Expression Levels Of Osteogenic Genes Research Articles

Chrysin induces osteogenic differentiation of human dental pulp stem cells

ObjectivesAs an ideal cell source for tissue engineering and bone defect repair, dental pulp stem cells (DPSCs) have good osteogenic differentiation potential. Chrysin, a flavonoid extracted from oroxylum seeds, has been proven to promote bone formation of bone marrow stem cells. However, the effect of chrysin on osteogenic differentiation of DPSCs remains unclear. This study aimed to investigate the role of Chrysin in promoting osteogenic differentiation of DPSCs and in DPSC-based bone formation. Material and methodsWe investigated the effects of chrysin on DPSCs from patients by CCK-8 assay, Alizarin Red S staining, qPCR and Western blotting. The effects of chrysin on DPSC-based bone formation in a heterotopic osteogenesis model in nude mice and a rat calvarial defect model were also performed. Finally, we investigated the mechanism of chrysin-treated DPSCs by proteomics. ResultsChrysin upregulated the expression of osteogenic proteins and induced osteogenic differentiation of DPSCs. Moreover, chrysin induced abundant β-TCP-induced formation of mineralized bone tissue and promoted DPSC-based bone formation in a heterotopic osteogenesis model in nude mice and a rat calvarial defect model. Proteomics showed that upregulation of the Smad3 was closely related to osteogenic differentiation. Inhibiting of Smad3 activation by a Smad3 inhibitor could reverse the chrysin-mediated increases in the expression levels of osteogenic genes and osteogenic induction of DPSCs. ConclusionsOur study implies the intriguing potential of chrysin-treated DPSCs in bone regeneration and bone defect repair.

Baghdadite coating formed by hybrid water-stabilized plasma spray for bioceramic applications: Mechanical and biological evaluations

This work studies the mechanical and biological properties of Baghdadite (BAG, Ca3ZrSi2O9) coating manufactured on Ti6Al4V substrates by hybrid water-stabilized plasma spray (WSP-H). Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) coating was produced by gas-stabilized atmospheric plasma spray and used as a reference material. Upon spraying, the BAG coating exhibited lower crystallinity than the HAp coating. Mechanical testing demonstrated superior properties of the BAG coating: its higher hardness, elastic modulus as well as a better resistance to scratch and wear. In the cell viability study, the BAG coating presented better human osteoblast attachment and proliferation on the coating surface after three days and seven days compared to the HAp counterpart. Furthermore, the gene expression study of human osteoblasts indicated that the BAG coating surface showed higher expression levels of osteogenic genes than those on the HAp coating. Overall, this study indicates that enhanced mechanical and bioactive properties can be achieved for the BAG coating compared to the benchmark HAp coating. It is therefore concluded here that the BAG coating is a potential candidate for coating orthopedic implants.

Biological evaluation of the modified nano-amorphous phosphate calcium doped with citrate/poly-amino acid composite as a potential candidate for bone repair and reconstruction

Large numbers of research works related to fabricating organic–inorganic composite materials have been carried out to mimic the natural structure of bone. In this study, a new modified n-ACP doped with citrate (n-ACP-cit)/poly (amino acids) (PAA) composite (n-ACP-cit/PAA) was synthesized by employing high bioactive n-ACP-cit and the biodegradable and biocompatible PAA copolymer. Its basic structure was characterized by X-ray diffraction spectroscopy, Fourier transformed infrared spectroscopy, and X-ray photoelectron spectroscopy. Moreover, the degradability, bioactivity, biocompatibility, and osteoconductivity of n-ACP-cit/PAA composite were evaluated in vitro and in vivo, using simulated body fluid (SBF) solution soaking test, mouse bone marrow mesenchymal stem cells proliferation and differentiation, morphological observation test, expression of genes associated with osteogenesis, and bone defect model repair test, respectively. The modified n-ACP-cit/PAA composite exhibited a much higher weight loss rate (36.01 wt.%) than that of PAA (23.99 wt.%) after immersing in SBF solution for 16 weeks and the pH values of local environment restored to neutral condition. Moreover, cells co-culturing with composites exhibited higher alkaline phosphatase activity, more calcium nodule-formation, and higher expression levels of osteogenic differentiation-related genes (Bmp-2, Colla I, OCN, OPN, and Runx-2) than that of PAA. Furthermore, the bone defect model repair test revealed that the composite could be intimately incorporated with the surrounding bone without causing any deleterious reaction and capable of guiding new bone formation. Together, these results indicated that the new modified bone repair n-ACP-cit/PAA composite material with specific characteristics may be designed for meeting diverse requirements from biomedical applications.

