Development of a Multifunctional Nano-Hydroxyapatite Platform (nHEA) for Advanced Treatment of Severely Infected Full-Thickness Skin Wounds.

The treatment of full-thickness skin injuries complicated by severe infection is hampered by the lack of comprehensive solutions that can regulate the various stages of wound healing. Consequently, there is an urgent need for a multifunctional dressing capable of multi-level regulation. In this study, we propose a novel solution by covalently integrating ε-poly-L-lysine-grafted gallic acid (EG) and in situ bioreduced silver nanoparticles (AgNPs) onto nano-hydroxyapatite (nHAP), thereby developing a multi-layered, multifunctional nanoplatform (nHEA). Cell experiments have shown that, compared to nHAP and nHAP loaded only with EG (nHEG), the addition of AgNPs to nHEA confers excellent antibacterial properties while maintaining optimal biocompatibility. The incorporation of EG onto nHEG and nHEA imparts antioxidation, anti-inflammatory, and pro-angiogenic functions, and the release of Ca2+ and EG further enhances fibroblast migration and collagen secretion. In a rat model of full-thickness skin injury with severe infection, nHEA demonstrates remarkable antibacterial and anti-inflammatory effects, along with promoting collagen remodeling and regeneration. Together, both cell experiments and animal studies confirm the significant potential of this innovative multifunctional nanoplatform in the treatment of full-thickness skin injuries with severe infection. STATEMENT OF SIGNIFICANCE: Treating infected full-thickness skin injuries poses a longstanding challenge due to the lack of comprehensive solutions that can regulate different stages of wound healing. This study introduces a novel multifunctional nanoplatform, nHEA, developed by covalently integrating ε-poly-L-lysine grafted with gallic acid (EG) and in situ bioreduced AgNPs onto nano-hydroxyapatite (nHAP). Cell experiments reveal that the integration of AgNPs enhances nHEA's antibacterial performance while maintaining optimal biocompatibility. The inclusion of EG bestows antioxidant, inflammation-regulating, and angiogenetic properties upon nHEA, and the release of Ca2+ and EG stimulates the migration and collagen secretion of fibroblast cells. Consequently, nHEA exhibits superior antibacterial and inflammation-regulating efficacy, and stimulates collagen remodeling and regeneration in vivo, making it a promising treatment for severely infected skin injuries.

Structure driven bio-responsive ability of injectable nanocomposite hydrogels for efficient bone regeneration.

Injectable hydrogels are promising for treatment of bone defects in clinic owing to their minimally invasive procedure. Currently, there is limited emphasis on how to utilize injectable hydrogels to mobilize body's regenerative potential for enhancing bone regeneration. Herein, an injectable bone-mimicking hydrogel (BMH) scaffold assembled from nanocomposite microgel building blocks was developed, in which a highly interconnected microporous structure and an inorganic/organic (methacrylated hydroxyapatite and methacrylated gelatin) interweaved nano structure were well-designed. Compared with hydrogels lacking micro-nano structures or only showing microporous structure, the BMH scaffold enhanced the ingrowth of vessels and promoted the formation of dense cellular networks (including stem cells and M2 macrophages), across the entire scaffold at early stage after subcutaneous implantation. Moreover, the BMH scaffold could not only directly trigger osteogenic differentiation of the infiltrated stem cells, but also provided an instructive osteo-immune microenvironment by inducing macrophages into M2 phenotype. Mechanistically, our results reveal that the nano-rough structure of the BMH plays an essential role in inducing macrophage M2 polarization through activating mechanotransduction related RhoA/ROCK2 pathway. Overall, this work offers an injectable hydrogel with micro-nano structure driven bio-responsive abilities, highlighting harnessing body's inherent regenerative potential to realize bone regeneration.

A Redox-Triggered Autophagy-Induced Nanoplatform with PD-L1 Inhibition for Enhancing Combined Chemo-Immunotherapy.

