Defect Rat Model Research Articles

Sequential delivery of IL-10 and icariin using nanoparticle/hydrogel hybrid system for prompting bone defect repair

The treatment of large bone defects remains challenging due to the lack of spatiotemporal management of the immune microenvironment, inflammation response and bone remodeling. To address these issues, we designed and developed a nanoparticle/hydrogel hybrid system that can achieve the combined and sequential delivery of an anti-inflammatory factor (IL-10) and osteogenic drug (icariin, ICA). A photopolymerizable composite hydrogel was prepared by combining gelatin methacryloyl (GelMA) and heparin-based acrylated hyaluronic acid (HA) hydrogels containing IL-10, and poly(dl-lactide-co-glycolide) (PLGA)-HA nanoparticles loaded with ICA were incorporated into the composite hydrogels. The nanoparticle/hydrogel hybrid system demonstrates an array of features including mechanical strength, injectability and photo-crosslinking. The rapid release of IL-10 from the hydrogel effectively exerts immunomodulatory activity, whereas the long-term sustained release of icariin from the PLGA-HA nanoparticles significantly triggers the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Notably, the combined delivery of IL-10 and ICA from the hybrid system exhibit a synergistic effect for bone remodeling in a critical cranial defect rat model. Our findings indicate the importance of the immunomodulatory microenvironment and osteogenic differentiation for high-quality skull remodeling, and thus the dual-factor releasing nanoparticle/hydrogel hybrid system could be a promising candidate for repairing bone defects.

Dental pulp stem cell‑derived extracellular vesicles loaded with hydrogels promote osteogenesis in rats with alveolar bone defects.

Alveolar bone defects caused by inflammation, trauma and tumors adversely affect periodontal health, causing tooth loosening or dentition defects, thus affecting denture or implant repair. Advancements in tissue engineering technology and stem cell biology have significantly improved the regenerative reconstruction of alveolar bone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells play important roles in exerting their therapeutic effects. Several studies have reported the role of dental pulp stem cells (DPSCs) in bone regeneration, but the regenerative effects of DPSC‑EVs on alveolar bone defects are unclear. In the present study, the osteogenic effects of DPSC‑EVs on Hertwig's epithelial root sheath (HERS) cells in vitro and their osteoinductive effects in an alveolar bone defect rat model were investigated. The results showed that DPSC‑EVs significantly promoted the expression of osteogenic genes, such as runt‑related transcription factor 2 and alkaline phosphatase, and increased the osteogenic differentiation capability of HERS. These findings suggested that transforming growth factor β1 inhibition decreased DPSC‑EV‑induced Smad, MAPK and ERK phosphorylation in HERS. In vivo, DPSC‑EV‑loaded hydrogels were transplanted into the alveolar sockets of Sprague‑Dawley rats and observed for eight weeks. The new bone grew concentrically in the DPSC‑EV or DPSC‑EV‑loaded hydrogel group, with greater bone mass than that in the control group, and the bone volume/total volume increased notably. The results confirmed the osteogenic and osteoinductive effects of DPSC‑EVs and DPSC‑Exo‑loaded hydrogels on alveolar bone defects. Due to their low immunogenicity, high stability, good biocompatibility and osteogenic propensity, DPSC‑EV‑loaded hydrogels are a safe cell‑free therapeutic approach for defective alveolar bone regeneration.

Multifunctional Dual Nano-MOF-Modified Decellularized Small Intestinal Submucosa Membrane Accelerates Healing of Infected Wound.

The treatment of complex or chronic skin wounds caused by burns, trauma, surgery, and genetic disorders has been a worldwide challenge. Small intestinal submucosa (SIS) is a biological material that is widely used in wound healing. How to further expand the wound healing application of SIS, especially in repairing infected wounds, remains a hot research topic for many tissue engineering and biomaterial scholars focusing on skin regeneration. This study uses nanometal-organic frameworks (nano-MOFs), which have not been applied to modify the SIS membrane before, to construct multifunctional dual nano-MOFs @ SIS membrane (dnMOF@SISm). Nano-MOFs are functionalized onto the nanofiber of SIS via in situ self-assembly under mild reaction conditions without any toxic reagent or complex instruments. The dnMOF@SISm can release Co2+, Zn2+, and bioactive factors, participating in the whole stage of the repair of infected wounds. In vitro, it can regulate the biological activities of various functional cells such as fibroblasts, endothelial cells, and macrophages and shows good antibacterial ability. In the infected full-thickness skin defect rat model, dnMOF@SISm can release metal ions and ligands, killing pathogenic bacteria colonized on the wound surface at the first stage, and then trigger and accelerate the skin repair process via angiogenesis, immune regulation, and collagen deposition. Above all, an efficient, nontoxic, mild self-assembly strategy realizes the functionalization of dual nano-MOFs on the nanofiber of SIS to expand its clinical application scenarios, especially in infected wounds.

