneration. Its use as a molecular delivery system may also serve as a useful tool for the investigation of biological processes and molecules during tissue regeneration processes.

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3D printed scaffolds composed of gelatin and ß-tri-calcium phosphate (ß-TCP) as a biomimetic bone material are fabricated, thereby providing an environment appropriate for bone regeneration. The Ca2+ in ß-TCP and COO- in gelatin form a stable electrostatic interaction, and the composite scaffold shows suitable rheological properties for bioprinting. The gelatin/ß-TCP scaffold is crosslinked with glutaraldehyde vapor and unreacted aldehyde groups which can cause toxicity to cells is removed by a glycine washing. The stable binding of the hydrogel is revealed as a result of FTIR and degradation rate. It is confirmed that the composite scaffold has compressive strength similar to that of cancellous bone and 60 wt% ß-TCP groups containing 40 wt% gelatin have good cellular activity with preosteoblasts. Also, in the animal experiments, the gelatin/ß-TCP scaffold confirms to induce bone formation without any inflammatory responses. This study suggests that these fabricated scaffolds can serve as a potential bone substitute for bone regeneration.

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Although biological therapies based on growth factors and transplanted cells have demonstrated some positive outcomes for intervertebral disc (IVD) regeneration, repeated injection of growth factors and cell leakage from the injection site remain considerable challenges for human therapeutic use. Herein, we prepare human bone marrow-derived mesenchymal stem cells (hBMSCs) and transforming growth factor-ß3 (TGF-ß3)-loaded porous particles with a unique leaf-stack structural morphology (LSS particles) as a combination bioactive delivery matrix for degenerated IVD. The LSS particles are fabricated with clinically acceptable biomaterials (polycaprolactone and tetraglycol) and procedures (simple heating and cooling). The LSS particles allow sustained release of TGF-ß3 for 18 days and stable cell adhesiveness without additional modifications of the particles. On the basis of in vitro and in vivo studies, it was observed that the hBMSCs/TGF-ß3-loaded LSS particles can provide a suitable milieu for chondrogenic differentiation of hBMSCs and effectively induce IVD regeneration in a beagle dog model. Thus, therapeutically loaded LSS particles offer the promise of an effective bioactive delivery system for regeneration of various tissues including IVD.

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Nuclear factor kappa B (NF-κB) regulates inflammatory gene expression and represents a likely target for novel disease treatment approaches, including skeletal disorders. Several plant-derived sesquiterpene lactones can inhibit the activation of NF-κB. Parthenolide (PTL) is an abundant sesquiterpene lactone, found in Mexican Indian Asteraceae family plants, with reported anti-inflammatory activity, through the inhibition of a common step in the NF-κB activation pathway. This study examined the effects of PTL on the enhanced, in vitro, osteogenic phenotypes of human periosteum-derived cells (hPDCs), mediated by the inflammatory cytokine tumor necrosis factor (TNF)-α. PTL had no significant effects on hPDC viability or osteoblastic activities, whereas TNF-α had positive effects on the in vitro osteoblastic differentiation of hPDCs. c-Jun N-terminal kinase (JNK) signaling played an important role in the enhanced osteoblastic differentiation of TNF-α-treated hPDCs. Treatment with 1 µM PTL did not affect TNF-α-treated hPDCs; however, 5 and 10 µM PTL treatment decreased the histochemical detection and activity of alkaline phosphatase (ALP), alizarin red-positive mineralization, and the expression of ALP and osteocalcin mRNA. JNK phosphorylation decreased significantly in TNF-α-treated hPDCs pretreated with PTL. These results suggested that PTL exerts negative effects on the increased osteoblastic differentiation of TNF-α-treated hPDCs by inhibiting JNK signaling.

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BACKGROUND: Osteoporosis is a metabolic bone disorder that leads to low bone mass and microstructural deterioration of bone tissue and increases bone fractures. Resveratrol, a natural polyphenol compound, has pleiotropic effects including anti-oxidative, anti-aging, and anti-cancer effects. Resveratrol also has roles in increasing osteogenesis and in upregulating mitochondrial biogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is still unclear that resveratrol can enhance osteogenic differentiation or mitochondrial biogenesis of periosteum-derived MSCs (PO-MSCs), which play key roles in bone tissue maintenance and fracture healing. Thus, in order to test a possible preventive or therapeutic effect of resveratrol on osteoporosis, this study investigated the effects of resveratrol treatments on osteogenic differentiation and mitochondrial biogenesis of PO-MSCs. METHODS: The optimal doses of resveratrol treatment on PO-MSCs were determined by cell proliferation and viability assays. Osteogenic differentiation of PO-MSCs under resveratrol treatment was assessed by alkaline phosphatase activities (ALP, an early biomarker of osteogenesis) as well as by extracellular calcium deposit levels (a late biomarker). Mitochondrial biogenesis during osteogenic differentiation of PO-MSCs was measured by quantifying both mitochondrial mass and mitochondrial DNA (mtDNA) contents. RESULTS: Resveratrol treatments above 10 µM seem to have negative effects on cell proliferation and viability of PO-MSCs. Resveratrol treatment (at 5 µM) on PO-MSCs during osteogenic differentiation increased both ALP activities and calcium deposits compared to untreated control groups, demonstrating an enhancing effect of resveratrol on osteogenesis. In addition, resveratrol treatment (at 5 µM) during osteogenic differentiation of PO-MSCs increased both mitochondrial mass and mtDNA copy numbers, indicating that resveratrol can bolster mitochondrial biogenesis in the process of PO-MSC osteogenic differentiation. CONCLUSION: Taken together, the findings of this study describe the roles of resveratrol in promoting osteogenesis and mitochondrial biogenesis of human PO-MSCs suggesting a possible application of resveratrol as a supplement for osteoporosis and/or osteoporotic fractures.