CircAKT3 positively regulates osteogenic differentiation of human dental pulp stromal cells via miR-206/CX43 axis

BackgroundHuman dental pulp stromal cells (hDPSCs) are promising sources of mesenchymal stem cells (MSCs) for bone tissue regeneration. Circular RNAs (circRNAs) have been demonstrated to play critical roles in stem cell osteogenic differentiation. Herein, we aimed to investigate the role of circAKT3 during osteogenesis of hDPSCs and the underlying mechanisms of its function.MethodsWe performed circRNA sequencing to investigate the expression profiles of circular RNAs during osteogenesis of hDPSCs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression pattern of circAKT3 and miR-206 in hDPSCs during osteogenesis. We knocked down circAKT3 and interfered the expression of miR-206 to verify their regulatory role in hDPSC osteogenesis. We detected hDPSCs mineralization by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining and used dual-luciferase reporter assay to validate the direct binding between circAKT3 and miR-206. To investigate in vivo mineralization, we performed subcutaneous transplantation in nude mice and used hematoxylin and eosin, Masson’s trichrome, and immunohistochemistry staining.ResultsTotally, 86 circRNAs were differentially expressed during hDPSC osteogenesis, in which 29 were downregulated while 57 were upregulated. circAKT3 was upregulated while miR-206 was downregulated during hDPSC osteogenesis. Knockdown of circAKT3 inhibited ALP/ARS staining and expression levels of osteogenic genes. circAKT3 directly interacted with miR-206, and the latter one suppressed osteogenesis of hDPSCs. Silencing miR-206 partially reversed the inhibitory effect of circAKT3 knockdown on osteogenesis. Connexin 43 (CX43), which positively regulates osteogenesis of stem cells, was predicted as a target of miR-206, and overexpression or knockdown of miR-206 could correspondingly decrease and increase the expression of CX43. In vivo study showed knockdown of circAKT3 suppressed the formation of mineralized nodules and expression of osteogenic proteins.ConclusionDuring osteogenesis of hDPSCs, circAKT3 could function as a positive regulator by directly sponging miR-206 and arresting the inhibitive effect of miR-206 on CX43 expression.

Interplay of Hydrogel Composition and Geometry on Human Mesenchymal Stem Cell Osteogenesis.

Microgels are emerging as an outstanding platform for tissue regeneration because they overcome issues associated with conventional bulk/macroscopic hydrogels such as limited cell-cell contact and cell communication and low diffusion rates. Owing to the enhanced mass transfer and injectability via a minimally invasive procedure, these microgels are becoming a promising approach for bone regeneration applications. Nevertheless, there still remains a huge gap between the understanding of how the hydrogel matrix composition can influence cell response and overall tissue formation when switching from bulk formats to microgel format, which is often neglected or rarely studied. Here, we fabricated polyethylene glycol-based microgels and bulk hydrogels incorporating gelatin and hyaluronic acid (HA), either individually or together, and assessed the impact of both hydrogel composition and format upon the osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs). Osteogenesis was significantly greater in microgels than bulk hydrogels for both gelatin alone (Gel) and gelatin HA composite (Gel:HA) hydrogels, as determined by the expression of Runt-related transcription factor (Runx2) and alkaline phosphatase (ALP) genes and mineral deposition. Interestingly, Gel and Gel:HA hydrogels behaved differently between bulk and microgel format. In bulk format, overall osteogenic outcomes were better in Gel:HA hydrogels, but in microgel format, while the level of osteogenic gene expression was equivalent between both compositions, the degree of mineralization was reduced in Gel:HA microgels. Investigation into the affinity of hydroxyapatite for the different matrix compositions indicated that the decreased mineralization of Gel:HA microgels was likely due to a low affinity of hydroxyapatite to bind to HA and support mineral deposition, which has a greater impact on microgels than bulk hydrogels. Together, these findings suggest that both hydrogel composition and format can determine the success of tissue formation and that there is a complex interplay of these two factors on both cell behavior and matrix deposition. This has important implications for tissue engineering, showing that hydrogel composition and geometry must be evaluated together when optimizing conditions for cell differentiation and tissue formation.

