Osteogenic Differentiation Research Articles

Mesenchymal stromal cells surface engineering for efficient hematopoietic reconstitution

Mesenchymal stromal cells (MSCs) are believed to migrate to injury sites, release chemical attractants, and either recruit local stem cells or modulate the immune system positively. Although MSCs are highly desired for their potential to reduce inflammation and promote tissue regeneration, their limited lifespan restricts their applications. This study presents a simple approach for protecting MSCs with epigallocatechin-3-gallate (EGCG) and magnesium (Mg) based metal-organic framework coatings (E-Mg@MSC). The layer strengthens MSCs resistant to harmful stresses and creates a favorable microenvironment for repair by providing Mg to facilitate MSCs' osteogenic differentiation and using EGCG to neutralize excessive reactive oxygen species (ROS). E-Mg@MSC serves as a treatment for hematopoietic injury induced by ionizing radiation (IR). Coated MSCs exhibit sustained secretion of hematopoietic growth factors and precise homing to radiation-sensitive tissues. In vivo studies show substantial enhancement in hematopoietic system recovery and multi-organ protection. Mechanistic investigations suggest that E-Mg@MSC mitigates IR-induced ROS, cell apoptosis, and ferroptosis, contributing to reduced radiation damage. The system represents a versatile and compelling strategy for cell-surface engineering with functional materials to advance MSCs therapy.

Microfluidic Fabrication of Gelatin-Nano Hydroxyapatite Scaffolds for Enhanced Control of Pore Size Distribution and Osteogenic Differentiation of Dental Pulp Stem Cells.

The combination of gelatin and hydroxyapatite (HA)has emerged as a promising strategy in dental tissue engineering due to its favorable biocompatibility, mechanical properties, and ability to support cellular activities essential for tissue regeneration, rendering them ideal components for hard tissue applications. Besides, precise control over interconnecting porosity is of paramount importance for tissue engineering materials. Conventional methods for creating porous scaffolds frequently encounter difficulties in regulating pore size distribution. This study demonstrates the fabrication of gelatin-nano HA scaffolds with uniform porosity using a T-type junction microfluidic device in a single-step process. Significant improvements in control over the pore size distribution are achieved by regulating the flow parameters, resulting in effective and time-efficient manufacturing comparable in quality to the innovative 3D bioprinting techniques. The overall porosity of the scaffolds exceeded 60%, with a remarkably narrow size distribution. The incorporation of nano-HAinto 3D porous gelatin scaffolds successfully induced osteogenic differentiation in stem cells at both the protein and gene levels, as evidenced by the significant increase in osteocalcin (OCN), an important marker of osteogenic differentiation. The OCN levels are 26 and 43 times higher for gelatin and gelatin-HA scaffolds, respectively, compared to the control group.

Role of in-situ electrical stimulation on early-stage mineralization and in-vitro osteogenesis of electroactive bioactive glass composites

Bioactive glass (BAG) has emerged as an effective bone graft substitute due to its diverse qualities of biocompatibility, bioactivity, osteoblast adhesion and enhanced revascularization. However, inferior osteogenic capacity of BAG compared to autologous bone grafts continues to limiting it's wide-spread clinical applications towards repairing of bone fractures and healing. In this study, we have fabricated BAG composites with 0.5 to 2 wt% bismuth ferrite (BF, a multiferroic material) with an aim to generate in-situ electrical charges pertinent to early-stage bone regeneration thus mimicking nature bone, which is a piezoelectric material. The fabricated BAG composites were characterised in terms of microstructures, phase analysis, remanent polarization, wettability and subsequently evaluated for in vitro cell proliferation and osteogenesis with and without magnetic field exposure (200 mT, 30 min./day). Pre-osteoblast cells from mice (MC3T3-E1) seeded on these composites exhibited excellent cell growth without any cytotoxicity, which is further supported by FITC/DAPI staining and a live/dead assay. The results of Alizarin Red S assay and increased levels of Alkaline Phosphatase (ALP) activity, at 21 days of culture, suggest that the BAG-BF composites promote in vitro osteogenic differentiation of pre-osteoblast cells. The enhanced osteogenesis of BAG-BF composites was also confirmed through qRT-PCR analysis, which showed rapid upregulation of osteoblastogenic specific genes namely RunX-2, Collagen-1, Bone Sialo Protein, and ALP after 21 days. Additionally, the osteogenic differentiation was assessed by the Western Blot technique, which revealed significantly higher band intensity of osteogenic markers in BAG-1.5 BF and BAG-2 BF composites than pure BAG. These findings clearly demonstrate that in-situ electrical stimulation and osteoconductive capacity of BF reinforced BAG composites have positive impact on osteoblast cell development, bone formation, and healing.