Development of Functionalized Poly(ε-caprolactone)/Hydroxyapatite Scaffolds via Electrospinning 3D for Enhanced Bone Regeneration.

Functionalized scaffolds based on biodegradable polymers are materials used in bone tissue engineering. This study presents the development of functionalized fibrous scaffolds, fabricated from poly(ε-caprolactone) (PCL) and hydroxyapatite (HA). To produce this material, a short-distance electrospinning (ES) system was developed by adapting a 3D printer. The morphology and chemical properties of the scaffolds were evaluated using scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The results confirmed the porous structure and the presence of hydroxyapatite throughout the entire scaffold area. Mechanical tests indicated good elasticity and tensile strength of the scaffolds, favorable for bone regeneration. In vitro tests showed high levels of cell viability. Furthermore, in vivo experiments using a calvarial defect model in rats demonstrated that the PCL/HA scaffold promoted enhanced bone regeneration. Therefore, the PCL/HA scaffold developed through the adapted electrospinning system shows promise for bone repair.

Enhancing osteogenesis and mandibular defect repair with magnesium-modified acellular bovine bone matrix

An acellular bovine bone matrix modified to release Magnesium ions (Mg2+) (ABBM-Mg) was prepared and evaluated for its potential in osteogenesis and mandibular defect repair. Mg2+ was incorporated into ABBM using an ion exchange method. The microstructure and mechanical properties of both ABBM and ABBM-Mg were analyzed using SEM and a biomechanical testing machine. Cytocompatibility, cell adhesion, and osteogenic differentiation were assessed using various methods including CCK-8, Live/Dead staining, SEM, ALP staining, and qPCR analysis in MC3T3-E1 cells. Additionally, a mandibular defect model in rats was established. The bone defect repair outcomes were evaluated using Micro-CT, histological HE staining, and Masson staining. The study showed that mineralization containing magnesium was redeposited on the surface of the three-dimensional porous ABBM, and the ABBM-Mg scaffold promoted cell proliferation and osteogenic differentiation compared to the ABBM scaffold. In the rat mandibular defect model, the ABBM-Mg scaffold demonstrated superior bone repair ability. This study successfully incorporated Mg2+ into ABBM without significantly affecting its microstructure and compressive strength. Furthermore, ABBM-Mg showed sustained release of Mg2+ which enhanced cell proliferation, adhesion, and osteogenic differentiation in vitro, and promoted mandibular defect healing in rats. This research opens up new possibilities for the clinical application of functionalized acellular bone matrix.Graphical

Comparison of the Effects of Microfracture, Soft Callus Implantation, and Matrix-Supported Chondrocyte Implantation in an Experimental Osteochondral Defect Model in Rats