Zingerone Attenuates Pi-induced Vascular Calcification via AMPK-mediated TIMP4 Expression.

ObjectiveVascular calcification requires the differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. This phenomenon can be enhanced by inflammation and oxidative stress. Zingerone is one of the active ingredients present in the ginger plant that has anti-inflammatory and antioxidant effects. Other functions include anti-obesity, anti-nausea effects. However, the functions of zingerone on vascular calcification has not yet been elucidated. This study investigated the effect of zingerone on vascular calcification and its molecular mechanism.MethodsReverse transcription-polymerase chain reaction (PCR), real-time PCR and Western blot analysis was used to measure expression levels of osteogenic marker genes and to investigate whether calcification was regulated by the expression of AMP-activated protein kinase (AMPK) and tissue inhibitor of metalloproteinase 4 (TIMP4). Alizarin red S staining was used to measure calcium deposition. Studies were carried out in VSMCs.ResultsZingerone induced the expression of 2 markers of VSMCs differentiation (α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α)) and decreased the expression of core-binding factor α-1 (CBFA1). Additionally, zingerone decreased inorganic phosphate (Pi)-induced expression of distal-less homeobox 5 and CBFA1. AMPK phosphorylation and TIMP4 expression were increased by zingerone. Importantly, zingerone protected VSMCs from calcification, and this protective effect was confirmed by increased TIMP4 via overexpression of AMPK, and inhibition of TIMP4 by Compound C. Zingerone upregulated AMPK/TIMP4 expression and recovered Pi-induced inhibition of TIMP4.ConclusionsTaken together, our results show that zingerone inhibits Pi-induced vascular calcification by regulating the AMPK/TIMP4 signaling cascade in VSMCs. These results suggest that the natural product zingerone could be useful for treating vascular and metabolic diseases.

Lanthanum-doped mesoporous bioglasses/chitosan composite scaffolds enhance synchronous osteogenesis and angiogenesis for augmented osseous regeneration

With the progressive research of fabrication methodologies of biomaterials, hierarchically porous structures and bioactive compositions have been identified to promote bone repair and regeneration. In this study, we firstly fabricated lanthanum-doped bioglasses/chitosan (La-BGs/CS) composite scaffolds for novel bone tissue engineering materials. The La-BG microspheres in the scaffolds possessed hierarchically porous structures combining hollow cores and mesoporous shells. The effects of La-BGs/CS scaffolds on osteogenesis and angiogenesis were evaluated both in vitro and in vivo. The as-doped lanthanum oxide in the scaffolds remarkably promoted osteogenic differentiation of bone marrow-derived mesenchymal stem cells via activating ERK signaling pathway, and stimulated migration and tube formation of human umbilical vein endothelial cells. As compared with the BGs/CS scaffolds, the La dopants in the La-BGs/CS scaffolds contributed to higher expression levels of both osteogenic and angiogenic-related genes. Moreover, the mesopores and macropores within the La-BGs/CS scaffolds enhanced the osteoconductivity and stimulated bone regeneration in cranial defect model of rats. The La-BGs/CS scaffolds could also promote vascularization in vivo as demonstrated by Microfil perfusion, which further augmented osseous regeneration. Therefore, the La-BGs/CS scaffolds provide an admirable application and novel platform for bone tissue engineering.

Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling.

