The role of lysophosphatidylcholine acyltransferase 2 in osteoblastic differentiation of C2C12 cells.

Glycerophospholipids, a primary component of cellular membranes, play important structural and functional roles in cells. In the remodelling pathway (Lands' cycle), the concerted actions of phospholipase As and lysophospholipid acyltransferases (LPLATs) contribute to the incorporation of diverse fatty acids in glycerophospholipids in an asymmetric manner, which differ between cell types. In this study, the role of LPLATs in osteoblastic differentiation of C2C12 cells was investigated. Gene and protein expression levels of lysophosphatidylcholine acyltransferase 2 (LPCAT2), one of the LPLATs, increased during osteoblastic differentiation in C2C12 cells. LPCAT2 knockdown in C2C12 cells downregulated the expression of osteoblastic differentiation markers and the number and size of lipid droplets (LDs) and suppressed the phosphorylation of Smad1/5/9. In addition, LPCAT2 knockdown inhibited Snail1 and the downstream target of Runx2 and vitamin D receptor (VDR). These results suggest that LPCAT2 modulates osteoblastic differentiation in C2C12 cells through the bone morphogenetic protein (BMP)/Smad signalling pathway.

Brain-Derived Neurotrophic Factor (BDNF) Enhances Osteogenesis and May Improve Bone Microarchitecture in an Ovariectomized Rat Model.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) has gained attention as a therapeutic agent due to its potential biological activities, including osteogenesis. However, the molecular mechanisms involved in the osteogenic activity of BDNF have not been fully understood. This study aimed to investigate the action of BDNF on the osteoblast differentiation in bone marrow stromal cells, and its influence on signaling pathways. In addition, to evaluate the clinical efficacy, an in vivo animal study was performed. METHODS: Preosteoblast cells (MC3T3-E1), bone marrow-derived stromal cells (ST2), and a direct 2D co-culture system were treated with BDNF. The effect of BDNF on cell proliferation was determined using the CCK-8 assay. Osteoblast differentiation was assessed based on alkaline phosphatase (ALP) activity and staining and the protein expression of multiple osteoblast markers. Calcium accumulation was examined by Alizarin red S staining. For the animal study, we used ovariectomized Sprague-Dawley rats and divided them into BDNF and normal saline injection groups. MicroCT, hematoxylin and eosin (H&E), and tartrate-resistant acid phosphatase (TRAP) stain were performed for analysis. RESULTS: BDNF significantly increased ALP activity, calcium deposition, and the expression of osteoblast differentiation-related proteins, such as ALP, osteopontin, etc., in both ST-2 and the MC3T3-E1 and ST-2 co-culture systems. Moreover, the effect of BDNF on osteogenic differentiation was diminished by blocking tropomyosin receptor kinase B, as well as inhibiting c-Jun N-terminal kinase and p38 MAPK signals. Although the animal study results including bone density and histology showed increased osteoblastic and decreased osteoclastic activity, only a portion of parameters reached statistical significance. CONCLUSIONS: Our study results showed that BDNF affects osteoblast differentiation through TrkB receptor, and JNK and p38 MAPK signal pathways. Although not statistically significant, the trend of such effects was observed in the animal experiment.

Porcine Mandibular Bone Marrow-Derived Mesenchymal Stem Cell (BMSC)-Derived Extracellular Vesicles Can Promote the Osteogenic Differentiation Capacity of Porcine Tibial-Derived BMSCs.