Epirubicin (EPI) alone can trigger mildly protective autophagy in residual tumor cells, resulting in an immunosuppressive microenvironment. This accelerates the recurrence of residual tumors and leads to antiprogrammed death ligand 1 (anti-PD-1)/PD-L1 therapy resistance, posing a significant clinical challenge in tumor immunotherapy. The combination of checkpoint inhibitors targeting the PD-1/PD-L1 pathway and amplifying autophagy presents an innovative approach to tumor treatment, which can prevent tumor immune escape and enhance therapeutic recognition. Herein, we aimed to synthesize a redox-triggered autophagy-induced nanoplatform with SA&EA-induced PD-L1 inhibition. The hyaluronic acid (HA) skeleton and arginine segment promoted active nanoplatform targeting, cell uptake, and penetration. The PLGLAG peptide was cleaved by overexpressing matrix metalloproteinase-2 (MMP-2) in the tumor microenvironment, and the PD-L1 inhibitor D-PPA was released to inhibit tumor immune escape. The intense autophagy inducers, STF-62247 and EPI, were released owing to the cleavage of disulfide bonds influenced by the high glutathione (GSH) concentration in tumor cells. The combination of EPI and STF induced apoptosis and autophagic cell death, effectively eliminating a majority of tumor cells. This indicated that the SA&EA nanoplatform has better therapeutic efficacy than the single STF@AHMPP and EPI@AHMPTP groups. This research provided a way to set up a redox-triggered autophagy-induced nanoplatform with PD-L1 inhibition to enhance chemo-immunotherapy.

3D printing of customized bioceramics for promoting bone tissue regeneration by regulating sympathetic nerve behavior.

Numerous studies have shown that there are multiple neural activities involved in the process of bone resorption and bone regeneration, and promoting osteogenesis by promoting neural network reconstruction is an effective strategy for repairing critical size bone defects. However, traumatic bone defects often cause activation of the sympathetic nervous system (SNS) in the damaged area, releasing excess catecholamines (CAs), resulting in a decrease in the rate of bone formation. Herein, a 3D-printed scaffold loaded with propranolol (PRN) is proposed to reduce CA concentrations in bone defect areas and promote bone regeneration through drug release. For this purpose, PRN-loaded methacrylated gelatin (GelMA) microspheres were mixed with low-concentration GelMA and perfused into a 3D-printed porous hydroxyapatite (HAp) scaffold. By releasing PRN, which can block ß-adrenergic receptors, it hinders the activation of sympathetic nerves and inhibits the release of excess CA by the SNS. At the same time, the composite scaffold recruits bone marrow mesenchymal stem cells (BMSCs) and promotes the differentiation of BMSCs in the direction of osteoblasts, which effectively promotes bone regeneration in the rabbit femoral condyle defect model. The results of the study showed that the release of PRN from the composite scaffold could effectively hinder the activation of sympathetic nerves and promote bone regeneration, providing a new strategy for the treatment of bone defects.

Advancements in Photothermal Therapy Using Near-Infrared Light for Bone Tumors.

Bone tumors, particularly osteosarcoma, are prevalent among children and adolescents. This ailment has emerged as the second most frequent cause of cancer-related mortality in adolescents. Conventional treatment methods comprise extensive surgical resection, radiotherapy, and chemotherapy. Consequently, the management of bone tumors and bone regeneration poses significant clinical challenges. Photothermal tumor therapy has attracted considerable attention owing to its minimal invasiveness and high selectivity. However, key challenges have limited its widespread clinical use. Enhancing the tumor specificity of photosensitizers through targeting or localized activation holds potential for better outcomes with fewer adverse effects. Combinations with chemotherapies or immunotherapies also present avenues for improvement. In this review, we provide an overview of the most recent strategies aimed at overcoming the limitations of photothermal therapy (PTT), along with current research directions in the context of bone tumors, including (1) target strategies, (2) photothermal therapy combined with multiple therapies (immunotherapies, chemotherapies, and chemodynamic therapies, magnetic, and photodynamic therapies), and (3) bifunctional scaffolds for photothermal therapy and bone regeneration. We delve into the pros and cons of these combination methods and explore current research focal points. Lastly, we address the challenges and prospects of photothermal combination therapy.