Filifactor alocis, an asaccharolytic anaerobic Gram-positive rod (AAGPR), is an emerging marker of periodontitis. Severe periodontitis causes destruction of the alveolar bone that supports teeth and can even lead to tooth loss. Based on our previous report that F. alocis-derived extracellular vesicles (FA EVs) contain various effector molecules and have immunostimulatory activity, we investigated the effect of FA EVs on osteogenesis using mouse bone-derived mesenchymal stromal cells (BMSCs). FA EVs dramatically inhibited bone mineralization similar to whole bacteria and reduced the expression levels of osteogenic marker genes. The osteogenic differentiation of TLR2-deficient BMSCs was not inhibited by FA EVs, suggesting that their inhibitory effect on osteogenesis is dependent on TLR2 signaling. FA EVs effectively activated TLR2 downstream signaling of the MAPK and NF-κB pathways. In addition, FA EVs regulated RANKL and OPG gene expression, increasing the RANKL/OPG ratio in BMSCs in a TLR2-dependent manner. Our study suggests that F. alocis-derived EVs interfere with bone metabolism via TLR2 activation, providing insight into the pathogenesis of bone loss associated with periodontitis.

Linking gene expression and phenotypic changes in the developmental and evolutionary origins of osteosclerosis in the ribs of bowhead whales (Balaena mysticetus).

Bowhead whales are among the longest-lived mammals with an extreme lifespan of about 211 years. During the first 25 years of their lives, rib bones increase in mineral density and the medulla transitions from compact to trabecular bone. Molecular drivers associated with these phenotypic changes in bone remain unknown. This study assessed expression levels of osteogenic genes from samples of rib bones of bowheads. Samples were harvested from prenatal to 86-year-old whales, representing the first third of the bowhead lifespan. Fetal to 2-year-old bowheads showed expression levels consistent with the rapid deposition of the bone extracellular matrix. Sexually mature animals showed expression levels associated with low rates of osteogenesis and increased osteoclastogenesis. After the first 25 years of life, declines in osteogenesis corresponded with increased expression of EZH2, an epigenetic regulator of osteogenesis. These findings suggest EZH2 may be at least one epigenetic modifier that contributes to the age-related changes in the rib bone phenotype along with the transition from compact to trabecular bone. Ancient cetaceans and their fossil relatives also display these phenotypes, suggesting EZH2 may have shaped the skeleton of whales in evolutionary history.

Low-magnitude vibration induces osteogenic differentiation of bone marrow mesenchymal stem cells via miR-378a-3p/Grb2 pathway to promote bone formation in a rat model of age-related bone loss.

The dysfunction of bone marrow mesenchymal stem cells (BMSCs) in osteogenic differentiation is one of the main causes of age-related bone loss. Our previous studies have shown that low-magnitude vibration (LMV) induces the osteogenic differentiation of BMSCs derived from ovariectomized osteoporotic rats. To investigate whether LMV promotes osteogenic differentiation of BMSCs and its underlying mechanisms in aged rats, 20-month-old female Sprague-Dawley rats (n=20) were randomly divided into LMV group (rats were vibrated at 0.3g and 90Hz for 30minutes, once daily, 5days a week until 12weeks for subsequent analysis, n=10), static group (rats were placed in the box on the vibration platform without vibration, n=10); 6-month-old female Sprague-Dawley rats were used as control (young group, n=10). The bone mineral density and bone strength of aged rats were significantly decreased compared with the young rats. Furthermore, the primary BMSCs isolated and cultured from the aged rats with the whole-bone marrow differential pasting method showed a decreased ability in osteogenic differentiation compared with that from the young rats. Then the differentially expressed miRNAs between the aged and young rat-derived BMSCs were screened by high-throughput sequencing and verified by qRT-PCR, and we found that miR-378a-3p was significantly downregulated in the aged rat-derived BMSCs compared with the young rat-derived BMSCs. By transfecting miRNA mimics and inhibitors, miR-378a-3p was confirmed to promote the expression levels of osteogenic genes (Runx2, ALP, Col I, and OCN) and ALP activity of the aged rat-derived BMSCs. Meanwhile, the expression levels of osteogenic genes and miR-378a-3p of aged rat-derived BMSCs were significantly upregulated by LMV (cells were vibrated at 0.3g and 90Hz for 30minutes a day, until 5days for subsequent analysis), while the LMV-induced osteogenic gene expression levels of aged rat-derived BMSCs were suppressed by miR-378a-3p inhibitors. Furthermore, the inhibition of growth factor receptor-bound protein 2 (Grb2) by miR-378a-3p and Grb2-siRNA promoted the LMV-induced osteogenic differentiation of aged rat-derived BMSCs. Additionally, LMV was found to promote bone mineral density and bone strength of aged rats in vivo, as well as upregulating the expression level of miR-378a-3p and downregulating the expression level of Grb2 of BMSCs from aged rats. These results suggest that LMV induces osteogenic differentiation of BMSCs through miR-378a-3p/Grb2 pathway to improve bone mineral density and mechanical properties in a rat model of age-related bone loss.

