Novel approach to soft tissue regeneration: in vitro study of compound hyaluronic acid and horizontal platelet-rich fibrin combination.

OBJECTIVE: This study aims to develop a compound biomaterial to achieve effective soft tissue regeneration. METHODOLOGY: Compound hyaluronic acid (CHA) and liquid horizontal-platelet-rich fibrin (H-PRF) were mixed at a ratio of 1:1 to form a CHA-PRF gel. Human gingival fibroblasts (HGFs) were used in this study. The effect of CHA, H-PRF, and the CHA-PRF gel on cell viability was evaluated by CCK-8 assays. Then, the effect of CHA, H-PRF, and the CHA-PRF gel on collagen formation and deposition was evaluated by qRT‒PCR and immunofluorescence analysis. Finally, qRT‒PCR, immunofluorescence analysis, Transwell assays, and scratch wound-healing assays were performed to determine how CHA, H-PRF, and the CHA-PRF gel affect the migration of HGFs. RESULTS: The combination of CHA and H-PRF shortened the coagulation time of liquid H-PRF. Compared to the pure CHA and H-PRF group, the CHA-PRF group exhibited the highest cell proliferation at all time points, as shown by the CCK-8 assay. Col1a and FAK were expressed at the highest levels in the CHA-PRF group, as shown by qRT‒PCR. CHA and PRF could stimulate collagen formation and HGF migration, as observed by fluorescence microscopy analysis of COL1 and F-actin and Transwell and scratch healing assays. CONCLUSION: The CHA-PRF group exhibited greater potential to promote soft tissue regeneration by inducing cell proliferation, collagen synthesis, and migration in HGFs than the pure CHA or H-PRF group. CHA-PRF can serve as a great candidate for use alone or in combination with autografts in periodontal or peri-implant soft tissue regeneration.

FOXO1 inhibits FSL-1 regulation of integrin ß6 by blocking STAT3 binding to the integrin ß6 gene promoter.

Integrin ß6 (ITGB6), an epithelial-specific receptor, is downregulated in the gingival epithelium of periodontitis and is associated with inflammation response and periodontitis development. However, the transcriptional regulatory mechanism of ITGB6 downregulation in the human gingival epithelium remains unclear. Fibroblast-stimulating lipopeptide-1 (FSL-1), an oral biofilm component, promotes an epithelial cell-driven proinflammatory response in periodontitis partially by suppressing ITGB6 expression. The aim of the current study was to investigate the transcriptional regulatory mechanism of ITGB6 inhibition by FSL-1 in human epithelial cells (HaCaT and primary human gingival epithelial cells), and to delineate the transcriptional mechanism of ITGB6 suppression in periodontitis. We found that FSL-1 inhibited ITGB6 transcription through increasing forkhead box protein O1 (FOXO1) expression and inhibiting signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, FOXO1 bound to STAT3 directly, leading to decreased STAT3 phosphorylation induced by FSL-1. Consequently, the binding of phosphorylated STAT3 to the ITGB6 promoter was decreased, and ITGB6 transcription was therefore downregulated following FSL-1 stimulation. The reciprocal action of STAT3 and FOXO1 on ITGB6 downregulation was also confirmed by the immunostaining of the inflammatory epithelium associated with periodontitis. Our findings suggest that the interaction of FOXO1-STAT3 may be a useful signal target for the treatment of periodontitis.

Oxygen Ion Implantation Improving Cell Adhesion on Titanium Surfaces through Increased Attraction of Fibronectin PHSRN Domain.

Mechanistic understanding of fibronectin (FN) adsorption which determines cell adhesion on cell-implant interfaces is significant for improving the osteoconduction and soft-tissue healing of implants. Here, it is shown that the adsorption behavior of FN on the titanium oxide surface (TiO2 ) is highly relative to its Pro-His-Ser-Arg-Asn (PHSRN) peptide. FN lacking PHSRN fails to bind to surfaces, resulting in inhibited cell adhesion and spreading. Molecular dynamics simulation shows higher affinity and greater adsorption energy of PHSRN peptide with TiO2 surface due to the stronger hydrogen bonds formed by the serine and arginine residues with O ion of the substrate. Finally, by increasing O content in TiO2 surfaces through O ion-beam implantation, improving the cell adhesion, cell differentiation, and the subsequent biomineralization on titanium implant is realized. This study reveals the vital role of PHSRN in FN-mediated cell adhesion on implant surfaces, providing a promising new target for further tissue integration and implant success.

Erratum: Minimally invasive implantation and decreased inflammation reduce osteoinduction of biomaterial: Erratum.

[This corrects the article DOI: 10.7150/thno.39507.].

An electroporation strategy to synthesize the membrane-coated nanoparticles for enhanced anti-inflammation therapy in bone infection.