Regulation of Osteogenic Differentiation of hBMSCs by the Overlay Angles of Bone Lamellae-like Matrices.

Oriented fibers in bone lamellae are recognized for their contribution to the anisotropic mechanical performance of the cortical bone. While increasing evidence highlights that such oriented fibers also exhibit osteogenic induction to preosteoblasts, little is known about the effect of the overlay angle between lamellae on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). In this study, bone lamellae-like fibrous matrices composed of aligned core-shell [core: polycaprolactone (PCL)/type I collagen (Col I) + shell: Col I] nanofibers were seeded with human BMSCs (hBMSCs) and then laid over on each other layer-by-layer (L-b-L) at selected angles (0 or 45°) to form three-dimensional (3D) constructs. Upon culture for 7 and 14 days, osteogenic differentiation of hBMSCs and mineralization within the lamellae assembly (LA) were characterized by real-time PCR, Western blot, immunofluorescent staining for osteogenic markers, and alizarin red staining for calcium deposition. Compared to those of random nanofibers (LA-RF) or aligned fibers with the overlay angle of 45° (LA-AF-45), the LA of aligned fibers at a 0° overlay angle (LA-AF-0) exhibited a noticeably higher osteogenic differentiation of hBMSCs, i.e., elevated gene expression of OPN, OCN, and RUNX2 and protein levels of ALP and RUNX2, while promoting mineral deposition as indicated by alizarin red staining and mechanical testing. Further analyses of hBMSCs within LA-AF-0 revealed an increase in both total and phosphorylated integrin β1, which subsequently increased total focal adhesion kinase (FAK), phosphorylated FAK (p-FAK), and phosphorylated extracellular signal kinase ERK1/2 (p-ERK1/2). Inhibition of integrin β1 and ERK1/2 activity effectively reduced the LA-AF-0-induced upregulation of p-FAK and osteogenic markers (OPN, OCN, and RUNX2), confirming the involvement of integrin β1-FAK-ERK1/2 signaling. Altogether, the overlay angle of aligned core-shell nanofiber membranes regulates the osteogenic differentiation of hBMSCs via integrin β1-FAK-ERK1/2 signaling, unveiling the effects of anisotropic fibers on bone tissue formation.

Effects of pulsed electromagnetic fields on bone fractures: a systematic review update.

Fractures are common bone injuries, which have a great burden on global health. Fracture healing is a long-term process that may be influenced by a number of factors. The 10-15% of all bone fractures may be complicated by an impaired healing (i.e. delayed union or non-union). The application of weak electromagnetic fields has been proposed to have different effects on bones such as enhancing proliferation - orientation - migration of osteoblast-like cells and supporting osteogenic differentiation in bone marrow-derived mesenchymal stem cells. Despite the amount of evidence on cellular and histological effects, to date the application of pulsed electromagnetic fields has not achieved univocal consensus in daily practice. The purpose of this systematic review update is to research, select, analyze and summarize the most recent scientific evidence regarding the effects of pulsed electromagnetic fields in the treatment of acute bone fractures. A systematic review using the following MeSH terms (Magnetic Field Therapy; Electromagnetic Fields; Bone and Bones; Fractures, Bone; Fractures Healing) and strings {("Magnetic Field Therapy [mh]" OR "Electromagnetic Fields [mh]) OR "magnetotherapy" OR "pulsed electromagnetic field") and ("Fractures, bone [mh]" OR "Bone and Bones [mh]") and ("Fractures Healing [mh]")} was conducted on PubMed, Cochrane Library, Epistemonikos and Scopus electronic databases. Only full articles published in English between January 2014 and December 2022 were considered. Eligibility criteria were defined according to the Population, Intervention, Comparison, Outcomes, and Study (PICOS) design framework. Case reports, case series, uncontrolled studies and expert opinions were excluded. All articles were checked for quality and risk of bias. Three randomized controlled trials were included (197 patients in total). None of the studies found significant effects of pulsed electromagnetic fields on the acute bone healing process. Contradictory results about pain relief emerged. Only one study showed a transitory positive effect of pulsed electromagnetic fields on strength and range of motion recovery in patients with acute bone fracture. This systematic review update does not support the use of pulsed electromagnetic fields for improving the bone healing process in patients with acute fractures. Controversial evidence was found about the effects of pulsed electromagnetic fields on pain and functional recovery. Considering the scant and heterogenous literature published to date, which represents a limit for our conclusion, further studies with rigorous and high-quality methodology are needed.