Different Responsiveness of Alveolar Bone and Long Bone to Epithelial-Mesenchymal Interaction-Related Factor.

ABSTRACTAlveolar bone is both morphologically and functionally different from other bones of the axial or peripheral skeleton. Because of its sensitive nature to external stimuli including mechanical stress, bone loss stimuli, and medication‐related osteonecrosis of the jaw, alveolar bone rendering is seen as an important factor in various dental surgical processes. Although multiple studies have validated the response of long bone to various factors, how alveolar bone responds to functional stimuli still needs further clarification. To examine the characteristics of bone in vitro, we isolated cells from alveolar, femur, and tibia bone tissue. Although primary cultured mouse alveolar bone‐derived cells (mABDCs) and mouse long bone‐derived cells (mLBDCs) exhibited similar osteoblastic characteristics, morphology, and proliferation rates, both showed distinct expression of neural crest (NC) and epithelial–mesenchymal interaction (EMI)‐related genes. Furthermore, they showed significantly different mineralization rates. RNA sequencing data demonstrated distinct transcriptome profiles of alveolar bone and long bone. Osteogenic, NC‐, and EMI‐related genes showed distinct expression between mABDCs and mLBDCs. When the gene expression patterns during osteogenic differentiation were analyzed, excluding several osteogenic genes, NC‐ and EMI‐related genes showed different expression patterns. Among EMI‐related proteins, BMP4 elevated the expression levels of osteogenic genes, Msx2, Dlx5, and Bmp2 the most, more noticeably in mABDCs than in mLBDCs during osteogenic differentiation. In in vivo models, the BMP4‐treated mABDC group showed massive bone formation and maturation as opposed to its counterpart. Bone sialoprotein expression was also validated in calcified tissues. Overall, our data suggest that alveolar bone and long bone have different responsiveness to EMI by distinct gene regulation. In particular, BMP4 has critical bone formation effects on alveolar bone, but not on long bone. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Different effects of total flavonoids from Arachniodes exilis on human umbilical cord mesenchymal stem cells in vitro.

Traditional Chinese medicines are used in promotion of fractured bone healing and bone diseases. Some studies reported total flavonoids from plant can be used as an auxiliary source of exogenous.Use different methods to identify and verify effects of total flavonoids from Arachniodes exilis (TFAE) on human umbilical cord mesenchymal stem cells (HUCMSCs) in vitro.Concentrations of 1 and 5 μg/mL TFAE significantly increased ALPase activity in HUCMSCs compared to the other concentrations at days 3 and 7 (P < .05). RT-PCR showed that expression levels of osteogenic genes (Col1a1, OPN, Runx2 and Osx) were remarkably enhanced in HUCMSCs following treatment with different concentrations of TFAE for 9 days compared with 0 μg/mL TFAE group (control). The results showed that concentration < 5 μg/mL of TFAE induced osteogenic differentiation in HUCMSCs Alizarin red staining assays revealed that both TFAE and S1191 was significantly decreased (7.80 ± 0.66) compared with the TFAE group (16.00 ± 0.97) (P < .01). ALPase activity on days 3 and 7 was relatively lower in HUCMSCs grown in media supplemented with both S1191 and TFAE than that of in TFAE group only. The results indicated that osteogenic markers (Col1a1, OPN, Runx2 and Osx) were significantly downregulated in the TFAE + S1191 group in comparison to the control group. The expressions of Col1a and OPN in the TFAE + S1191 group decreased significantly (P < .01) by Western blotting.TFAE promotes the odonto/osteogenic differentiation of human UCMSCs via activation of ER.

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.