The cell membrane-coated nanoparticles (MNPs) showed great potential in treating infectious disease due to their superior biofunctions in improving biocompatibility of nanoparticles and neutralization of pathogen or toxins. However, bone infection is accompanied with severe inflammation and bone loss, which also requires anti-inflammatory and osteoconductive treatment. The conventional membrane coating method has to undergo ultrasonication and extrusion procedures, which reduces the functionality of cell membrane and limits the choice of nanoparticles. In this study, we proposed an electroporation-based membrane coating strategy to facilitate the synthesis of MNPs to tackle those problems. Methods: Magnetic composite nanoparticles with osteoconductive Ca3(PO4)2 and bactericidal TiO2 were assembled into macrophages through phagocytosis and then collected to expose in electric field for obtaining macrophage membrane-coating nanoparticles. By using molecular dynamics simulation and materials characterizations, the cell membrane coating efficiency was confirmed. The in vitro anti-bacterial and anti-inflammatory abilities were tested by bacteria culturing and immune cells activation. Then drug-resistant bacteria induced bone infection model was established to verify its in vivo therapeutic effects. Results: The coated membrane prepared through electroporation reserved the integrality of membrane structure and right-sidedness, with more functional proteins. Those led to the superior properties of recognition and adsorption with bacteria, toxins and inflammatory cytokines. Owing to the benefits of electroporation, the MNPs exhibited significant better antibacterial and anti-inflammatory abilities for enhancing the tissue repair process. Conclusion: This study provides a novel self-assembly cell membrane coating strategy by electroporation to construct multifunctional membrane-coating nanoparticles for bone infection treatment. This strategy not only improves the functions of coated membrane, but is also proved to be universal for varies nanoparticles or cells, indicating a great potential for future applications in the bioengineering field.

Cell-Membrane-Display Nanotechnology.

Advances in material science have set the stage for nanoparticle-based research with potent applications for the diagnosis, bioimaging, and precise treatment of diseases. Despite the wide range of biomaterials developed, the rational design of biomaterials with predictable bioactivity and safety remains a critical challenge. In recent years, the field of cell-membrane-based therapeutics has emerged as a promising platform for addressing unmet medical needs. The utilization of natural cell membranes endows biomaterials with a remarkable ability to serve as biointerfaces that interact with the host environment. To improve the function and efficacy of cell-membrane-based therapeutics, a series of novel strategies is developed as cell-membrane-display nanotechnology, which utilizes various methods to selectively display therapeutic molecules of cell membranes on nanoparticles. Although cell-membrane-display nanotechnology remains in the early phases, considerable work is currently being conducted in the field. This review discusses details of innovative strategies for displaying cell-membrane molecules, including the following: 1) displaying molecules of cell membranes on biomaterials, 2) pretreating cell membranes to induce increased expression of inherent molecules of cell membranes and enhance their function, and 3) inserting additional functional molecules on cell membranes. For each area, the theoretical basis, application scenarios, and potential development are highlighted.

Antibacterial effects of platelet-rich fibrin produced by horizontal centrifugation.

Platelet-rich fibrin (PRF) has been widely used owing to its ability to stimulate tissue regeneration. To date, few studies have described the antibacterial properties of PRF. Previously, PRF prepared by horizontal centrifugation (H-PRF) was shown to contain more immune cells than leukocyte- and platelet-rich fibrin (L-PRF). This study aimed to compare the antimicrobial effects of PRFs against Staphylococcus aureus and Escherichia coli in vitro and to determine whether the antibacterial effects correlated with the number of immune cells. Blood samples were obtained from eight healthy donors to prepare L-PRF and H-PRF. The sizes and weights of L-PRF and H-PRF were first evaluated, and their antibacterial effects against S. aureus and E. coli were then tested in vitro using the inhibition ring and plate-counting test methods. Flow-cytometric analysis of the cell components of L-PRF and H-PRF was also performed. No significant differences in size or weight were observed between the L-PRF and H-PRF groups. The H-PRF group contained more leukocytes than the L-PRF group. While both PRFs had notable antimicrobial activity against S. aureus and E. coli, H-PRF demonstrated a significantly better antibacterial effect than L-PRF. Furthermore, the antimicrobial ability of the PRF solid was less efficient than that of wet PRF. In conclusion, H-PRF exhibited better antibacterial activity than L-PRF, which might have been attributed to having more immune cells.

Graphdiyne-modified TiO2 nanofibers with osteoinductive and enhanced photocatalytic antibacterial activities to prevent implant infection.