Bone quality relies on hyaluronan synthesis – Insights from mice with complete knockout of hyaluronan synthase expression

Bone consists of a complex mineralised matrix that is maintained by a controlled equilibrium of synthesis and resorption by different cell types. Hyaluronan (HA) is an important glycosaminoglycan in many tissues including bone.Previously, the importance of HA synthesis for bone development during embryogenesis has been shown. We therefore investigated whether HA synthesis is involved in adult bone turnover and whether abrogation of HA synthesis in adult mice would alter bone quality.To achieve complete abrogation of HA synthesis in adult mice, we generated a novel Has-total knockout (Has-tKO) mouse model in which a constitutive knockout of Has1 and Has3 was combined with an inducible, Ubc-Cre-driven Has2 knockout.By comparing bone tissue from wild-type, Has1,3 double knockout and Has-tKO mice, we demonstrate that Has2-derived HA mainly contributes to the HA content in bone. Furthermore, Has-tKO mice show a significant decrease of bone integrity in trabecular and cortical bone, as shown by µ-CT analysis. These effects are detectable as early as five weeks after induced Has2 deletion, irrespective of sex and progress with age.Mesenchymal stem cells (MSC) during osteogenic differentiation in vitro showed that Has2 expression is increased while Has3 expression is decreased during differentiation. Furthermore, the complete abrogation of HA synthesis results in significantly reduced osteogenic differentiation as indicated by reduced marker gene expression (Runx-2, Tnalp, Osterix) as well as alizarin red staining. RNAseq analysis revealed that MSC from Has-tKO are characterised by decreased expression of genes annotated for bone and organ development, whereas expression of genes associated with chemokine related interactions and cytokine signalling is increased.Taken together, we present a novel mouse model with complete deletion of HA synthases in adult mice which has the potential to study HA function in different organs and during age-related HA reduction. With respect to bone, HA synthesis is important for maintaining bone integrity, presumably based on the strong effect of HA on osteogenic differentiation.

Pre-vascularized porous gelatin-coated β-tricalcium phosphate scaffolds for bone regeneration: an in vivo and in vitro investigation.

Vascularization is vital in bone tissue engineering, supporting development, remodeling, and regeneration. Lack of vascularity leads to cell death, necessitating vascularization strategies. Angiogenesis, forming new blood vessels, provides crucial nutrients and oxygen. Pre-vascularized gelatin-coated β-tricalcium phosphate (G/β-TCP) scaffolds show promise in bone regeneration and vascularization. Our study evaluates G/β-TCP scaffolds' osteogenic and angiogenic potential in vitro and a canine model with vascular anastomosis. Channel-shaped G/β-TCP scaffolds were fabricated using foam casting and sintering of a calcium phosphate/silica slurry-coated polyurethane foam, then coated with cross-linked gelatin. Buccal fat pad-derived stem cells (BFPdSCs) were seeded onto scaffolds and assessed over time for adhesion, proliferation, and osteogenic capacity using scanning electron microscopy (SEM), 4,6-diamidino-2-phenylindole (DAPI) staining, Alamar blue, and alkaline phosphatase (ALP) assays. Scaffolds were implanted in a canine model to evaluate osteogenesis and angiogenesis by histology and CT scans at 12 wk. Our studies showed preliminary results for G/β-TCP scaffolds supporting angiogenesis and bone regeneration. In vitro analyses demonstrated excellent proliferation/viability, with BFPdSCs adhering and increasing on the scaffolds. ALP activity and protein levels increased, indicating osteogenic differentiation. Examination of tissue samples revealed granulation tissue with a well-developed vascular network, indicating successful angiogenesis and osteogenesis was further confirmed by a CT scan. In vivo, histology revealed scaffold resorption. However, scaffold placement beneath muscle tissue-restricted bone regeneration. Further optimization is needed for bone regeneration applications.