An injectable cartilage-coating composite with long-term protection, effective lubrication and chondrocyte nourishment for osteoarthritis treatment.

The osteoarthritic (OA) environment within articular cartilage poses significant challenges, resulting in chondrocyte dysfunction and cartilage matrix degradation. While intra-articular injections of anti-inflammatory drugs, biomaterials, or bioactive agents have demonstrated some effectiveness, they primarily provide temporary relief from OA pain without arresting OA progression. This study presents an injectable cartilage-coating composite, comprising hyaluronic acid and decellularized cartilage matrix integrated with specific linker polymers. It enhances the material retention, protection, and lubrication on the cartilage surface, thereby providing an effective physical barrier against inflammatory factors and reducing the friction and shear force associated with OA joint movement. Moreover, the composite gradually releases nutrients, nourishing OA chondrocytes, aiding in the recovery of cellular function, promoting cartilage-specific matrix production, and mitigating OA progression in a rat model. Overall, this injectable cartilage-coating composite offers promising potential as an effective cell-free treatment for OA. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) in the articular cartilage leads to chondrocyte dysfunction and cartilage matrix degradation. This study introduces an intra-articular injectable composite material (HDC), composed of decellularized cartilage matrix (dECMs), hyaluronan (HA), and specially designed linker polymers to provide an effective cell-free OA treatment. The linker polymers bind HA and dECMs to form an integrated HDC structure with an enhanced degradation rate, potentially reducing the need for frequent injections and associated trauma. They also enable HDC to specifically coat the cartilage surface, forming a protective and lubricating layer that enhances long-term retention, acts as a barrier against inflammatory factors, and reduces joint movement friction. Furthermore, HDC nourishes OA chondrocytes through gradual nutrient release, aiding cellular function recovery, promoting cartilage-specific matrix production, and mitigating OA progression.

An injectable photocuring silk fibroin-based hydrogel for constructing an antioxidant microenvironment for skin repair.

Skin has a protein microenvironment dominated by functional collagen fibers, while oxidative stress caused by injury can greatly slow down the progress of wound healing. Here, methacrylated dopamine was incorporated into methacrylated silk fibroin molecule chains to develop an injectable hydrogel with photocuring properties for constructing an antioxidant skin protein microenvironment. This silk fibroin-based hydrogel (SF-g-SDA) showed good tensile and adhesion properties for adapting to the wound shape and skin movement, exhibited stable mechanical properties, good biodegradability and cytocompatibility, and promoted cell adhesion and vascularization in vitro. In addition, its phenolic hydroxyl-mediated antioxidant properties effectively protected cells from damage caused by oxidative stress and supported normal cellular life activities. In animal experiments, SF-g-SDA achieved better skin repair effects in comparison to commercial Tegaderm™ in vivo, showing its ability to accelerate wound healing, improve collagen deposition and alignment in newly fabricated tissues, and promote neovascularization and hair follicle formation. These experimental results indicated that the SF-g-SDA hydrogel is a promising wound dressing.

A facile Immunoregulatory Constructional Design by Proanthocyanidin Optimizing Directional Chitosan Microchannel.

Improving the interconnected structure and bioregulatory function of natural chitosan is beneficial for optimizing its performance in bone regeneration. Here, a facile immunoregulatory constructional design is proposed for developing instructive chitosan by directional freezing and alkaline salting out. The molecular dynamics simulation confirmed the assembly kinetics and structural features of various polyphenols and chitosan molecules. Along with the in vitro anti-inflammatory, antioxidative, promoting bone mesenchymal stem cell (BMSC) adhesion and proliferation performance, proanthocyanidin optimizing chitosan (ChiO) scaffold presented an optimal immunoregulatory structure with the directional microchannel. Transcriptome analysis in vitro further revealed the cytoskeleton- and immune-regulation effect of ChiO are the key mechanism of action on BMSC. The rabbit cranial defect model (Φ = 10 mm) after 12 weeks of implantation confirmed the significantly enhanced bone reconstitution. This facile immunoregulatory directional microchannel design provides effective guidance for developing inducible chitosan scaffolds.