Titanium implants have been widely used in bone tissue engineering for decades. However, orthopedic implant-associated infections increase the risk of implant failure and even lead to amputation in severe cases. Although TiO2 has photocatalytic activity to produce reactive oxygen species (ROS), the recombination of generated electrons and holes limits its antibacterial ability. Here, we describe a graphdiyne (GDY) composite TiO2 nanofiber that combats implant infections through enhanced photocatalysis and prolonged antibacterial ability. In addition, GDY-modified TiO2 nanofibers exert superior biocompatibility and osteoinductive abilities for cell adhesion and differentiation, thus contributing to the bone tissue regeneration process in drug-resistant bacteria-induced implant infection.

Minimally invasive implantation and decreased inflammation reduce osteoinduction of biomaterial.

Surgical trauma of biomaterial implantation significantly influences the immune system and the biological effects of biomaterials. Minimally invasive surgery has become a trend of clinical development but violating the concept of osteoimmunomodulation will hinder the biological effects of materials. Our study focused on biphasic calcium phosphate (BCP), the ectopia osteoinductive materials, filling the research blank of the significance of adaptive immunity crosstalk with bone biomaterials, and improving the interaction mechanism between bone biomaterials and immune response. Methods: The BCP bioceramics were implanted by conventional and minimally invasive methods in the gastrocnemius wild-type or T cells depleted mice to test the effect of ectopia osteoinduction. Moreover, flow cytometry was used to detect immune responses, T cell sorting and Western Blot molecular biology experiments, and transwell assays migration of mesenchymal stem cells (MSCs). Results: We found that BCP, an implantable osteoinductive material, could not activate the adaptive immune response mediated by T cells after minimally invasive surgery. Further studies revealed that under the conventional non-minimally invasive BCP implantation, a positive correlation existed between T cell recruitment and the infiltration and osteogenic differentiation of MSCs. Interestingly, after BCP was implanted by minimally invasive surgery or implanted in T cell depleted mice, MSCs infiltration and osteogenic differentiation were significantly reduced, and BCP could not achieve the biological effects of ectopia ossification. Finally, we confirmed that a certain extent inflammatory stimulation activated the adaptive immune response mediated by T cells, up-regulated the nuclear factor-κB (NF-κB) signal in T cells, released a large amount of chemokine C-C motif chemokine ligand 5(CCL5) to recruit MSCs to the surrounding material, and finally achieved the ideal effect of osteoinduction. Conclusion: From experimental research and clinical surgery, this study discovered that the T cells are indispensable in the ectopia ossification mediated by osteoinductive materials, put forward and confirmed the surgery method as a key variable factor restricting the application effect of biological materials, enriched the key mechanism of adaptive immunity in osteoimmunomodulation, and laid a theoretical foundation for the development of osteoinductive materials and bone tissue regeneration.

Dual-Wavelength Photosensitive Nano-in-Micro Scaffold Regulates Innate and Adaptive Immune Responses for Osteogenesis.

The immune response of a biomaterial determines its osteoinductive effect. Although the mechanisms by which some immune cells promote regeneration have been revealed, the biomaterial-induced immune response is a dynamic process involving multiple cells. Currently, it is challenging to accurately regulate the innate and adaptive immune responses to promote osteoinduction in biomaterials. Herein, we investigated the roles of macrophages and dendritic cells (DCs) during the osteoinduction of biphasic calcium phosphate (BCP) scaffolds. We found that osteoinductive BCP directed M2 macrophage polarization and inhibited DC maturation, resulting in low T cell response and efficient osteogenesis. Accordingly, a dual-targeting nano-in-micro scaffold (BCP loaded with gold nanocage, BCP-GNC) was designed to regulate the immune responses of macrophages and DCs. Through a dual-wavelength photosensitive switch, BCP-GNC releases interleukin-4 in the early stage of osteoinduction to target M2 macrophages and then releases dexamethasone in the later stage to target immature DCs, creating a desirable inflammatory environment for osteogenesis. This study demonstrates that biomaterials developed to have specific regulatory capacities for immune cells can be used to control the early inflammatory responses of implanted materials and induce osteogenesis.

Dual Functional Monocytes Modulate Bactericidal and Anti-Inflammation Process for Severe Osteomyelitis Treatment.

Osteomyelitis is an inflammatory bone disease caused by infection microorganisms which leads to progressive bone destruction and loss. Drug resistance and inflammatory damage make it urgent to develop new dual-functional therapies. Based on the powerful bactericidal effect of monocyte/macrophage cells by nature, a functional monocyte with programed anti-inflammatory ability is promising for osteomyelitis treatment. Herein, gold nanocage (GNC)-modified monocytes are developed which contain aspirin to realize the controlled antibacterial and anti-inflammatory process for bone infection treatment effectively. Aspirin@GNC-laden monocytes inherit the biological functions of origin monocytes such as chemotaxis to bacteria, differentiation potential, and phagocytic ability. The controlled release of aspirin from GNC has a beneficial effect on improving the rate and amount of bone regeneration after the anti-infection stage due to its ability to suppress the activity of natural immunity and induce osteoblast differentiation during the treatment of osteomyelitis. The present work described here is the first to utilize living monocytes to achieve a dual effect to antibacteria and anti-inflammation in a time-oriented and programed way, and provides an inspiration for future therapy based on this concept.