Polydopamine-based surface coating fabrication on titanium implant by combining a photothermal agent and TiO2 nanosheets for efficient photothermal antibacterial therapy and promoted osteogenic activity

Developing titanium-based dental implants with both excellent antibacterial properties and good osseointegration is crucial for the success of the implant operation and the long-term durability of the implant. In this study, a polydopamine-based coating was created by attaching TiO2 nanosheets-cyanine composites onto the titanium surface, enabling the integration of effective photothermal antibacterial therapy with osseointegration. The exceptional dual-photothermal conversion abilities of polydopamine and cyanine in the coating resulted in outstanding photothermal antibacterial and antibiofilm therapy against four types of bacteria. Furthermore, TiO2 nanosheets promoted the adhesion, proliferation and early osteogenic differentiation of osteoblasts. In an infected dental implant model in rats, the developed coating exhibited potent antibacterial activity and remarkable osteogenic differentiation in the bone, leading to increased bone formation around the implants. This innovative approach, combining photothermal therapy with osteogenic two-dimensional nanomaterials, presents a novel method for surface functionalization of implants to achieve effective antibacterial and osseointegration capabilities.

Spatial Transcriptomics Unravel the Tissue Complexity of Oral Pathogenesis.

Spatial transcriptomics (ST) is a cutting-edge methodology that enables the simultaneous profiling of global gene expression and spatial information within histological tissue sections. Traditional transcriptomic methods lack the spatial resolution required to sufficiently examine the complex interrelationships between cellular regions in diseased and healthy tissue states. We review the general workflows for ST, from specimen processing to ST data analysis and interpretations of the ST dataset using visualizations and cell deconvolution approaches. We show how recent studies used ST to explore the development or pathogenesis of specific craniofacial regions, including the cranium, palate, salivary glands, tongue, floor of mouth, oropharynx, and periodontium. Analyses of cranial suture patency and palatal fusion during development using ST identified spatial patterns of bone morphogenetic protein in sutures and osteogenic differentiation pathways in the palate, in addition to the discovery of several genes expressed at critical locations during craniofacial development. ST of salivary glands from patients with Sjögren's disease revealed co-localization of autoimmune antigens with ductal cells and a subpopulation of acinar cells that was specifically depleted by the dysregulated autoimmune response. ST of head and neck lesions, such as premalignant leukoplakia progressing to established oral squamous cell carcinomas, oral cancers with perineural invasions, and oropharyngeal lesions associated with HPV infection spatially profiled the complex tumor microenvironment, showing functionally important gene signatures of tumor cell differentiation, invasion, and nontumor cell dysregulation within patient biopsies. ST also enabled the localization of periodontal disease-associated gene expression signatures within gingival tissues, including genes involved in inflammation, and the discovery of a fibroblast subtype mediating the transition between innate and adaptive immune responses in periodontitis. The increased use of ST, especially in conjunction with single-cell analyses, promises to improve our understandings of craniofacial development and pathogenesis at unprecedented tissue-level resolution in both space and time.

Selenium-doped carbon dots inhibit ferroptosis by multi-hierarchy iron chelation and mitochondrial homeostasis regulation to control inflammation