Advanced glycation end products inhibit the osteogenic differentiation potential of adipose-derived stem cells by modulating Wnt/β-catenin signalling pathway via DNA methylation.

ObjectivesAdvanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose‐derived stem cells (ASCs) are widely used in the research of bone regeneration, the mechanisms of the osteogenic differentiation of ASCs from diabetic osteoporosis model remain unclear. This work aimed to investigate the influence and the molecular mechanisms of AGEs on the osteogenic potential of ASCs.Materials and methodsEnzyme‐linked immunosorbent assay was used to measure the change of AGEs in diabetic osteoporotic and control C57BL/6 mice. ASCs were obtained from the inguinal fat of C57BL/6 mice. AGEs, 5‐aza2′‐deoxycytidine (5‐aza‐dC) and DKK‐1 were used to treat ASCs. Real‐time cell analysis and cell counting kit‐8 were used to monitor the proliferation of ASCs within and without AGEs. Real‐time PCR, Western blot and Immunofluorescence were used to analyse the genes and proteins expression of osteogenic factors, DNA methylation factors and Wnt/β‐catenin signalling pathway among the different groups.ResultsThe AGEs and DNA methylation were increased in the adipose and bone tissue of the diabetic osteoporosis group. Untreated ASCs had higher cell proliferation activity than AGEs‐treatment group. The expression levels of osteogenic genes, Opn and Runx2, were lower, and mineralized nodules were less in AGEs‐treatment group. Meanwhile, DNA methylation was increased, and the Wnt signalling pathway markers, including β‐Catenin, Lef1 and P‐GSK‐3β, were inhibited. After treatment with 5‐aza‐dC, the osteogenic differentiation capacity of ASCs in the AGEs environment was restored and the Wnt signalling pathway was activated during this process.ConclusionsAdvanced glycation end products inhibit the osteogenic differentiation ability of ASCs by activating DNA methylation and inhibiting Wnt/β‐catenin pathway in vitro. Therefore, DNA methylation may be promising targets for the bone regeneration of ASCs with diabetic osteoporosis.

Extracellular Matrix/Amorphous Magnesium Phosphate Bioink for 3D Bioprinting of Craniomaxillofacial Bone Tissue.

Bioprinting, a promising field in regenerative medicine, holds great potential to create three-dimensional, defect-specific vascularized bones with tremendous opportunities to address unmet craniomaxillofacial reconstructive challenges. A cytocompatible bioink is a critical prerequisite to successfully regenerate functional bone tissue. Synthetic self-assembling peptides have a nanofibrous structure resembling the native extracellular matrix (ECM), making them an excellent bioink component. Amorphous magnesium phosphates (AMPs) have shown greater levels of resorption while maintaining high biocompatibility, osteoinductivity, and low inflammatory response, as compared to their calcium phosphate counterparts. Here, we have established a novel bioink formulation (ECM/AMP) that combines an ECM-based hydrogel containing 2% octapeptide FEFEFKFK and 98% water with AMP particles to realize high cell function with desirable bioprintability. We analyzed the osteogenic differentiation of dental pulp stem cells (DPSCs) encapsulated in the bioink, as well as in vivo bone regeneration, to define the potential of the formulated bioink as a growth factor-free bone-forming strategy. Cell-laden AMP-modified bioprinted constructs showed an improved cell morphology but similar cell viability (∼90%) compared to their AMP-free counterpart. In functional assays, the cell-laden bioprinted constructs modified with AMP exhibited a high level of mineralization and osteogenic gene expression without the use of growth factors, thus suggesting that the presence of AMP-triggered DPSCs' osteogenic differentiation. Cell-free ECM-based bioprinted constructs were implanted in vivo. In comparison with the ECM group, bone volume per total volume for ECM/1.0AMP was approximately 1.7- and 1.4-fold higher at 4 and 8 weeks, respectively. Further, a significant increase in the bone density was observed in ECM/1.0AMP from 4 to 8 weeks. These results demonstrate that the presence of AMP in the bioink significantly increased bone formation, thus showing promise for in situ bioprinting strategies. We foresee significant potential in translating this innovative bioink toward the regeneration of patient-specific bone tissue for regenerative dentistry.