Engineered Microchannel Scaffolds with Instructive Niches Reinforce Endogenous Bone Regeneration by Regulating CSF-1/CSF-1R Pathway.

Structural and physiological cues provide guidance for the directional migration and spatial organization of endogenous cells. Here, a microchannel scaffold with instructive niches is developed using a circumferential freeze-casting technique with an alkaline salting-out strategy. Thereinto, polydopamine-coated nano-hydroxyapatite is employed as a functional inorganic linker to participate in the entanglement and crystallization of chitosan molecules. This scaffold orchestrates the advantage of an oriented porous structure for rapid cell infiltration and satisfactory immunomodulatory capacity to promote stem cell recruitment, retention, and subsequent osteogenic differentiation. Transcriptomic analysis as well as its in vitro and in vivo verification demonstrates that essential colony-stimulating factor-1 (CSF-1) factor is induced by this scaffold, and effectively bound to the target colony-stimulating factor-1 receptor (CSF-1R) on the macrophage surface to activate the M2 phenotype, achieving substantial endogenous bone regeneration. This strategy provides a simple and efficient approach for engineering inducible bone regenerative biomaterials.

Hierarchical Scaffold with Directional Microchannels Promotes Cell Ingrowth for Bone Regeneration.

Bone regenerative scaffolds with a bionic natural bone hierarchical porous structure provide a suitable microenvironment for cell migration and proliferation. Here, a bionic scaffold (DP-PLGA/HAp) with directional microchannels is prepared by combining 3D printing and directional freezing technology. The 3D printed framework provides structural support for new bone tissue growth, while the directional pore embedded in the scaffolds provides an express lane for cell migration and nutrition transport, facilitating cell growth and differentiation. The hierarchical porous scaffolds achieve rapid infiltration and adhesion of bone marrow mesenchymal stem cells (BMSCs) and improve the expression of osteogenesis-related genes. The rabbit cranial defect experiment presents significant new bone formation, demonstrating that DP-PLGA/HAp offers an effective means to guide cranial bone regeneration. The combination of 3D printing and directional freezing technology might be a promising strategy for developing bone regenerative biomaterials.

Molecular co-assembled strategy tuning protein conformation for cartilage regeneration.

The assembly of oligopeptide and polypeptide molecules can reconstruct various ordered advanced structures through intermolecular interactions to achieve protein-like biofunction. Here, we develop a "molecular velcro"-inspired peptide and gelatin co-assembly strategy, in which amphiphilic supramolecular tripeptides are attached to the molecular chain of gelatin methacryloyl via intra-/intermolecular interactions. We perform molecular docking and dynamics simulations to demonstrate the feasibility of this strategy and reveal the advanced structural transition of the co-assembled hydrogel, which brings more ordered ß-sheet content and 10-fold or more compressive strength improvement. We conduct transcriptome analysis to reveal the role of co-assembled hydrogel in promoting cell proliferation and chondrogenic differentiation. Subcutaneous implantation evaluation confirms considerably reduced inflammatory responses and immunogenicity in comparison with type I collagen. We demonstrate that bone mesenchymal stem cells-laden co-assembled hydrogel can be stably fixed in rabbit knee joint defects by photocuring, which significantly facilitates hyaline cartilage regeneration after three months. This co-assembly strategy provides an approach for developing cartilage regenerative biomaterials.

Injectable Microgels with Hybrid Exosomes of Chondrocyte-Targeted FGF18 Gene-Editing and Self-Renewable Lubrication for Osteoarthritis Therapy.

Abnormal silencing of fibroblast growth factor (FGF) signaling significantly contributes to joint dysplasia and osteoarthritis (OA); However, the clinical translation of FGF18-based protein drugs is hindered by their short half-life, low delivery efficiency and the need for repeated articular injections. This study proposes a CRISPR/Cas9-based approach to effectively activate the FGF18 gene of OA chondrocytes at the genome level in vivo, using chondrocyte-affinity peptide (CAP) incorporated hybrid exosomes (CAP/FGF18-hyEXO) loaded with an FGF18-targeted gene-editing tool. Furthermore, CAP/FGF18-hyEXO are encapsulated in methacrylic anhydride-modified hyaluronic (HAMA) hydrogel microspheres via microfluidics and photopolymerization to create an injectable microgel system (CAP/FGF18-hyEXO@HMs) with self-renewable hydration layers to provide persistent lubrication in response to frictional wear. Together, the injectable CAP/FGF18-hyEXO@HMs, combined with in vivo FGF18 gene editing and continuous lubrication, have demonstrated their capacity to synergistically promote cartilage regeneration, decrease inflammation, and prevent ECM degradation both in vitro and in vivo, holding great potential for clinical translation.