Ferroptosis has been identified as a prevalent pathological process in both infectious and non-infectious inflammation. Multiregional iron accumulation within extracellular, cytoplasmic, and subcellular compartments, particularly into mitochondria, can trigger lipid peroxidation and subsequent ferroptosis. Meanwhile, mitochondrial dysfunction, as a hallmark feature of ferroptosis, also exacerbates ferroptosis and inflammation. Here, a strategy of multi-hierarchy iron chelation coupled with mitochondrial homeostasis regulation is proposed to inhibit ferroptosis and relieve inflammation based on selenium-doped carbon dots (SeCDs). SeCDs with reactive oxygen species (ROS) scavenging capacity can be efficiently internalized into cells and enriched in mitochondria. Through the multi-hierarchy chelation for extracellular, cytoplasmic, and mitochondrial iron, iron overload is remarkably inhibited. Meanwhile, promoting glutathione peroxidase 4 (GPX4) expression and activating the AMPK pathway can restrain the metabolism of glycerophospholipids and polyunsaturated fatty acids and lipid peroxidation. Importantly, SeCDs restore mitochondrial homeostasis by chelating mitochondrial iron, scavenging mtROS, and increasing the level of GPX4, thereby enhancing ferroptosis suppression. Further, SeCDs rescue the osteogenic differentiation potential of stem cells, which is generally weakened by ferroptosis. In vivo experiments prove that SeCDs can significantly inhibit ferroptosis and inflammation, and facilitate tissue regeneration. Thus, this multifunctional nanosystem provides a promising therapeutic strategy to inhibit ferroptosis and ease inflammation.

An immunomodulatory and osteogenic bacterial cellulose scaffold for bone regeneration via regulating the immune microenvironment

Creating a bone homeostasis microenvironment that balances osteogenesis and immunity is a substantial challenge for bone regeneration. Here, we prepared an immunomodulatory and osteogenic bacterial cellulose scaffold (FOBS) via a facile one-pot approach. The aldehyde groups were generated via selective oxidation of the hydroxyl groups of bacterial cellulose, offering the bonding sites for dopamine through a Schiff base reaction. At the same time, the deposition of Ca2+ and PO43− was promoted on the aldehyde cellulose scaffold because of the high affinity of the catechol moiety for Ca2+. Compared with that of the unmodified scaffold, the hydroxyapatite content of FOBS increased by 47.1 % according to the ICP results. Interestingly, FOBS regulated the immune microenvironment to accelerate the conversion of M1 to M2 macrophages. The expressions of ARG-1 and Dectin-1 (M2) in the FOBS group increased by >100 %. The expression of osteogenic differentiation of BMSCs was also upregulated. In a rat cranial defect model, the BV/TV of FOBS was significantly increased. Further immunohistochemical analysis revealed that an improved immune microenvironment promoted the osteogenic differentiation of stem cells in vivo. This work provides an effective and easy-to-operate strategy for the development of the bone tissue engineering scaffolds.

Macrophage-derived amphiregulin promoted the osteogenic differentiation of chondrocytes through EGFR/Yap axis and TGF-β activation

Endochondral ossification represents a crucial biological process in skeletal development and bone defect repair. Macrophages, recognized as key players in the immune system, are now acknowledged for their substantial role in promoting endochondral ossification within cartilage. Concurrently, the epidermal growth factor receptor (EGFR) ligand amphiregulin (Areg) has been documented for its contributory role in restoring bone tissue homeostasis post-injury. However, the mechanism by which macrophage-secreted Areg facilitates bone repair remains elusive. In this study, the induction of macrophage depletion through in vivo administration of clodronate liposomes was employed in a standard open tibial fracture mouse model to assess bone healing using micro-computed tomography (micro-CT) analysis, histomorphology, and ELISA serum evaluations. The investigation revealed sustained expression of Areg during the fracture healing period in wild-type mice. Macrophage depletion significantly reduced the number of macrophages on the local bone surface and vital organs. This reduction led to diminished Areg secretion, decreased collagen production, and delayed fracture healing. However, histological and micro-CT assessments at 7 and 21 days post-local Areg treatment exhibited a marked improvement of bone healing compared to the vehicle control. In vitro studies demonstrated an increase of Areg secretion by the Raw264.7 cells upon ATP stimulation. Indirect co-culture of Raw264.7 and ATDC5 cells indicated that Areg overexpression enhanced the osteogenic potential of chondrocytes, and vice versa. This osteogenic promotion was attributed to Areg's activation of the membrane receptor EGFR in the ATDC5 cell line, the enhanced phosphorylation of transcription factor Yap, and the facilitation of the expression of bioactive TGF-β by chondrocytes. Collectively, this research elucidates the direct mechanistic effects of macrophage-secreted Areg in promoting bone homeostasis following bone injury.