Background: The treatment of cartilage defects remains challenging due to the avascular nature of cartilage. Aim: This study investigates the therapeutic effect of soft callus in osteochondral defects and explores the ability of multipotent and pluripotent cells within the callus to form fibrous or hyaline cartilage in the defective area. Methods: Twenty-one rats were divided into three equal groups: Group 1 received only microfracture (MF), group 2 received microfracture with autologous chondrocyte implantation (MF+ACI), and group 3 received microfracture with soft callus implantation (MF+SCI). All rats underwent diaphyseal fracture in their left tibias, which was stabilized with a Kirshner wire. One week later, osteochondral defects were created in the right knees of all rats. For group 1, microfracture alone was applied to initiate healing in the defects. In group 2, heterologous chondrocytes, previously harvested from the lateral condyle of a rat’s left femur and cultivated in a laboratory environment, were implanted into the microfracture site. In group 3, soft callus tissue obtained from the left tibial fracture was compressed and implanted into the defective area. All groups were sacrificed at the 6th week, and the healing status of the osteochondral defect areas was histopathologically evaluated. Results: Macroscopic examination at the end of the study revealed comparable ICRS-1 scores for MF+ACI (group 2) (11.28 ± 1.25) and MF+SCI (group 3) (11.14 ± 0.37), while MF alone (group 1) (4.28 ± 1.25) showed significantly lower results. Microscopic examination yielded similar outcomes. Regarding histological scores, ICRS-2 scores for MF (group 1) (35.30 ± 1.13), MF+ACI (group 2) (47.09 ± 1.63), and MF+SCI (group 3) (43.97 ± 1.49) were statistically significantly lower. Conclusion: Defects treated with soft callus implantation demonstrated comparable outcomes to the widely used and gold-standard autologous chondrocyte implantation. When compared to microfracture alone, better macroscopic and microscopic results were achieved with soft callus implantation.

Polydatin accelerates osteoporotic bone repair by inducing the osteogenesis-angiogenesis coupling of bone marrow mesenchymal stem cells via the PI3K/AKT/GSK-3β/β-catenin pathway.

Polydatin (POL), a natural stilbenoid, has multiple pharmacological activities. However, its effect on osteoporotic bone defect has not yet been examined. This study was designed to explore the unknown role of POL on osteoporotic bone repair. The effect of POL on osteogenesis and angiogenesis were investigated firstly. Then a series of angiogenesis-related assays were carried out to explore the relationship between osteogenesis and angiogenesis of POL, and the underlying mechanism was further explored. Whereafter, ovariectomy-induced osteoporosis rats with bone defect were treated with POL or placebo, the imageological and histological examinations were conducted to assess the effect of POL on osteoporotic bone repair. The moderate concentrations (1μM and 10μM) of POL enhanced osteogenesis of bone marrow mesenchymal stem cells (BMSCs) and elevated the expression of angiogenic-specific markers. Further research found that POL induced human umbilical vein endothelial cells migration and tube formation through the osteogenesis-angiogenesis coupling of BMSCs, and the POL-induced osteogenesis-angiogenesis coupling was reversed after co-cultured with LY294002, Mechanistically, this was conducted via activating PI3K/AKT/GSK-3β/β-catenin pathway. After that, using osteoporotic bone defect rat model, we observed that POL facilitated osteoporotic bone repair through enhancing osteogenesis and CD31hiEMCNhi type H-positive vessels formation via the PI3K/AKT/GSK-3β/β-catenin pathway. The data above indicated that POL could accelerate osteoporotic bone repair by inducing the osteogenesis-angiogenesis coupling of BMSCs via the PI3K/AKT/GSK-3β/β-catenin pathway, which provided new insight and strategy for osteoporotic bone repair.

2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside–stimulated dental pulp stem cells-derived exosomes for wound healing and bone regeneration

Background/purpose-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is a bioactive component in the Chinese herb Polygonum multiflorum, recognized for its anti-inflammatory and lipid-lowering properties. Human dental pulp stem cells (hDPSCs) have excellent capabilities in tooth regeneration, wound healing, and neural repair. The exosomes (Exo) released by hDPSCs contain bioactive molecules that influence cell proliferation, differentiation, and immune responses. Therefore, we aimed to unveil the potential of THSG-Exo and evaluate its regenerative capabilities through the in vitro experiment and rat bone defect model. Materials and methodsThe effects of hDPSC-derived exosomes, with or without THSG treatment, on repair and bone regeneration were evaluated through in vitro and in vivo studies. Finally, we conducted a proteomic analysis to meticulously compare the compositional contents of the two types of exosomes. ResultsIn vitro data showed that 10 and 100 μM THSG-Exo enhanced cell proliferation and osteogenic differentiation, reducing wound size to 40 % of its original size. In our maxillary bone defect rat model, THSG-Exo significantly increased bone volume, trabecular thickness, and bone density in the bone defect area. In addition, proteomic analysis of THSG-Exo revealed diverse proteins linked to bone differentiation and tissue repair, including bone morphogenetic protein-1 (BMP-1) and tumor necrosis factor (TNF)-α-stimulated gene 6 (TNFAIP6). Our searches in functional databases revealed that THSG-Exo is involved in numerous biological pathways. ConclusionTHSG-Exo enhanced cell proliferation, wound healing, and osteogenesis in vitro, while also expediting tissue repair and bone regeneration in vivo. The protein diversity of THSG-Exo contributes significant value in both basic and regenerative medicine.