OBJECTIVE: Existing research suggests that bone marrow-derived mesenchymal stem cells (BMSCs) may promote endogenous bone repair. This may be through the secretion of factors that stimulate repair processes or directly through differentiation into osteoblast-progenitor cells. However, the osteogenic potential of BMSCs varies among different tissue sources (e.g., mandibular versus long BMSCs). The main aim of this study was to investigate the difference in osteogenic differentiation capacity between mandibular BMSCs (mBMSCs) and tibial BMSCs (tBMSCs). MATERIALS AND METHODS: Bioinformatics analysis of the GSE81430 dataset taken from the Gene Expression Omnibus (GEO) database was performed using GEO2R. BMSCs were isolated from mandibular and tibial bone marrow tissue samples. Healthy pigs (n = 3) (registered at the State Office for Nature, Environment, and Consumer Protection, North Rhine-Westphalia (LANUV) 81-02.04.2020.A215) were used for this purpose. Cell morphology and osteogenic differentiation were evaluated in mBMSCs and tBMSCs. The expression levels of toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively. In addition, mBMSC-derived extracellular vesicles (mBMSC-EVs) were gained and used as osteogenic stimuli for tBMSCs. Cell morphology and osteogenic differentiation capacity were assessed after mBMSC-EV stimulation. RESULTS: Bioinformatic analysis indicated that the difference in the activation of the TLR4/NF-κB pathway was more pronounced compared to all other examined genes. Specifically, this demonstrated significant downregulation, whereas only 5-7 upregulated genes displayed significant variances. The mBMSC group showed stronger osteogenic differentiation capacity compared to the tBMSC group, confirmed via ALP, ARS, and von Kossa staining. Furthermore, qPCR and WB analysis revealed a significant decrease in the expression of the TLR4/NF-κB pathway in the mBMSC group compared to the tBMSC group (TLR4 fold changes: mBMSCs vs. tBMSCs p < 0.05; NF-κB fold changes: mBMSCs vs. tBMSCs p < 0.05). The osteogenic differentiation capacity was enhanced, and qPCR and WB analysis revealed a significant decrease in the expression of TLR4 and NF-κB in the tBMSC group with mBMSC-EVs added compared to tBMSCs alone (TLR4 fold changes: p < 0.05; NF-κB fold changes: p < 0.05). CONCLUSION: Our results indicate that mBMSC-EVs can promote the osteogenic differentiation of tBMSCs in vitro. The results also provide insights into the osteogenic mechanism of mBMSCs via TLR4/NF-κB signaling pathway activation. This discovery promises a fresh perspective on the treatment of bone fractures or malunions, potentially offering a novel therapeutic method.

SIRT6-mediated Runx2 downregulation inhibits osteogenic differentiation of human aortic valve interstitial cells in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a progressive cardiovascular disorder involving multiple pathogenesis. Effective pharmacological therapies are currently unavailable. Sirtuin6 (SIRT6) has been shown to protect against aortic valve calcification in CAVD. The exact regulatory mechanism of SIRT6 in osteoblastic differentiation remains to be determined, although it inhibits osteogenic differentiation of aortic valve interstitial cells. We demonstrated that SIRT6 was markedly downregulated in calcific human aortic valves. Mechanistically, SIRT6 suppressed osteogenic differentiation in human aortic valve interstitial cells (HAVICs), as confirmed by loss- and gain-of-function experiments. SIRT6 directly interacted with Runx2, decreased Runx2 acetylation levels, and facilitated Runx2 nuclear export to inhibit the osteoblastic phenotype transition of HAVICs. In addition, the AKT signaling pathway acted upstream of SIRT6. Together, these findings elucidate that SIRT6-mediated Runx2 downregulation inhibits aortic valve calcification and provide novel insights into therapeutic strategies for CAVD.

CRP inhibits the osteoblastic differentiation of OPCs via the up-regulation of primary cilia and repression of the Hedgehog signaling pathway.

Inflammation disrupts bone metabolism and leads to bone damage. C-reactive protein (CRP) is a typical inflammation marker. Although CRP measurement has been conducted for many decades, how osteoblastic differentiation influences molecular mechanisms remains largely unknown. The present study attempted to investigate the effects of CRP on primary cultured osteoblast precursor cells (OPCs) while elucidating the underlying molecular mechanisms. OPCs were isolated from suckling Sprague-Dawleyrats. Fewer OPCs were observed after recombinant C-reactive protein treatment. In a series of experiments, CRP inhibited OPC proliferation, osteoblastic differentiation, and the OPC gene expression of the hedgehog (Hh) signaling pathway. The inhibitory effect of CRP on OPC proliferation occurred via blockade of the G1-S transition of the cell cycle. In addition, the regulation effect of proto cilium on osteoblastic differentiation was analyzed using the bioinformatics p. This revealed the primary cilia activation of recombinant CRP effect on OPCs through in vitro experiments. A specific Sonic Hedgehog signaling agonist (SAG) rescued osteoblastic differentiation inhibited by recombinant CRP. Moreover, chloral hydrate, which removes primary cilia, inhibited the Suppressor of Fused (SUFU) formation and blocked Gli2 degradation. This counteracted osteogenesis inhibition caused by CRP. Therefore, these data depict that CRP can inhibit the proliferation and osteoblastic differentiation of OPCs. The underlying mechanism could be associated with primary cilia activation and Hh pathway repression.

FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis.

Bone defects have remained a clinical problem in current orthopedics. Bone marrow mesenchymal stem cells (BM-MSCs) with multi-directional differentiation ability have become a research hotspot for repairing bone defects. In vitro and in vivo models were constructed, respectively. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to detect osteogenic differentiation ability. Western blotting (WB) was used to detect the expression of osteogenic differentiation-related proteins. Serum inflammatory cytokine levels were detected by ELISA. Fracture recovery was evaluated by HE staining. The binding relationship between FOXC1 and Dnmt3b was verified by dual-luciferase reporter assay. The relationship between Dnmt3b and CXCL12 was explored by MSP and ChIP assays. FOXC1 overexpression promoted calcium nodule formation, upregulated osteogenic differentiation-related protein expression, promoted osteogenic differentiation, and decreased inflammatory factor levels in BM-MSCs, and promoted callus formation, upregulated osteogenic differentiation-related protein expression, and downregulated CXCL12 expression in the mouse model. Furthermore, FOXC1 targeted Dnmt3b, with Dnmt3b knockdown decreasing calcium nodule formation and downregulating osteogenic differentiation-related protein expression. Additionally, inhibiting Dnmt3b expression upregulated CXCL12 protein expression and inhibited CXCL12 methylation. Dnmt3b could be binded to CXCL12. CXCL12 overexpression attenuated the effects of FOXC1 overexpression and inhibited BM-MSCs osteogenic differentiation. This study confirmed that the FOXC1-mediated regulation of the Dnmt3b/CXCL12 axis had positive effects on the osteogenic differentiation of BM-MSCs.

Dehydrocostus lactone (DHC) promotes osteoblastic differentiation and mineralization through p38/RUNX-2 signaling.

Dysregulation of osteoblastic differentiation is an important risk factor of osteoporosis, the therapy of which is challenging. Dehydrocostus lactone (DHC), a sesquiterpene isolated from medicinal plants, has displayed anti-inflammatory and antitumor properties. In this study, we investigated the effects of DHC on osteoblastic differentiation and mineralization of MC3T3-E1 cells. Interestingly, we found that DHC increased the expression of marker genes of osteoblastic differentiation, such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Additionally, DHC increased the expressions of collagen type I alpha 1 (Col1a1) and collagen type I alpha 2 (Col1a2). We also demonstrate that DHC increased ALP activity. Importantly, the Alizarin Red S staining assay revealed that DHC enhanced osteoblastic differentiation of MC3T3-E1 cells. Mechanistically, it is shown that DHC increased the expression of Runx-2, a central regulator of osteoblastic differentiation. Treatment with DHC also increased the levels of phosphorylated p38, and its blockage using its specific inhibitor SB203580 abolished the effects of DHC on runt-related transcription factor 2 (Runx-2) expression and osteoblastic differentiation, suggesting the involvement of p38. Based on these findings, we concluded that DHC might possess a capacity for the treatment of osteoporosis by promoting osteoblastic differentiation.

Pro-osteogenic role of interleukin-22 in calcific aortic valve disease.

BACKGROUND AND AIMS: Although calcific aortic valve disease (CAVD) is a common valvular disease among elderly populations and its incidence has markedly increased in recent decades, the pathogenesis of CAVD remains unclear. In this study, we explored the potential role of interleukin (IL)-22 and the underlying molecular mechanism in CAVD. METHODS AND RESULTS: Our results showed that IL-22 was upregulated in calcific aortic valves from CAVD patients, and its main sources were CD3+ T cells and CD68+ macrophages. Human aortic valve interstitial cells (VICs) expressed the IL-22-specific receptor IL-22R1, and IL-22R1 expression also was elevated in calcified valves. Treatment of cultured human VICs with recombinant human IL-22 resulted in markedly increased expression of osteogenic proteins Runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), as well as increased matrix calcium deposition. Moreover, siRNA silencing of IL-22R1 blocked the pro-osteogenic effect of IL-22 in VICs. In IL-22-treated VICs, we also observed increased phosphorylation of JAK3 and STAT3 and nuclear translocation of STAT3. Pretreatment with a specific JAK3 inhibitor, WHIP-154, or siRNA knockout of STAT3 effectively mitigated the IL-22-induced osteoblastic trans-differentiation of human VICs. CONCLUSIONS: Together, these data indicate that IL-22 promotes osteogenic differentiation of VICs by activating JAK3/STAT3 signaling. Based on our results demonstrating a pro-osteogenic role of IL-22 in human aortic valves, pharmacological inhibition of IL-22 signaling may represent a potential strategy for alleviating CAVD.