IMPC-based screening revealed that ROBO1 can regulate osteoporosis by inhibiting osteogenic differentiation.

The utilization of denosumab in treating osteoporosis highlights promising prospects for osteoporosis intervention guided by gene targets. While omics-based research into osteoporosis pathogenesis yields a plethora of potential gene targets for clinical transformation, identifying effective gene targets has posed challenges. We first queried the omics data of osteoporosis clinical samples on PubMed, used International Mouse Phenotyping Consortium (IMPC) to screen differentially expressed genes, and conducted preliminary functional verification of candidate genes in human Saos2 cells through osteogenic differentiation and mineralization experiments. We then selected the candidate genes with the most significant effects on osteogenic differentiation and further verified the osteogenic differentiation and mineralization functions in mouse 3T3-E1 and bone marrow mesenchymal stem cells (BMSC). Finally, we used RNA-seq to explore the regulation of osteogenesis by the target gene. We identified PPP2R2A, RRBP1, HSPB6, SLC22A15, ADAMTS4, ATP8B1, CTNNB1, ROBO1, and EFR3B, which may contribute to osteoporosis. ROBO1 was the most significant regulator of osteogenesis in both human and mouse osteoblast. The inhibitory effect of Robo1 knockdown on osteogenic differentiation may be related to the activation of inflammatory signaling pathways. Our study provides several novel molecular mechanisms involved in the pathogenesis of osteoporosis. ROBO1 is a potential target for osteoporosis intervention.

ALKBH5 regulates etoposide-induced cellular senescence and osteogenic differentiation in osteoporosis through mediating the m6A modification of VDAC3.

Osteoporosis, a common bone disease in older individuals, involves the progression influenced by N6-methyladenosine (m6A) modification. This study aimed to elucidate the effects of VDAC3 m6A modification on human bone mesenchymal stromal cell (BMSC) senescence and osteogenic differentiation. BMSCs were treated with etoposide to induce senescence. Senescence was assessed by β-galactosidase staining and quantitative real-time PCR (qPCR), and osteogenic differentiation was evaluated using Western blot, alkaline phosphatase, and alizarin red S staining. VDAC3 and ALKBH5 expression were quantified by qPCR, and their interaction was assessed by RNA immunoprecipitation (RIP) and luciferase reporter assay. m6A methylation was analyzed using the Me-RIP assay. VDAC3 expression was significantly decreased in etoposide-treated BMSCs (1.00 ± 0.13 vs. 0.26 ± 0.06). VDAC3 overexpression reduced etoposide-induced senescence and promoted osteogenic differentiation. ALKBH5 overexpression inhibited VDAC3 m6A modification (1.00 ± 0.095 vs. 0.233 ± 0.177) and its stability. ALKBH5 knockdown decreased etoposide-induced senescence and promoted osteogenic differentiation, effects that were reversed by VDAC3 knockdown. YTHDF1 was identified as the m6A methylation reader, and its overexpression inhibited VDAC3 stability. We demonstrated that ALKBH5 inhibited osteogenic differentiation of etoposide-induced senescent cells through the inhibition of VDAC3 m6A modification, and YTHDF1 acted as the m6A methylation reader. These findings provide a novel theoretical basis for the treatment of osteoporosis.

Mechanism research of elastic fixation promoting fracture healing based on proteomics and fracture microenvironment.

This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process. Mean callus volume was larger in the elastic fixation group (1,755 mm3 (standard error of the mean (SEM) 297)) than in the stiff fixation group (258 mm3 (SEM 65)). Pathological observation found that the expression levels of osterix (OSX), collagen, type I, alpha 1 (COL1α1), and alkaline phosphatase (ALP) in the callus of the elastic fixation group were higher than those of the stiff fixation group. The protein sequence of the callus revealed 199 DEPs, 124 of which were highly expressed in the elastic fixation group. In the in vitro study, it was observed that a stress of 200 g led to upregulation of thrombospondin 1 (THBS1) and osteoglycin (OGN) expression in bone marrow mesenchymal stem cells (BMSCs). Additionally, these genes were found to be upregulated during the osteogenic differentiation process of the BMSCs. Elastic fixation can promote fracture healing and osteoblast differentiation in callus, and the ability of elastic fixation to promote osteogenic differentiation of BMSCs may be achieved by upregulating genes such as THBS1 and OGN.