Biomimetic anti-inflammatory nano-capsule serves as a cytokine blocker and M2 polarization inducer for bone tissue repair.

Controlling of pro-inflammation induced by pro-inflammatory cytokines and anti-inflammatory response induced by M2 macrophages is important for osteogenesis in the process of bone tissue repair. Thus, we fabricated biomimetic anti-inflammatory nano-capsule (BANC) that can block cytokines and promote M2 macrophage polarization, presenting a positive role for bone tissue repair. The BANC is a biomimic nanosystem, coated with lipopolysaccharide-treated macrophage cell membranes with cytokine receptors enveloping gold nanocage (AuNC) as "cytokine blocker", and loaded with resolvin D1 inside into AuNC as "M2 polarization inducer" whose controlled-release could be triggered under near-infrared laser irradiation in sequence, and these chronological events were consistent with the healing process of bone tissue repair. Moreover, in vivo application of femoral bone defects revealed that the BANC composite boron-containing mesoporous bioactive glass scaffolds improved the final effects of bone tissue repair through preventing inflammatory response, promoting M2 polarization in sequence in accord with the in vitro investigation. Hence, cytokine neutralization and M2 macrophage polarization enables the BANC to enhance the bone tissue repair as a biomimetic anti-inflammation effector. Therefore, this study provides potential therapeutic strategies for trauma-mediated or inflammation-related bone defects based on a biomimetic nanomaterial with weakened pro-inflammatory and enhanced anti-inflammatory effects. STATEMENT OF SIGNIFICANCE: Cell membrane-mimic nanomaterials have been popular for blocking natural cell responses for some infection diseases, yet their role in biological process of bone repair is unknown. Here, we fabricated Biomimetic Anti-inflammatory Nano-Capsule (BANC), coated with cell membrane with cytokines receptors on the surface which could neutralize the pro-inflammatory cytokine receptor to block activated pro-inflammation, loaded with Resolvin D1 inside which could be controllably released by NIR irradiation to promote M2 macrophage polarization for the following bone formation during the process of bone repair. Administration of BANC as cytokines blocker and M2 polarization inducer to enhance the bone regeneration, thus presenting a promising potential for the treatment of bone repair and regeneration.

Fe3O4@TiO2-Laden Neutrophils Activate Innate Immunity via Photosensitive Reactive Oxygen Species Release.

Although a variety of advanced sterilization materials and treatments have emerged, the complete elimination of bacterial infection, especially drug-resistant bacterial infection, remains an immense challenge. Here, we demonstrate the use of neutrophils loaded with photocatalytic nanoparticles to reduce bacterial infection. This method activates the immune system to achieve an anti-infection response. We prepared the photocatalytic nanoparticle-laden neutrophils in vivo through neutrophil phagocytosis. The resulting loaded cells retained the cell membrane functionality of the source cell, as well as the complete immune cell function of neutrophils, particularly the ability to recruit macrophages to the target area. Photocatalytic nanoparticle-laden neutrophils can target infection sites and release reactive oxygen species to induce the secretion of chemokines, leading to the targeted recruitment of macrophages and enhancing a powerful immune cascade. In a severe mouse infection model induced by pathogenic bacteria, small doses of photocatalytic nanoparticle-laden neutrophils showed a remarkable therapeutic effect by enhancing macrophage recruitment and the immune cascade.

Near-Infrared Light-Sensitive Nano Neuro-Immune Blocker Capsule Relieves Pain and Enhances the Innate Immune Response for Necrotizing Infection.

Sensory neurons promote profound suppressive effects on neutrophils during Streptococcus pyogenes infection and contribute to the pathogenesis of necrotizing infection ("flesh-eating disease"). Thus, the development of new antibacterial agents for necrotizing infection is promising because of the clear streptococcal neuro-immune communication. Herein, based on the immune escape membrane exterior and competitive membrane functions of the glioma cell membrane, a novel nano neuro-immune blocker capsule was designed to prevent neuronal activation and improve neutrophil immune responses for necrotizing infection. These nano neuro-immune blockers could neutralize streptolysin S, suppress neuron pain conduction and calcitonin gene-related peptide release, and recruit neutrophils to the infection site, providing a strong therapeutic effect against necrotizing infection. Furthermore, nano neuro-immune blockers could serve as an effective inflammatory regulator and antibacterial agent via photothermal effects under near-infrared irradiation. In the Streptococcus pyogenes-induced necrotizing fasciitis mouse model, nano neuro-immune blockers showed significant therapeutic efficacy by ameliorating sensitivity to pain and promoting the antibacterial effect of neutrophils.