MiR-224-5p inhibits osteoblast differentiation and impairs bone formation by targeting Runx2 and Sp7.

Osteoporosis is a complicated multifactorial disorder characterized by low bone mass and deteriorated bone microarchitecture with an elevated fracture risk. MicroRNAs play important roles in osteoblastic differentiation. In the present study, we found that miR-224-5p was markedly downregulated during the osteogenic differentiation of C2C12 cells. Overexpression of miR-224-5p in C2C12 cells inhibited osteoblast activity, as indicated by reduced ALP activity, matrix mineralization and the expression of osteogenic marker genes. Moreover, we demonstrated that Runx2 and Sp7 were direct targets of miR-224-5p. Furthermore, the specific inhibition of miR-224-5p by femoral bone marrow cavity injection with miR-224-5p antagomir prevented ovariectomy-induced bone loss. Finally, we found that the levels of miR-224-5p were markedly elevated in the sera of patients with osteoporosis. Collectively, this study revealed that miR-224-5p negatively regulates osteogenic differentiation by targeting Runx2 and Sp7. It also highlights the potential use of miR-224-5p as a therapeutic target and diagnostic biomarker for osteoporosis. Supplementary information: The online version contains supplementary material available at 10.1007/s10616-023-00593-z.

Tanshinone IIA attenuates valvular interstitial cells' calcification induced by oxidized low density lipoprotein via reducing endoplasmic reticulum stress.

Recent studies revealed that endoplasmic reticulum (ER) stress played an emerging role of in valve calcification. Tanshinone IIA (TanIIA) has been a research hotspot in cardiovascular diseases. Previously we found that sodium TanIIA dampened the pathological phenotype transition of valvular interstitial cells (VICs) by affecting ER stress published in Chinese Journal. Here, we test the hypothesis that TanIIA attenuates the pro-osteogenic effects of oxidized low-density lipoprotein (oxLDL) in VICs by reducing induction of ER stress. Patients' aortic valve (AV) was collected, and porcine VICs were cultured for in vitro model. ER stress markers were tested in human leaflets by immunostaining. Immunoblotting were used to test the osteoblastic factors such as Runx2, osteocalcin, and ER stress markers GRP78, CHOP, XBP1, etc. Alkakine phosphate (ALP) activity assay were used to test the activity of ALP kinase. Pro-inflammatory gene expression was detected by polymerase chain reaction. As a result, ER stress markers were elevated in patients' calcified AVs. OxLDL induced osteogenesis and inflammation via promoting ER stress. TanIIA attenuated oxLDL induced ER stress. TanIIA also inhibited theosteoblastic factors and inflammatory cytokine expressions in VICs. In conclusion, our data provide evidence that TanIIA exerts anti-inflammation and anti-osteogenic effects in VICs by attenuating ER stress, and ER stress acts as an important regulator in oxLDL induced VICs' phenotype transition.

Arl4c is involved in tooth germ development through osteoblastic/ameloblastic differentiation.

Murine tooth germ development proceeds in continuous sequential steps with reciprocal interactions between the odontogenic epithelium and the adjacent mesenchyme, and several growth factor signaling pathways and their activation are required for tooth germ development. The expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby involved in epithelial morphogenesis and tumorigenesis. In contrast, the other functions of Arl4c (in addition to cellular growth) are largely unknown. Although we recently demonstrated the involvement of the upregulated expression of Arl4c in the proliferation of ameloblastomas, which have the same origin as odontogenic epithelium, its effect on tooth germ development remains unclear. In the present study, single-cell RNA sequencing (scRNA-seq) analysis revealed that the expression of Arl4c, among 17 members of the Arf-family, was specifically detected in odontogenic epithelial cells, such as those of the stratum intermedium, stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in non-ameloblast and inner enamel epithelium, which include immature cells, of P7 mouse incisors. In the mouse tooth germ rudiment culture, treatment with SecinH3 (an inhibitor of the ARNO/Arf6 pathway) reduced the size, width and cusp height of the tooth germ and the thickness of the eosinophilic layer, which would involve the synthesis of dentin and enamel matrix organization. In addition, loss-of-function experiments using siRNAs and shRNA revealed that the expression of Arl4c was involved in cell proliferation and osteoblastic cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis with a gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that osteoblastic differentiation-related gene sets and/or GO terms were downregulated in shArl4c-expressing odontogenic epithelial cells. These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes to tooth germ development through osteoblastic/ameloblastic differentiation.