Zeolitic Imidazolate Framework-8 Encapsulating Risedronate Synergistically Enhances Osteogenic and Antiresorptive Properties for Bone Regeneration.

Bisphosphonates (BPs) are routinely administered for the treatment of turnover bone diseases. To avoid the undesirable adverse effects of long-term usage of bisphosphonates and improve their bioavailability in the bone microenvironment, we initially encapsulated risedronate (RIS) molecules inside nanoscale zeolitic imidazolate framework-8 particles (nZIF-8) by a one-step synthesis method to generate RIS@ZIF-8 nanoparticles. RIS@ZIF-8 nanoparticles displayed high loading encapsulation efficiency (64.21 ± 2.48%), good biocompatibility, controlled drug release capacity, and dual effects for bone regeneration. This work explored the potential of RIS@ZIF-8 nanoparticles, which could not only enhance ATP production, induce extracellular matrix (ECM) mineralization, and upregulate the expression levels of osteogenic genes but also effectively inhibit the formation of multinucleated giant osteocasts and decrease the Rankl/Opg ratio. Overall, RIS@ZIF-8 nanoparticles could be a very promising approach to synergistically enhance osteogenic and antiresorptive properties for bone regeneration, which could be utilized for the local treatment of bone defects.

PERK-eIF2\u03b1-ATF4 signaling contributes to osteogenic differentiation of periodontal ligament stem cells

Protein kinase-like endoplasmic reticulum kinase (PERK) is a type I transmembrane protein located in the endoplasmic reticulum (ER). The PERK-eukaryotic initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway has been proved to be involved in osteoblast differentiation, but the involvement of the PERK-eIF2α-ATF4 signaling pathway in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) has remained unclear. Therefore, the aim of this study was to explore the role of PERK in osteogenic differentiation of hPDLSCs and to assess whether PERK-eIF2α-ATF4 contributes to the process of osteogenic differentiation in hPDLSCs. In our study, we constructed PERK-overexpressed and PERK-silenced hPDLSCs by lentiviral transduction. Furthermore, lentivirus-transfected cells were induced to differentiate into osteoblast cells for different days. Alkaline phosphatase (ALP) activity and Alizarin Red staining were used to evaluate the mineralization capacity, and the expression levels of related genes-ATF4, ALP, bone sialoprotein, runt-related transcription factor 2 (Runx2), and osteocalcin were measured to evaluate the osteogenic differentiation of hPDLSCs. The results showed that over-expression of PERK greatly increased ALP activity and the expression levels of related osteogenic genes, which displayed the strongest osteogenesis capacity. However, suppression of PERK caused decreased ALP activity and the weakest osteogenesis capacity, and the levels of ATF4 and p-eIF2α in PERK-silenced hPDLSCs were also decreased. Our results indicated that the PERK gene plays an important role in the differentiation of hPDLSCs to osteoblast-like cells. The PERK-eIF2α-ATF4 signaling pathway contributes to osteoblast differentiation of hPDLSCs.

The molecular mechanism of platelet lysate promotes transformation of non-union cells into osteoblasts.

BackgroundPlatelet lysate (PL) had a remarkable therapeutic effect on bone repair related diseases, such as delayed fracture healing, femoral head necrosis and meniscal tear. In this study, we investigated the effect of PL on patients with nonunion, cartilage repair and osteonecrosis, and to evaluate the effect of PL on nonunion cells proliferation and the effect of PL on OPG/RANKL signaling pathway in nonunion cell of male rats. To reveal the molecular mechanism of PL for bone healing.MethodsWe used different concentrations of PL to treat nonunion cells, then detected cell proliferation and protein expression levels of osteoprotegerin (OPG), RANKL, osteopontin (OPN), osteocalcin (OCN) and alkaline phosphatase (ALP).ResultsThe proliferation rate of nonunion cells treated by 5% PL, was significantly higher than that of the control group (P<0.05). Surprisingly, there were no significant difference among the proliferation rates of nonunion cells treated by 8% PL, 10% FBS and the control group (P>0.05). the results of western blot analysis and immunofluorescence analysis showed that PL improved the expression of OPG, OPN, OCN and ALP proteins in nonunion cells, but PL had no effect on the expression of nuclear factor-κB ligand (RANKL) protein.ConclusionsWe found that PL had a remarkable therapeutic effect on bone repair related diseases; 5% PL significantly improved the proliferation rate of the nonunion cells; 10% PL had a significantly positive effect on improving the expression levels of osteogenic related genes.