GO/Cu Nanosheet-Integrated Hydrogel Platform as a Bioactive and Biocompatible Scaffold for Enhanced Calvarial Bone Regeneration.

The treatment of craniofacial bone defects caused by trauma, tumors, and infectious and degenerative diseases is a significant issue in current clinical practice. Following the rapid development of bone tissue engineering (BTE) in the last decade, bioactive scaffolds coupled with multifunctional properties are in high demand with regard to effective therapy for bone defects. Herein, an innovative bone scaffold consisting of GO/Cu nanoderivatives and GelMA-based organic-inorganic hybrids was reported for repairing full-thickness calvarial bone defect. In this study, motivated by the versatile biological functions of nanomaterials and synthetic hydrogels, copper nanoparticle (CuNP)-decorated graphene oxide (GO) nanosheets (GO/Cu) were combined with methacrylated gelatin (GelMA)-based organic-inorganic hybrids to construct porous bone scaffolds that mimic the extracellular matrix (ECM) of bone tissues by photocrosslinking. The material characterizations, in vitro cytocompatibility, macrophage polarization and osteogenesis of the biohybrid hydrogel scaffolds were investigated, and two different animal models (BALB/c mice and SD rats) were established to further confirm the in vivo neovascularization, macrophage recruitment, biocompatibility, biosafety and bone regenerative potential. We found that GO/Cu-functionalized GelMA/β-TCP hydrogel scaffolds exhibited evidently promoted osteogenic activities, M2 type macrophage polarization, increased secretion of anti-inflammatory factors and excellent cytocompatibility, with favorable surface characteristics and sustainable release of Cu2+. Additionally, improved neovascularization, macrophage recruitment and tissue integration were found in mice implanted with the bioactive hydrogels. More importantly, the observations of microCT reconstruction and histological analysis in a calvarial bone defect model in rats treated with GO/Cu-incorporated hydrogel scaffolds demonstrated significantly increased bone morphometric values and newly formed bone tissues, indicating accelerated bone healing. Taken together, this BTE-based bone repair strategy provides a promising and feasible method for constructing multifunctional GO/Cu nanocomposite-incorporated biohybrid hydrogel scaffolds with facilitated osteogenesis, angiogenesis and immunoregulation in one system, with the optimization of material properties and biosafety, it thereby demonstrates great application potential for correcting craniofacial bone defects in future clinical scenarios.

T. gondii excretory proteins promote the osteogenic differentiation of human bone mesenchymal stem cells via the BMP/Smad signaling pathway

BackgroundBone defects, resulting from substantial bone loss that exceeds the natural self-healing capacity, pose significant challenges to current therapeutic approaches due to various limitations. In the quest for alternative therapeutic strategies, bone tissue engineering has emerged as a promising avenue. Notably, excretory proteins from Toxoplasma gondii (TgEP), recognized for their immunogenicity and broad spectrum of biological activities secreted or excreted during the parasite’s lifecycle, have been identified as potential facilitators of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Building on our previous findings that TgEP can enhance osteogenic differentiation, this study investigated the molecular mechanisms underlying this effect and assessed its therapeutic potential in vivo.MethodsWe determined the optimum concentration of TgEP through cell cytotoxicity and cell proliferation assays. Subsequently, hBMSCs were treated with the appropriate concentration of TgEP. We assessed osteogenic protein markers, including alkaline phosphatase (ALP), Runx2, and Osx, as well as components of the BMP/Smad signaling pathway using quantitative real-time PCR (qRT-PCR), siRNA interference of hBMSCs, Western blot analysis, and other methods. Furthermore, we created a bone defect model in Sprague-Dawley (SD) male rats and filled the defect areas with the GelMa hydrogel, with or without TgEP. Microcomputed tomography (micro-CT) was employed to analyze the bone parameters of defect sites. H&E, Masson and immunohistochemical staining were used to assess the repair conditions of the defect area.ResultsOur results indicate that TgEP promotes the expression of key osteogenic markers, including ALP, Runx2, and Osx, as well as the activation of Smad1, BMP2, and phosphorylated Smad1/5—crucial elements of the BMP/Smad signaling pathway. Furthermore, in vivo experiments using a bone defect model in rats demonstrated that TgEP markedly promoted bone defect repair.ConclusionOur results provide compelling evidence that TgEP facilitates hBMSC osteogenic differentiation through the BMP/Smad signaling pathway, highlighting its potential as a therapeutic approach for bone tissue engineering for bone defect healing.