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The purpose of this study is to develop a bioactive bone graft based on polycaprolactone (PCL, synthetic polymer; used in clinical practices as a grafting material for craniofacial bone defects) and hyaluronic acid (HA, bioactive natural polymer; known as a promoting substance for bone regeneration) that would be fabricated by clinically available procedures (mild condition without toxic chemicals) and provide bioactivity for sufficient period, and thus effectively induce bone reconstruction. For this, PCL/HA hybrid microspheres were produced by a spray-precipitation technique using clinically adapted solvents. The HA was stably and evenly entrapped in the PCL/HA hybrid microspheres. It was demonstrated that the PCL/HA hybrid microspheres provide an appropriate environment for proliferation and osteogenic differentiation of human periosteum-derived cells (hPDCs) (in vitro) and allow significantly enhanced bone regeneration (in vivo) compared with PCL microspheres without HA. The PCL/HA hybrid microspheres can be a simple but clinically applicable bioactive bone graft for large-sized bone defects.

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The ultimate purpose of this study was to develop a bioactive filler system that would allow volume restoration (passive property) and continuous release of signaling molecules to recruit soft tissues (bioactive property) and thus effectively correct facial aging. To achieve this, we prepared porous particles with a leaf-stacked structure throughout the entire particle volume (LSS particles) using a simple heating-cooling technique. LSS particles were loaded with insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) separately, by immersing the particles in signaling molecule-containing solutions for target tissue recruitment (adipose by IGF-1 and blood vessels by VEGF). IGF-1 and VEGF were continuously released from LSS particles for 28 and 21 days in vitro, respectively, even without additional chemical/physical modifications, because of the unique morphology of the particles. Signaling molecules preserved their bioactivity in vitro (induction of adipogenic and angiogenic differentiation) and in vivo (recruitment of fat and blood vessels) for a sufficient period. Moreover, it was observed that the LSS particles themselves have stable volume retention characteristics in the body. Thus, we suggest that the signaling molecule-loaded LSS particles can function as a bioactive filler system for volume retention and target tissue regeneration.

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Due to a rapidly expanding aging population, the incidence of age-related or degenerative diseases has increased, and efforts to handle the issue with regenerative medicine via adult stem cells have become more important. And it is now clear that the mitochondrial energy metabolism is important for stem cell differentiation. When stem cells commit to differentiate, glycolytic metabolism is being shifted to mitochondrial oxidative phosphorylation (OXPHOS) to meet an increased cellular energy demand required for differentiated cells. However, the nature of cellular metabolisms during the differentiation process of periosteum-derived mesenchymal stem cells (POMSC) is still unclear. In the present study, we investigated mitochondrial biogenesis during the adipogenic, chondrogenic, and osteogenic differentiation of POMSCs. Both mitochondrial DNA (mtDNA) contents and mitochondrial proteins (VDAC and mitochondrial OXPHOS complex subunits) were increased during all of these mesenchymal lineage differentiations of POMSCs. Interestingly, glycolytic metabolism is reduced as POMSCs undergo osteogenic differentiation. Furthermore, reducing mtDNA contents by ethidium bromide treatments prevents osteogenic differentiation of POMSCs. In conclusion, these results indicate that mitochondrial biogenesis and OXPHOS metabolism play important roles in the differentiation of POMCS and suggest that pharmaceutical modulation of mitochondrial biogenesis and/or function can be a novel regulation for POMSC differentiation and regenerative medicine.

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Background: Despite the development of progressive surgical techniques and antibiotics, osteomyelitis is a big challenge for orthopedic surgeons. The main aim of this study is to fabricate an in situ gelling hydrogel that permits sustained release of antibiotic (for control of infection) and growth factor (for induction of new bone formation) for effective treatment of osteomyelitis. Methods: An in situ gelling alginate (ALG)/hyaluronic acid (HA) hydrogel containing vancomycin (antibiotic) and bone morphogenetic protein-2 (BMP-2; growth factor) was prepared by simple mixing of ALG/HA/Na2HPO4 solution and CaSO4/vancomycin/BMP-2 solution. The release behaviors of vancomycin and BMP-2, anti-bacterial effect (in vitro); and therapeutic efficiency for osteomyelitis and bone regeneration (in vivo, osteomyelitis rat model) of the vancomycin and BMP-2-incorporated ALG/HA hydrogel were investigated. Results: The gelation time of the ALG/HA hydrogel was controlled into approximately 4 min, which is sufficient time for handling and injection into osteomyelitis lesion. Both vancomycin and BMP-2 were continuously released from the hydrogel for 6 weeks. From the in vitro studies, the ALG/HA hydrogel showed an effective anti-bacterial activity without significant cytotoxicity for 6 weeks. From an in vivo animal study using Sprague-Dawley rats with osteomyelitis in femur as a model animal, it was demonstrated that the ALG/HA hydrogel was effective for suppressing bacteria (Staphylococcus aureus) proliferation at the osteomyelitis lesion and enhancing bone regeneration without additional bone grafts. Conclusions: From the results, we suggest that the in situ gelling ALG/HA hydrogel containing vancomycin and BMP-2 can be a feasible therapeutic tool to treat osteomyelitis.

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Background: Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, three-dimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional mono-layer cultured MSCs. Methods: In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties. Results: In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis. Conclusion: To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.

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