CARM1 deficiency inhibits osteoblastic differentiation of bone marrow mesenchymal stem cells and delays osteogenesis in mice.

Bone repair remains a clinical challenge due to low osteogenic capacity. Coactivator associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that mediates arginine methylation and endochondral ossification. However, the roles of CARM1 in osteoblastic differentiation and bone remodeling have not been explored. In our study, heterozygous CARM1-knockout (KO) mice were generated using the CRISPR-Cas9 system and a model of femoral defect was created. At day 7 postsurgery, CARM1-KO mice exhibited obvious bone loss compared with wild type (WT) mice, as evidenced by reduced bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Deletion of CARM1 in mice lowered synthesis and accumulation of collagen at the injury sites. The alkaline phosphatase (ALP) activity and osteogenic-related gene expression were declined in CARM1-KO mice. To further understand the role of CARM1 in osteoblastic differentiation, bone marrow mesenchymal stem cells (BMSCs) were isolated from the tibia and femur of WT or CARM1-KO mice. CARM1 deletion decreased histone arginine methylation and inhibited osteoblastic differentiation and mineralization. The mRNA sequencing of CARM1-KO BMSCs revealed the possible regulatory molecules by CARM1, which could deepen our understanding of CARM1 regulatory mechanisms. These data could be of interest to basic researchers and provide the direction for future research into bone-related disorders.

A nonobstructive condition: Medial arterial calcification. / ä¸å¼•èµ·ç®¡è ”ç‹­çª„çš„è¡€ç®¡ä¸­è†œé’™åŒ–.

Vascular calcification, including intimal and medial calcification, is closely associated with a significant increase in cardiovascular diseases. Although increased understandings were achieved, people still know much more about intimal calcification than medial calcification because the latter doesn't obstruct the arterial lumen, commonly considered as a non-significant finding. We clarified the pathologic characteristic of medial calcification, its difference from intimal calcification, principally focused on its clinical relevance, such as diagnosis, nosogenesis, and hemodynamics. We underline the importance of identifying and distinguishing medial calcification, understanding its effect to local/systematic arterial compliance, and relationship to diabetic neuropathy. Recent studies emphasize do not ignore its predictive role in cardiovascular mortality. It is of great clinical significance to summarize the mechanisms of occurrence, lesion characteristics, diagnostic methods, pathogenic mechanisms, hemodynamic changes, and the distinction as well as association of intimal calcification with intimal calcification.

Functional ß-TCP/MnO2 /PCL artificial periosteum promoting osteogenic differentiation of BMSCs by reducing locally reactive oxygen species level.

Segmental bone defects caused by trauma, tumor resection or congenital malformations are often reconstructed with autologous, allogeneic bone grafts or artificial bone materials, of which, about 5% ~ 10% will have delayed healing or even nonunion of fractures. The loss of periosteum and excessive accumulation of ROS in fracture site leads to the aging of osteoblasts and the decline of their proliferation and differentiation, thus affecting the fracture healing process. In this study, we prepared a functional modified artificial periosteum ß-TCP/MnO2 /PCL(ß-TMP) by electrospinning with a function of catalyzing decomposition of H2 O2 . We examined the surface morphology of ß-TMP, the concentration of Ca, P and Mn of ß-TMP, as well as the diameter distribution range of nanofibers on ß-TMP. Through X-ray diffraction patterns and Fourier transform infrared spectra, ß-TMP was characterized and its hydrophilicity was tested. The release of Mn2+ and Ca2+ of 0.1 and 0.05% ß-TMP in different pH values (7.4 and 5.5) determined by ICP. We also identified that ß-TMP could reduce the level of ROS in cells by lowering the level of H2 O2 . 0%, 0.05% and 0.1% ß-TMP displayed good cell compatibility, cell adhesion and cellular morphology in the condition with or without H2 O2 . 0.5% ß-TMP showed compromised cell compatibility in normal condition, however, the compromised phenotypes could be partially rescued in the present of H2 O2 . Compared with 0%, 0.05% and 0.1% ß-TMP displayed higher osteoblastic differentiation with or without H2 O2 in BMSCs as well as in MG-63. In sum, ß-TMP helped osteogenesis and promoted repair of bone defects.