Mesoporous Silicon with Strontium-Powered Poly(Lactic-Co-Glycolic acid)/Gelatin-Based Dressings Facilitate Skin Tissue Repair.

Functional inorganic nanomaterials (NMs) are widely exploited as bioactive materials and drug depots. The lack of a stable form of application of NMs at the site of skin injury, may impede the removal of the debridement, elevate pH, induce tissue toxicity, and limit their use in skin repair. This necessitates the advent of innovative wound dressings that overcome the above limitations. The overarching objective of this study was to exploit strontium-doped mesoporous silicon particles (PSiSr) to impart multifunctionality to poly(lactic-co-glycolic acid)/gelatin (PG)-based fibrous dressings (PG@PSiSr) for excisional wound management. Mesoporous silicon particles (PSi) and PSiSr were synthesized using a chemo-synthetic approach. Both PSi and PSiSr were incorporated into PG fibers using electrospinning. A series of structure, morphology, pore size distribution, and cumulative pH studies on the PG@PSi and PG@PSiSr membranes were performed. Cytocompatibility, hemocompatibility, transwell migration, scratch wound healing, and delineated angiogenic properties of these composite dressings were tested in vitro. The biocompatibility of composite dressings in vivo was assessed by a subcutaneous implantation model of rats, while their potential for wound healing was discerned by implantation in a full-thickness excisional defect model of rats. The PG@PSiSr membranes can afford the sustained release of silicon ions (Si4+) and strontium ions (Sr2+) for up to 192 h as well as remarkably promote human umbilical vein endothelial cells (HUVECs) and NIH-3T3 fibroblasts migration. The PG@PSiSr membranes also showed better cytocompatibility, hemocompatibility, and significant formation of tubule-like networks of HUVECs in vitro. Moreover, PG@PSiSr membranes also facilitated the infiltration of host cells and promoted the deposition of collagen while reducing the accumulation of inflammatory cells in a subcutaneous implantation model in rats as assessed for up to day 14. Further evaluation of membranes transplanted in a full-thickness excisional wound model in rats showed rapid wound closure (PG@SiSr vs control, 96.1% vs 71.7%), re-epithelialization, and less inflammatory response alongside skin appendages formation (eg, blood vessels, glands, hair follicles, etc.). To sum up, we successfully fabricated PSiSr particles and prepared PG@PSiSr dressings using electrospinning. The PSiSr-mediated release of therapeutic ions, such as Si4+ and Sr2+, may improve the functionality of PLGA/Gel dressings for an effective wound repair, which may also have implications for the other soft tissue repair disciplines.

3D-printed biomimetic scaffold with liposome-encapsulated SB431542 promotes scarless wound healing

Abnormal wound scarring often leads to functional impairments and cosmetic deformities, primarily driven by the prolonged activation of the TGF-β/Smad signaling pathway. Addressing this challenge, we developed a biomimetic scaffold aimed at facilitating rapid and scarless wound healing. This highly integrated 3D-printed dermal scaffold comprised modified recombinant human type III collagen (rhCOLIII-MA), gelatin methacrylate (GelMA), and liposomes encapsulating SB431542 to target TGF-β1 (Lip@SB). The rhCOLIII-MA/GelMA (CG) scaffold retained inherent biomaterial characteristics, exhibited tailored physicochemical properties, and demonstrated favorable biocompatibility. Moreover, the Lip@SB-loaded CG scaffold (CGL) effectively promoted in vitro wound healing, while enabling controlled release of SB431542 to inhibit pathological collagen deposition. In a full-thickness skin defect rat model, the CGL dermal scaffold combined with split-thickness skin graft (STSG) minimized scar contraction, stimulated functional neovascularization, and enhanced graft aesthetics comparable to normal skin. Remarkably, the performance of the CGL scaffold surpassed that of commercially available anti-scarring alternatives. This innovative strategy presents a straightforward approach toward scarless skin regeneration and holds promise in alleviating the prolonged, painful postoperative rehabilitation.