Bone regeneration capacities of alveolar bone mesenchymal stem cells sheet in rabbit calvarial bone defect

Mesenchymal stem cells sheets have been verified as a promising non-scaffold strategy for bone regeneration. Alveolar bone marrow mesenchymal stem cells, derived from neural crest, have the character of easily obtained and strong multi-differential potential. However, the bone regenerative features of alveolar bone marrow mesenchymal stem cells sheets in the craniofacial region remain unclear. The purpose of the present study was to compare the osteogenic differentiation and bone defect repairment characteristics of bone marrow mesenchymal stem cells sheets derived from alveolar bone (alveolar bone marrow mesenchymal stem cells) and iliac bone (Lon-bone marrow mesenchymal stem cells) in vitro and in vivo. Histology character, osteogenic differentiation, and osteogenic gene expression of human alveolar bone marrow mesenchymal stem cells and Lon-bone marrow mesenchymal stem cells were compared in vitro. The cell sheets were implanted in rabbit calvarial defects to evaluate tissue regeneration characteristics. Integrated bioinformatics analysis was used to reveal the specific gene and pathways expression profile of alveolar bone marrow mesenchymal stem cells. Our results showed that alveolar bone marrow mesenchymal stem cells had higher osteogenic differentiation than Lon-bone marrow mesenchymal stem cells. Although no obvious differences were found in the histological structure, fibronectin and integrin β1 expression between them, alveolar-bone marrow mesenchymal stem cells sheet exhibited higher mineral deposition and expression levels of osteogenic marker genes. After being transplanted in the rabbit calvarial defects area, the results showed that greater bone volume and trabecular thickness regeneration were found in bone marrow mesenchymal stem cells sheet group compared to Lon-bone marrow mesenchymal stem cells group at both 4 weeks and 8 weeks. Finally, datasets of bone marrow mesenchymal stem cells versus Lon-bone marrow mesenchymal stem cells, and periodontal ligament mesenchymal stem cells (another neural crest derived mesenchymal stem cells) versus umbilical cord mesenchymal stem cells were analyzed. Total 71 differential genes were identified by overlap between the 2 datasets. Homeobox genes, such as LHX8, MKX, PAX9, MSX, and HOX, were identified as the most significantly changed and would be potential specific genes in neural crest mesenchymal stem cells. In conclusion, the Al-bone marrow mesenchymal stem cells sheet-based tissue regeneration appears to be a promising strategy for craniofacial defect repair in future clinical applications.

Strontium Ameliorates Glucocorticoid Inhibition of Osteogenesis Via the ERK Signaling Pathway.

Glucocorticoid (GC) has been widely used in clinical work due to its anti-inflammatory and immune-inhibitory properties. However, long-term or high-dose administration is associated with side effects, such as GC-induced osteoporosis (GIOP), which causes great pain for and poses a heavy financial burden on patients. We sought to investigate the potential effects of strontium on GIOP and further explore its underlying mechanisms, including its reversal of the inhibitory effect of GC on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). We incubated BMSCs with Dexamethasone (DEX) in combination with or without strontium and then measured osteogenic and adipogenic gene expression levels by RT-qPCR and Western blot. We added a specific ERK signaling pathway inhibitor, U0126, to evaluate the involvement of that pathway. Strontium promoted osteogenic differentiation and matrix mineralization in DEX-treated BMSCs, accompanied by upregulation of RUNX2, Osx, ALP, BSP, COL1A1, and OCN. DEX blocked the expression of several osteogenesis-related marker genes by activating the ERK signaling pathway. U0126 attenuated the suppression of osteogenesis in DEX-treated BMSCs. These results suggested that strontium could enhance osteogenic differentiation and matrix mineralization by counteracting DEX's inhibitory effect on osteogenesis via the ERK signaling pathway. Therefore, strontium might be a promising therapeutic agent for GIOP.