Fibrous topology promoted pBMP2-activated matrix on titanium implants boost osseointegration.

Titanium (Ti) implants have been extensively used after surgical operations. Its surface bioactivity is of importance to facilitate integration with surrounding bone tissue, and ultimately ensure stability and long-term functionality of the implant. The plasmid DNA-activated matrix (DAM) coating on the surface could benefit osseointegration but is still trapped by poor transfection for further application, especially on the bone marrow mesenchymal stem cells (BMSCs) in vivo practical conditions. Herein, we constructed a DAM on the surface of fibrous-grained titanium (FG Ti) composed of phase-transition lysozyme (P) as adhesive, cationic arginine-rich lipid (RLS) as the transfection agent and plasmid DNA (pDNA) for bone morphology protein 2 (BMP2) expression. The cationic lipid RLS improved up to 30-fold higher transfection than that of commercial reagents (Lipofectamine 2000 and polyethyleneimine) on MSC. And importantly, Ti surface topology not only promotes the DAM to achieve high transfection efficiency (∼75.7% positive cells) on MSC due to the favorable combination but also reserves its contact induction effect for osteoblasts. Upon further exploration, the fibrous topology on FG Ti could boost pDNA uptake for gene transfection, and cell migration in MSC through cytoskeleton remodeling and induce contact guidance for enhanced osteointegration. At the same time, the cationic RLS together with adhesive P were both antibacterial, showing up to 90% inhibition rate against Escherichia coli and Staphylococcus aureus with reduced adherent microorganisms and disrupted bacteria. Finally, the FG Ti-P/pBMP2 implant achieved accelerated bone healing capacities through highly efficient gene delivery, aligned surface topological structure and increased antimicrobial properties in a rat femoral condylar defect model.

Unveiling protein corona composition: predicting with resampling embedding and machine learning.

Biomaterials with surface nanostructures effectively enhance protein secretion and stimulate tissue regeneration. When nanoparticles (NPs) enter the living system, they quickly interact with proteins in the body fluid, forming the protein corona (PC). The accurate prediction of the PC composition is critical for analyzing the osteoinductivity of biomaterials and guiding the reverse design of NPs. However, achieving accurate predictions remains a significant challenge. Although several machine learning (ML) models like Random Forest (RF) have been used for PC prediction, they often fail to consider the extreme values in the abundance region of PC absorption and struggle to improve accuracy due to the imbalanced data distribution. In this study, resampling embedding was introduced to resolve the issue of imbalanced distribution in PC data. Various ML models were evaluated, and RF model was finally used for prediction, and good correlation coefficient (R2) and root-mean-square deviation (RMSE) values were obtained. Our ablation experiments demonstrated that the proposed method achieved an R2 of 0.68, indicating an improvement of approximately 10%, and an RMSE of 0.90, representing a reduction of approximately 10%. Furthermore, through the verification of label-free quantification of four NPs: hydroxyapatite (HA), titanium dioxide (TiO2), silicon dioxide (SiO2) and silver (Ag), and we achieved a prediction performance with an R2 value >0.70 using Random Oversampling. Additionally, the feature analysis revealed that the composition of the PC is most significantly influenced by the incubation plasma concentration, PDI and surface modification.

Hollow Copper Sulfide Photothermal Nanodelivery Platform Boosts Angiogenesis of Diabetic Wound by Scavenging Reactive Oxygen Species.