Dipeptidyl peptidase 3 is essential for maintaining osteoblastic differentiation under a high-glucose environment by inhibiting apoptosis, oxidative stress and inflammation through the modulation of the Keap1-Nrf2 pathway.

Dipeptidyl peptidase 3 (Dpp3) has emerged as a pivotal mediator of bone homeostasis and bone loss pathology. However, whether Dpp3 plays a role in diabetic osteoporosis has not been addressed. Therefore, this work explored the possible role of Dpp3 in osteoblast dysfunction evoked by high glucose (HG), a cellular model for studying diabetic osteoporosis in vitro. Dpp3 expression was decreased in the pre-osteoblast MC3T3-E1 during osteoblastic differentiation under the HG environment. The osteoblastic differentiation impaired by HG was reversed in Dpp3-overexpressing MC3T3-E1 cells. The migration and invasion of MC3T3-E1 cells impeded by HG were reversed by Dpp3 overexpression. Moreover, HG-evoked apoptosis, oxidative stress and inflammation were ameliorated in Dpp3-overexpressing MC3T3-E1 cells. A mechanistic study showed that Dpp3 up-regulated the activation of nuclear factor E2-related factor 2 (Nrf2) depending on Kelch-like ECH-associated protein 1 (Keap1). The blockade of Nrf2 reversed Dpp3-mediated effects on osteoblastic differentiation, apoptosis, oxidative stress and inflammation of HG-stimulated MC3T3-E1 cells. Therefore, Dpp3 plays an essential role in maintaining osteoblastic differentiation under a HG environment associated with the regulation of the Keap1-Nrf2 pathway. This work indicates a possible relationship between Dpp3 and diabetic osteoporosis.

Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

Mineralization of Bone Extracellular Matrix-like Scaffolds Fabricated as Silk Sericin-Functionalized Dense Collagen-Fibrin Hybrid Hydrogels.

The design of hydrogels that combine both the biochemical cues needed to direct seeded cellular functions and mineralization to provide the structural and mechanical properties approaching those of mineralized native bone extracellular matrix (ECM) represents a significant challenge in bone tissue engineering. While fibrous hydrogels constituting of collagen or fibrin (and their hybrids) can be considered as scaffolds that mimic to some degree native bone ECM, their insufficient mechanical properties limit their application. In the present study, an automated gel aspiration-ejection (automated GAE) method was used to generate collagen-fibrin hybrid gel scaffolds with micro-architectures and mechanical properties approaching those of native bone ECM. Moreover, the functionalization of these hybrid scaffolds with negatively charged silk sericin accelerated their mineralization under acellular conditions in simulated body fluid and modulated the proliferation and osteoblastic differentiation of seeded MC3T3-E1 pre-osteoblastic cells. In the latter case, alkaline phosphatase activity measurements indicated that the hybrid gel scaffolds with seeded cells showed accelerated osteoblastic differentiation, which in turn led to increased matrix mineralization. In summary, the design of dense collagen-fibrin hybrid gels through an automated GAE process can provide a route to tailoring specific biochemical and mechanical properties to different types of bone ECM-like scaffolds, and can provide a model to better understand cell-matrix interactions in vitro for bioengineering purposes.

Osteoblast derived extracellular vesicles induced by dexamethasone: A novel biomimetic tool for enhancing osteogenesis in vitro.

Extracellular vesicles (EVs) are newly appreciated communicators involved in intercellular crosstalk, and have emerged as a promising biomimetic tool for bone tissue regeneration, overcoming many of the limitations associated with cell-based therapies. However, the significance of osteoblast-derived extracellular vesicles on osteogenesis has not been fully established. In this present study, we aim to investigate the therapeutic potential of extracellular vesicles secreted from consecutive 14 days of dexamethasone-stimulated osteoblasts (OB-EVDex) to act as a biomimetic tool for regulating osteogenesis, and to elucidate the underlying mechanisms. OB-EVdex treated groups are compared to the clinically used osteo-inductor of BMP-2 as control. Our findings revealed that OB-EVDex have a typical bilayer membrane nanostructure of, with an average diameter of 178 ± 21 nm, and that fluorescently labeled OB-EVDex were engulfed by osteoblasts in a time-dependent manner. The proliferation, attachment, and viability capacities of OB-EVDex-treated osteoblasts were significantly improved when compared to untreated cells, with the highest proliferative rate observed in the OB-EVDex + BMP-2 group. Notably, combinations of OB-EVDex and BMP-2 markedly promoted osteogenic differentiation by positively upregulating osteogenesis-related gene expression levels of RUNX2, BGLAP, SPP1, SPARC, Col 1A1, and ALPL relative to BMP-2 or OB-EVDex treatment alone. Mineralization assays also showed greater pro-osteogenic potency after combined applications of OB-EVDex and BMP-2, as evidenced by a notable increase in mineralized nodules (calcium deposition) revealed by Alkaline Phosphatase (ALP), Alizarin Red Alizarin Red staining (ARS), and von Kossa staining. Therefore, our findings shed light on the potential of OB-EVDex as a new therapeutic option for enhancing osteogenesis.