Photoactivated Hydrogel Therapeutic System with MXene-Based Nanoarchitectonics Potentiates Endogenous Bone Repair Through Reshaping the Osteo-Vascularization Network.

The repair and reconstruction of large-scale bone defects face enormous challenges because of the failure to reconstruct the osteo-vascularization network. Herein, a near-infrared (NIR) light-responsive hydrogel system is reported to achieve programmed tissue repair and regeneration through the synergetic effects of on-demand drug delivery and mild heat stimulation. The spatiotemporal hydrogel system (HG/MPa) composed of polydopamine-coated Ti3C2Tx MXene (MP) nanosheets decorated with acidic fibroblast growth factor (aFGF, a potent angiogenic drug) and hydroxypropyl chitosan/gelatin (HG) hydrogel is developed to orchestrate the reconstruction of the osteo-vascularization network and boost bone regeneration. Upon exposure to NIR light irradiation, the engineered HG/MPa hydrogel can achieve the initial complete release of aFGF to induce rapid angiogenesis and provide sufficient blood supply, maximizing its biofunction in the defect area. This integrated hydrogel system demonstrated good therapeutic efficacy in promoting cell adhesion, proliferation, migration, angiogenesis, and osteogenic differentiation through periodic NIR irradiation. In vivo, animal experiments further revealed that the spatiotemporalized hydrogel platform synergized with mild photothermal treatment significantly accelerated critical-sized bone defect healing by increasing the osteo-vascularization network density, recruiting endogenous stem cells, and facilitating the production of osteogenesis/angiogenesis-related factors. Overall, smart-responsive hydrogel couldenhance the reconstruction of the osteo-vascularization network in bone regeneration.

SLAMF8 regulates osteogenesis and adipogenesis of bone marrow mesenchymal stem cells via S100A6/Wnt/β-catenin signaling pathway

BackgroundThe inflammatory microenvironment plays an essential role in bone healing after fracture. The signaling lymphocytic activation molecule family (SLAMF) members deeply participate in inflammatory response and make a vast difference.MethodsWe identified SLAMF8 in GEO datasets (GSE129165 and GSE176086) and co-expression analyses were performed to define the relationships between SLAMF8 and osteogenesis relative genes (RUNX2 and COL1A1). In vitro, we established SLAMF8 knockdown and overexpression mouse bone marrow mesenchymal stem cells (mBMSCs) lines. qPCR, Western blot, ALP staining, ARS staining, Oil Red O staining and Immunofluorescence analyses were performed to investigate the effect of SLAMF8 in mBMSCs osteogenesis and adipogenesis. In vivo, mice femoral fracture model was performed to explore the function of SLAMF8.ResultsSLAMF8 knockdown significantly suppressed the expression of osteogenesis relative genes (RUNX2, SP7 and COL1A1), ALP activity and mineral deposition, but increased the expression of adipogenesis relative genes (PPARγ and C/EBPα). Additionally, SLAMF8 overexpression had the opposite effects. The role SLAMF8 played in mBMSCs osteogenic and adipogenic differentiation were through S100A6 and Wnt/β-Catenin signaling pathway. Moreover, SLAMF8 overexpression mBMSCs promoted the healing of femoral fracture.ConclusionsSLAMF8 promotes osteogenesis and inhibits adipogenesis of mBMSCs via S100A6 and Wnt/β-Catenin signaling pathway. SLAMF8 overexpression mBMSCs effectively accelerate the healing of femoral fracture in mice.

Inflammatory priming of human mesenchymal stem cells induces osteogenic differentiation via the early response gene IER3.