Sharply rising oxidative stress and ineffectual angiogenesis have imposed restrictions on diabetic wound healing. Here, a photothermal-responsive nanodelivery platform (HHC) was prepared by peroxidase (CAT)-loaded hollow copper sulfide dispersed in photocurable methacrylamide hyaluronan. The HHC could scavenge reactive oxygen species (ROS) and promote angiogenesis by photothermally driven CAT and Cu2+ release. Under near-infrared light irradiation, the HHC presented safe photothermal performance (<43 °C), efficient bacteriostatic ability against E. coli and S. aureus. It could rapidly release CAT into the external environment for decomposing H2O2 and oxygen generation to alleviate oxidative stress while promoting fibroblast migration and VEGF protein expression of endothelial cells by reducing intracellular ROS levels. The nanodelivery platform presented satisfactory therapeutic effects on murine diabetic wound healing by modulating tissue inflammation, promoting collagen deposition and increasing vascularization in the neodermis. This HHC provided a viable strategy for diabetic wound dressing design.

A drug-free cardiovascular stent functionalized with tailored collagen supports in-situ healing of vascular tissues.

Drug-eluting stent implantation suppresses the excessive proliferation of smooth muscle cells to reduce in-stent restenosis. However, the efficacy of drug-eluting stents remains limited due to delayed reendothelialization, impaired intimal remodeling, and potentially increased late restenosis. Here, we show that a drug-free coating formulation functionalized with tailored recombinant humanized type III collagen exerts one-produces-multi effects in response to injured tissue following stent implantation. We demonstrate that the one-produces-multi coating possesses anticoagulation, anti-inflammatory, and intimal hyperplasia suppression properties. We perform transcriptome analysis to indicate that the drug-free coating favors the endothelialization process and induces the conversion of smooth muscle cells to a contractile phenotype. We find that compared to drug-eluting stents, our drug-free stent reduces in-stent restenosis in rabbit and porcine models and improves vascular neointimal healing in a rabbit model. Collectively, the one-produces-multi drug-free system represents a promising strategy for the next-generation of stents.

Exploring the potential mechanism of Heng-Gu-Gu-Shang-Yu-He-Ji therapy for osteoporosis based on network pharmacology and transcriptomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Heng-Gu-Gu-Shang-Yu-He-Ji (Osteoking, OK) is a well-known formula for fracture therapy. In clinic, OK is effective in treating fractures while alleviating osteoporosis (OP) symptoms. However, active components of OK and the associated molecular mechanisms remain not fully elucidated. AIM OF THE STUDY: This study aims to systematically evaluate the anti-osteoporosis efficacy of OK and for the first time combine network pharmacology with high-throughput whole gene transcriptome sequencing to study its underlying mechanism. MATERIALS AND METHODS: In this study, the osteoporosis model was established by the castration of both ovaries. The level of serum bone turnover factor was detected by enzyme-linked immunosorbent assay. Micro-CT and HE staining were used to observe the changes of bone histopathology, and nano-indentation technique was used to detect the biomechanical properties of rat bone. The main active Chemical components of OK were identified using UPLC-DAD. Efficacy verification and mechanism exploration were conducted by network pharmacology, molecular docking, whole gene transcriptomics and in vivo experiments. RESULTS: In our study, OK significantly improved bone microarchitecture and bone biomechanical parameters in OVX rats, reduced osteoclast indexes such as C-telopeptide of type I collage (CTX-I) and increased Osteoprotegerin (OPG)/Receptor activator of NF-κB ligand (RANKL) levels. Mechanistically, PI3K/AKT pathway was a common pathway for genome enrichment analysis (KEGG) of both network pharmacology and RNA-seq studies. G protein-ß-like protein (GßL), Ribosomal-protein S6 kinase homolog 2 (S6K2), and Phosphoinositide 3-kinase (PI3K) appeared differentially expression in the PI3K-AKT signaling pathway. These results were also confirmed by qRT-PCR and immunohistochemistry. CONCLUSIONS: OK may be used to treat osteoporosis, at least partly by activating PI3K/AKT/mTORC1 signaling pathway.

Nucleation Domains in Biomineralization: Biomolecular Sequence and Conformational Features.