Bone-targeting polyphosphodiesters that promote osteoblastic differentiation.

Polymers for pharmaceutical use have been attractive in medical treatments because of the conjugation of multifunctional components and their long circulation time in the blood stream. Bone-targeted drug delivery systems are also no exceptional, and several polymers have been proposed for the treatment of bone diseases, such as cancer metastasis and osteoporosis. Herein, we report that polyphosphodiesters (PPDEs) have a potential to enhance osteoblastic differentiation, and they have a targeting ability to bone tissues in vivo. Two types of PPDEs, poly (ethylene sodium phosphate) (PEP•Na) and poly (propylene sodium phosphate) (PPP•Na), have been synthesized. Regardless of the alkylene structure in the main chain of PPDEs, the gene expression of osteoblast-specific transcription factors and differentiation markers of mouse osteoblastic-like cells (MC3T3-E1 cells) cultured in a differentiation medium was significantly upregulated by the addition of PPDEs. Moreover, it was also clarified that the signaling pathway related to cytoplasmic calcium ions was activated by PPDEs. The mineralization of MC3T3-E1 cells has a similar trend with its gene expression and is synergistically enhanced by PPDEs with ß-glycerophosphate. The biodistribution of fluorescence-labeled PPDEs was also determined after intravenous injection in mice. PPDEs accumulated well in the bone through the blood stream, whereas polyphosphotriesters (PPTEs) tended to be excreted from the kidneys. Hydrophilic PEP•Na showed a superior bone affinity as compared with PPP•Na. PPDEs could be candidate polymers for the restoration of bone remodeling and bone-targeting drug delivery platforms.

A dual synergistic effect of titanium and curcumin co-embedded on extracellular matrix hydrogels of decellularized bone: Potential application in osteoblastic differentiation of adipose-derived mesenchymal stem cells.

This research aims to design and fabricate a novel hydrogel-based composite as a functional biomimetic and biocompatible scaffold for amended osteoblastic differentiation of adipose-derived mesenchymal stem cells (ADMSCs). The extracellular matrix (ECM) hydrogel is an ideal scaffold in tissue engineering in terms of its structure mimics natural tissue. In this study, the fresh bovine femur was demineralized and decellularized; next, ECM hydrogel was obtained by digesting these matrices. Then, TiO2 and curcumin-loaded hydrogel (Hy/Ti/Cur) was fabricated besides TiO2-loaded hydrogel (Hy/Ti) and curcumin-loaded hydrogel (Hy/Cur). Comparing the scanning electron microscopy (SEM) images of the pure network hydrogel and the rough morphology of Hy/Ti/Cur revealed that curcumin and titanium dioxide were successfully loaded into the hydrogel. In addition, FTIR spectroscopy and X-ray diffraction (XRD) validated these findings. The findings of the hydrogels' swelling test indicated the favourable impact of curcumin and titanium dioxide in hydrogels, which enhances water absorption capacity. Our results showed that the hydrogels were cytocompatible, and the cell viability on the hydrogels was elevated compared to the control. The synergistic effect of TiO2 and Cur co-embedded on ECM hydrogel (Hy/Ti/Cur) stimulates bone differentiation markers, such as Runt-related transcription factor 2 (RUNX-2) and osteocalcin (OCN) in ADMSCs cultured in normal and osteogenic medium. Moreover, Alkaline Phosphatase (ALP) activity and calcium deposition of ADMSCs cultured on engineered hydrogels were increased. These experiments showed that newly fabricated hydrogel has the potential to induce osteogenesis, which is recommended as an attractive scaffold in bone tissue engineering.