A Gelatin/Alginate Double Network Hydrogel Nerve Guidance Conduit Fabricated by a Chemical-Free Gamma Radiation for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) are widely developed using various materials for the functional repair of injured or diseased peripheral nerves. Especially, hydrogels are considered highly suitable for the fabrication of NGCs due to their beneficial tissue-mimicking characteristics (e.g., high water content, softness, and porosity). However, the practical applications of hydrogel-based NGCs are hindered due to their poor mechanical properties and complicated fabrication processes. To bridge this gap, a novel double-network (DN) hydrogel using alginate and gelatin by a two-step crosslinking process involving chemical-free gamma irradiation and ionic crosslinking, is developed. DN hydrogels (1% alginate and 15% gelatin), crosslinked with 30 kGy gamma irradiation and barium ions, exhibit substantially improved mechanical properties, including tensile strength, elastic modulus, and fracture stain, compared to single network (SN) gelatin hydrogels. Additionally, the DN hydrogel NGC exhibits excellent kink resistance, mechanical stability to successive compression, suture retention, and enzymatic degradability. In vivo studies with a sciatic defect rat model indicate substantially improved nerve function recovery with the DN hydrogel NGC compared to SN gelatin and commercial silicone NGCs, as confirm footprint analysis, electromyography, and muscle weight measurement. Histological examination reveals that, in the DN NGC group, the expression of Schwann cell and neuronal markers, myelin sheath, and exon diameter are superior to the other controls. Furthermore, the DN NGC group demonstrates increased muscle fiber formation and reduced fibrotic scarring. These findings suggest that the mechanically robust, degradable, and biocompatible DN hydrogel NGC can serve as a novel platform for peripheral nerve regeneration and other biomedical applications, such as implantable tissue constructs.

The effect of mofettes and natural carbonated mineral water on accelerating bone healing in a femoral defect model in rats

Abstract: (1) Background: The aim of the study was to investigate the effecs of mofettes and car-bonated mineral water baths on bone healing using a rat femoral defect model; (2) Methods: A 2mm diameter unicortical defect in the left femoral diaphysis in 40 Wistar Rats was surgically created. Furthermore, the subjects were divided into 4 treatment-groups: control, mofette therapy, mofette therapy and carbonated mineral water bath, and carbonated mineral water bath. At the end of the 2-week treatment and at 4 and 6 weeks, the animals were evaluated through Micro-CT analysis of the bone defect and histological analysis of bone tissue and skin; (3) Results: The pro-cesses of bone consolidation and repair are not completed at 6 weeks in all groups. However, comparing the proliferated bone tissue in the created orifice and the degree of thickening of the femoral wall, it can be affirmed that at 6 weeks, the best results are present in Group 4, treated with carbonated mineral water baths, followed by the combination of mofettes and carbonated water. (4) The use of these treatments could open a new possibility for shortening the healing time in patients with bone defects, as it is non-invasive and accessible.

The Impact of Defect Size on Bone Healing in Critical-Size Bone Defects Investigated on a Rat Femur Defect Model Comparing Two Treatment Methods.

Critical-size bone defects up to 25 cm can be treated successfully using the induced membrane technique established by Masquelet. To shorten this procedure, human acellular dermis (HAD) has had success in replacing this membrane in rat models. The aim of this study was to compare bone healing for smaller and larger defects using an induced membrane and HAD in a rat model. Using our established femoral defect model in rats, the animals were placed into four groups and defects of 5 mm or 10 mm size were set, either filling them with autologous spongiosa and surrounding the defect with HAD or waiting for the induced membrane to form around a cement spacer and filling this cavity in a second operation with a cancellous bone graft. Healing was assessed eight weeks after the operation using µ-CT, histological staining, and an assessment of the progress of bone formation using an established bone healing score. The α-smooth muscle actin used as a signal of blood vessel formation was stained and counted. The 5 mm defects showed significantly better bone union and a higher bone healing score than the 10 mm defects. HAD being used for the smaller defects resulted in a significantly higher bone healing score even than for the induced membrane and significantly higher blood vessel formation, corroborating the good results achieved by using HAD in previous studies. In comparison, same-sized groups showed significant differences in bone healing as well as blood vessel formation, suggesting that 5 mm defects are large enough to show different results in healing depending on treatment; therefore, 5 mm is a viable size for further studies on bone healing.