Biomolecules play a vital role in the regulation of biomineralization. However, the characteristics of practical nucleation domains are still sketchy. Herein, the effects of the representative biomolecular sequence and conformations on calcium phosphate (Ca-P) nucleation and mineralization are investigated. The results of computer simulations and experiments prove that the line in the arrangement of dual acidic/essential amino acids with a single interval (Bc (Basic) -N (Neutral) -Bc-N-Ac (Acidic)- NN-Ac-N) is most conducive to the nucleation. 2α-helix conformation can best induce Ca-P ion cluster formation and nucleation. "Ac- × × × -Bc" sequences with α-helix are found to be the features of efficient nucleation domains, in which process, molecular recognition plays a non-negligible role. It further indicates that the sequence determines the potential of nucleation/mineralization of biomolecules, and conformation determines the ability of that during functional execution. The findings will guide the synthesis of biomimetic mineralized materials with improved performance for bone repair.

Accelerated osteogenesis of bone graft by optimizing the bone microenvironment formed by electrical signals dependent on driving micro vibration stimulation.

The strategy of coupling the micro-vibration mechanical field with Ca/P ceramics to optimize the osteogenic microenvironment and enhance the functional activity of the cells can significantly improve the bone regeneration of the graft. However, the regulation mode and mechanism of this coupling strategy are not fully understood at present. This study investigated the influence of different waveforms of the electrical signals driving Microvibration Stimulation (MVS) on this coupling effect. The results showed that there were notable variances in calcium phosphate dissolution and redeposition, protein adsorption, phosphorylation of ERK1/2 and FAK signal pathways and activation of calcium channels such as TRPV1/Piezo1/Piezo2 in osteogenic microenvironment under the coupling action of hydroxyapatite (HA) ceramics and MVS driven by different electrical signal waveforms. Ultimately, these differences affected the osteogenic differentiation process of cells by a way of time-sequential regulation. Square wave-MVS coupled with HA ceramic can significantly delay the high expression time of characteristic genes (such as Runx2, Col-I and OCN) in MC3T3-E1 cells during in vitro the early, middle and late stage of differentiation, while maintain the high proliferative activity of MC3T3-E1 cells. Triangle wave signal-MVS coupled with HA ceramic promoted the osteogenic differentiation of cells in the early and late stages. Sine wave-MVS shows the effect on the process of osteogenic differentiation in the middle stage (such as the up-regulation of ALP synthesis and Col-I gene expression in the early stage of stimulation). In addition, Square wave-MVS showed the best coupling effect. The bone graft constructed under square wave-MVS formed new bone tissue and mature blood vessels only 2 weeks after subcutaneous implantation in nude mice. Our study provides a new non-invasive regulation model for precisely optimizing the osteogenic microenvironment, which can accelerate bone regeneration in bone grafts more safely, accurately and reliably.

Cationic Biopolymeric Scaffold of Chelating Nanohydroxyapatite Self-Regulates Intraoral Microenvironment for Periodontal Bone Regeneration.

Periodontal bone defect is a common but longstanding healthcare issue since traditional bone grafts have limited functionalities in regulating complex intraoral microenvironments. Here, a porous cationic biopolymeric scaffold (CSC-g-nHAp) with microenvironment self-regulating ability was synthesized by chitosan-catechol chelating the Ca2+ of nanohydroxyapatite and bonding type I collagen. Chitosan-catechol's inherent antibacterial and antioxidant abilities endowed this scaffold with desirable abilities to eliminate periodontal pathogen infection and maintain homeostatic balances between free radical generation and elimination. Meanwhile, this scaffold promoted rat bone marrow stromal cells' osteogenic differentiation and achieved significant ectopic mineralization after 4 weeks of subcutaneous implantation in nude mice. Moreover, after 8 weeks of implantation in the rat critical-sized periodontal bone defect model, CSC-g-nHAp conferred 5.5-fold greater alveolar bone regeneration than the untreated group. This cationic biopolymeric scaffold could regulate the local microenvironment through the synergistic effects of its antibacterial, antioxidant, and osteoconductive activities to promote solid periodontal bone regeneration.