Mesenchymal stem cells (MSCs) have gained tremendous interest due to their overall potent pro-regenerative and immunomodulatory properties. In recent years, various invitro and preclinical studies have investigated different priming ("licensing") approaches to enhance MSC functions for specific therapeutic purposes. In this study, we primed bone marrow-derived human MSCs (hMSCs) with an inflammation cocktail designed to mimic the elevated levels of inflammatory mediators found in serum of patients with severe injuries, such as bone fractures. We observed a significantly enhanced osteogenic differentiation potential of primed hMSCs compared to untreated controls. By RNA-sequencing analysis, we identified the immediate early response 3 (IER3) gene as one of the top-regulated genes upon inflammatory priming. Small interfering RNA knockdown experiments established IER3 as a novel positive regulator of osteogenic differentiation. Mechanistic analysis further revealed that IER3 deletion significantly downregulated bone marrow stromal cell antigen 2 (BST2) expression and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in hMSCs, suggesting that IER3 regulates osteogenic differentiation through BST2 and ERK1/2 signaling pathway activation. On the basis of these findings, we propose IER3 as a novel therapeutic target to promote hMSC osteoblastogenesis, which might be of high clinical relevance, for example, in patients with osteoporosis or compromised fracture healing.

NEDD4L affects stability of the CHEK2/TP53 axis through ubiquitination modification to enhance osteogenic differentiation of periodontal ligament stem cells

ABSTRACT Background Checkpoint kinase 2 (CHEK2) and its regulated tumor protein p53 (TP53) have been correlated with osteogenic differentiation of osteoblast-like cells. Based on bioinformatics predictions, this study aims to investigate the effect of the CHEK2/TP53 axis on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to explore the regulatory mechanism. Methods PDLSCs were isolated from human impacted wisdom teeth, and they were cultured in normal medium (NM) or osteogenic medium (OM). Protein levels of CHEK2 and TP53 were examined using western blot analysis. Osteogenic differentiation ability of PDLSCs was analyzed by measuring marker proteins (RUNX2, OCN, and OSX), ALP activity, and ALP staining. Molecular interaction between NEDD4 like E3 ubiquitin protein ligase (NEDD4L) and CHEK2 was examined by ubiquitination and co-immunoprecipitation assays. Gain- and loss-of function assays of NEDD4L, CHEK2, and TP53 were performed to analyze their function in osteogenic differentiation of PDLSCs. A rat model of mandibular bone defect was generated for in vivo validation. Results NEDD4L was upregulated, while CHEK2 and TP53 were downregulated in PDLSCs cultured in OM. CHEK2 protected TP53 from degradation, while NEDD4L reduced CHEK2 protein level by ubiquitination modification. NEDD4L silencing reduced osteogenic differentiation ability of PDLSCs both in vitro and in vivo, which was restored by CHEK2 silencing. By contrast, CHEK2 overexpression blocked the osteogenic differentiation of PDLSCs in vitro. Conclusion This study demonstrates that NEDD4L affects protein stability of the CHEK2/TP53 axis through ubiquitination modification, thus increasing osteogenic differentiation of PDLSCs.

Biomimetic Scaffolds Regulating the Iron Homeostasis for Remolding Infected Osteogenic Microenvironment.

The treatment of infected bone defects (IBDs) needs simultaneous elimination of infection and acceleration of bone regeneration. One mechanism that hinders the regeneration of IBDs is the iron competition between pathogens and host cells, leading to an iron deficient microenvironment that impairs the innate immune responses. In this work, an in situ modification strategy is proposed for printing iron-active multifunctional scaffolds with iron homeostasis regulation ability for treating IBDs. As a proof-of-concept, ultralong hydroxyapatite (HA) nanowires are modified through in situ growth of a layer of iron gallate (FeGA) followed by incorporation in the poly(lactic-co-glycolic acid) (PLGA) matrix to print biomimetic PLGA based composite scaffolds containing FeGA modified HA nanowires (FeGA-HA@PLGA). The photothermal effect of FeGA endows the scaffolds with excellent antibacterial activity. The released iron ions from the FeGA-HA@PLGA help restore the iron homeostasis microenvironment, thereby promoting anti-inflammatory, angiogenesis and osteogenic differentiation. The transcriptomic analysis shows that FeGA-HA@PLGA scaffolds exert anti-inflammatory and pro-osteogenic differentiation by activating NF-κB, MAPK and PI3K-AKT signaling pathways. Animal experiments confirm the excellent bone repair performance of FeGA-HA@PLGA scaffolds for IBDs, suggesting the promising prospect of iron homeostasis regulation therapy in future clinical applications.