Silk fibroin/methacrylated gelatine/hydroxyapatite biomimetic nanofibrous membranes for guided bone regeneration

By mimicking in vivo bionic microenvironment and promoting osteogenic differentiation, the hybrid organic-inorganic nanofibrous membranes provide promising potential for guided bone regeneration (GBR) in the treatment of clinical bone defects. To develop a degradable and osteogenic membrane for GBR by combining the natural biomacromolecule silk fibroin (SF) and gelatine with the bioactive nano hydroxyapatite (nHA), the anhydride-modified gelatine-nano hydroxyapatite (GelMA-nHA) composites were synthesized in situ and introduced into silk fibroin to prepare nanofibrous membranes with different ratios using electrospinning and photocrosslinking. The nanofibrous membranes, particularly those with a mass ratio of 7:2:1, were found to exhibit satisfactory elongation at break up to 110 %, maintain the nanofibrous structure for up to 28 days, and rapidly form bone-like apatite within 3 days, thus offering advantages when it comes to guided bone regeneration. In vitro cell results showed that the SF/GelMA/nHA membranes had excellent biocompatibility and enhanced osteogenic differentiation of hBMSCs. In vivo studies revealed that the hybrid composite membranes can improve bone regeneration of critical-sized calvarial defects in rat model. Therefore, the novel hybrid nanofibrous membrane is proposed to be a alternative candidate for creating a bionic microenvironment that promotes bone regeneration, indicating their potential application to bone injury treatment.

3D-printed bioink loading with stem cells and cellular vesicles for periodontitis-derived bone defect repair

The suitable microenvironment of bone regeneration is critically important for periodontitis-derived bone defect repair. Three major challenges in achieving a robust osteogenic reaction are the exist of oral inflammation, pathogenic bacteria invasion and unaffluent seed cells. Herein, a customizable and multifunctional 3D-printing module was designed with glycidyl methacrylate (GMA) modified epsilon-poly-L-lysine (EPLGMA) loading periodontal ligament stem cells (PDLSCs) and myeloid-derived suppressive cells membrane vesicles (MDSCs-MV) bioink (EPLGMA/PDLSCs/MDSCs-MVs, abbreviated as EPM) for periodontitis-derived bone defect repair. The EPM showed excellent mechanical properties and physicochemical characteristics, providing a suitable microenvironment for bone regeneration. In vitro, EPMs presented effectively kill the periodontopathic bacteria depend on the natural antibacterial properties of the EPL. Meanwhile, MDSCs-MV was confirmed to inhibit T cells through CD73/CD39/adenosine signal pathway, exerting an anti-inflammatory role. Additionally, seed cells of PDLSCs provide an adequate supply for osteoblasts. Moreover, MDSCs-MV could significantly enhance the mineralizing capacity of PDLSCs-derived osteoblast. In the periodontal bone defect rat model, the results of micro-CT and histological staining demonstrated that the EPM scaffold similarly had an excellent anti-inflammatory and bone regeneration efficacy in vivo. This biomimetic and multifunctional 3D-printing bioink opens new avenues for periodontitis-derived bone defect repair and future clinical application.

Osteoking promotes bone formation and bone defect repair through ZBP1-STAT1-PKR-MLKL-mediated necroptosis.

Osteoking has been used for fracture therapy with a satisfying clinical efficacy. However, its therapeutic properties and the underlying mechanisms remain elusive. A bone defect rat model was established to evaluate the pharmacological effects of Osteoking by the dynamic observation of X-ray, micro-CT and histopathologic examination. Transcriptome profiling was performed to identify bone defect-related genes and Osteoking effective targets. Then, a "disease-related gene-drug target" interaction network was constructed and a list of key network targets were screened, which were experimentally verified. Osteoking effectively promoted bone defect repair in rats by accelerating the repair of cortical bone and the growth of trabeculae. Histopathologically, the bone defect rats displayed lower histopathologic scores in cortical bone, cancellous bone and bone connection than normal controls. In contrast, Osteoking exerted a favorable effect with a dose-dependent manner. The abnormal serum levels of bone turnover markers, bone growth factors and bone metabolism-related biochemical indexes in bone defect rats were also reversed by Osteoking treatment. Following the transcriptome-based network investigation, we hypothesized that osteoking might attenuate the levels of ZBP1-STAT1-PKR-MLKL-mediated necroptosis involved into bone defect. Experimentally, the expression levels of ZBP1, STAT1, PKR and the hallmark inflammatory cytokines for the end of necroptosis were distinctly elevated in bone defect rats, but were all effectively reversed by Osteoking treatment, which were also suppressed the activities of RIPK1, RIPK3 and MLKL in bone tissue supernatants. Osteoking may promote bone formation and bone defect repair by regulating ZBP1-STAT1-PKR axis, leading to inhibit RIPK1/RIPK3/MLKL activation-mediated necroptosis.

Comparison of the ability of exosomes and ectosomes derived from adipose-derived stromal cells to promote cartilage regeneration in a rat osteochondral defect model

BackgroundExtracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) offer promising prospects for stimulating cartilage regeneration. The different formation mechanisms suggest that exosomes and ectosomes possess different biological functions. However, little attention has been paid to the differential effects of EV subsets on cartilage regeneration.MethodsOur study compared the effects of the two EVs isolated from adipose-derived MSCs (ASCs) on chondrocytes and bone marrow–derived MSCs (BMSCs) in vitro. Additionally, we loaded the two EVs into type I collagen hydrogels to optimize their application for the treatment of osteochondral defects in vivo.ResultsIn vitro experiments demonstrate that ASC-derived exosomes (ASC-Exos) significantly promoted the proliferation and migration of both cells more effectively than ASC-derived ectosomes (ASC-Ectos). Furthermore, ASC-Exos facilitated a stronger differentiation of BMSCs into chondrogenic cells than ASC-Ectos, but both inhibited chondrocyte apoptosis to a similar extent. In the osteochondral defect model of rats, ASC-Exos promoted cartilage regeneration in situ better than ASC-Ectos. At 8 weeks, the hydrogel containing exosomes group (Gel + Exo group) had higher macroscopic and histological scores, a higher value of trabecular bone volume fraction (BV/TV), a lower value of trabecular thickness (Tb.Sp), and a better remodeling of extracellular matrix than the hydrogel containing ectosomes group (Gel + Ecto group). At 4 and 8 weeks, the expression of CD206 and Arginase-1 in the Gel + Exo group was significantly higher than that in the Gel + Ecto group.ConclusionOur findings indicate that administering ASC-Exos may be a more effective EV strategy for cartilage regeneration than the administration of ASC-Ectos.

Functionalized 3D Hydroxyapatite Scaffold by Fusion Peptides-Mediated Small Extracellular Vesicles of Stem Cells for Bone Tissue Regeneration.

3D printing technology offers extensive applications in tissue engineering and regenerative medicine (TERM) because it can create a three-dimensional porous structure with acceptable porosity and fine mechanical qualities that can mimic natural bone. Hydroxyapatite (HA) is commonly used as a bone repair material due to its excellent biocompatibility and osteoconductivity. Small extracellular vesicles (sEVs) derived from bone marrow mesenchymal stem cells (BMSCs) can regulate bone metabolism and stimulate the osteogenic differentiation of stem cells. This study has designed a functionalized bone regeneration scaffold (3D H-P-sEVs) by combining the biological activity of BMSCs-sEVs and the 3D-HA scaffold to improve bone regeneration. The scaffold utilizes the targeting of fusion peptides to increase the loading efficiency of sEVs. The composition, structure, mechanical properties, and in vitro degradation performance of the 3D H-P-sEVs scaffolds were examined. The composite scaffold demonstrated good biocompatibility, substantially increased the expression of osteogenic-related genes and proteins, and had a satisfactory bone integration effect in the critical skull defect model of rats. In conclusion, the combination of EVs and 3D-HA scaffold via fusion peptide provides an innovative composite scaffold for bone regeneration and repair, improving osteogenic performance.