Fish Collagen and Hydroxyapatite Reinforced Poly(lactide- co-glycolide) Fibrous Membrane for Guided Bone Regeneration.

The purpose of this study was to fabricate a low-immunogenicity fish collagen (FC) and bioactive nanohydroxyapatite (n-HA) enhanced poly(lactide- co-glycolide) (PLGA) nanofibrous membrane for guided bone regeneration (GBR) via electrospinning. The physicochemical properties and morphology study revealed that FC and n-HA particles were homogeneously dispersed in the PLGA fibrous matrix. Notably, the formation of enhanced polymeric chain network due to the interaction between FC and PLGA significantly improved the tensile strength of the PLGA membrane. The incorporation of FC altered the degradation behavior of fibers and accelerated the degradation rate of the PLGA-based membranes. Moreover, the membranes exhibited favorable cytocompatibility with bone mesenchymal stem cells (BMSCs) and human gingiva fibroblasts (HGF) cells. More importantly, the optimized membrane satisfied the requirements of the 'Biological evaluation of medical devices' during the incipient biosafety evaluation. All the results indicate that this composite fibrous membrane exhibits significant potential for guided bone or tissue regeneration.

M2 macrophage-derived exosome-functionalized topological scaffolds regulate the foreign body response and the coupling of angio/osteoclasto/osteogenesis.

Designing scaffolds that can regulate the innate immune response and promote vascularized bone regeneration holds promise for bone tissue engineering. Herein, electrospun scaffolds that combined physical and biological cues were fabricated by anchoring reparative M2 macrophage-derived exosomes onto topological pore structured nanofibrous scaffolds. The topological pore structure of the fiber and the immobilization of exosomes increased the nanoscale roughness and hydrophilicity of the fibrous scaffold. In vitro cell experiments showed that exosomes could be internalized by target cells to promote cell migration, tube formation, osteogenic differentiation, and anti-inflammatory macrophage polarization. The activation of fibrosis, angiogenesis, and macrophage was elucidated during the exosome-functionalized fibrous scaffold-mediated foreign body response (FBR) in subcutaneous implantation in mice. The exosome-functionalized nanofibrous scaffolds also enhanced vascularized bone formation in a critical-sized rat cranial bone defect model. Importantly, histological analysis revealed that the biofunctional scaffolds regulated the coupling effect of angiogenesis, osteoclastogenesis, and osteogenesis by stimulating type H vessel formation. This study elaborated on the complex processes within the cell microenvironment niche during fibrous scaffold-mediated FBR and vascularized bone regeneration to guide the design of implants or devices used in orthopedics and maxillofacial surgery. STATEMENT OF SIGNIFICANCE: How to design scaffold materials that can regulate the local immune niche and truly achieve functional vascularized bone regeneration still remain an open question. Here, combining physical and biological cues, we proposed new insight to cell-free and growth factor-free therapy, anchoring reparative M2 macrophage-derived exosomes onto topological pore structured nanofibrous scaffolds. The exosomes functionalized-scaffold system mitigated foreign body response, including excessive fibrosis, tumor-like vascularization, and macrophage activation. Importantly, the biofunctional scaffolds regulated the coupling effect of angiogenesis, osteoclastogenesis, and osteogenesis by stimulating type H vessel formation.

A sequential macrophage activation strategy for bone regeneration: A micro/nano strontium-releasing composite scaffold loaded with lipopolysaccharide.

Effective tissue repair relies on the orchestration of different macrophage phenotypes, both the M2 phenotype (promotes tissue repair) and M1 phenotype (pro-inflammatory) deserve attention. In this study, we propose a sequential immune activation strategy to mediate bone regeneration, by loading lipopolysaccharide (LPS) onto the surface of a strontium (Sr) ions -contained composite scaffold, which was fabricated by combining Sr-doped micro/nano-hydroxyapatite (HA) and dual degradable matrices of polycaprolactone (PCL) and poly (lactic-co-glycolic acid) (PLGA). Our strategy involves the sequential release of LPS to promote macrophage homing and induce the expression of the pro-inflammatory M1 phenotype, followed by the release of Sr ions to suppress inflammation. In vitro and in vivo experiments demonstrated that, the appropriate pro-inflammatory effects at the initial stage of implantation, along with the anti-inflammatory effects at the later stage, as well as the structural stability of the scaffolds conferred by the composition, can synergistically promote the regeneration and repair of bone defects.

Plant-Derived Polyphenol and LL-37 Peptide-Modified Nanofibrous Scaffolds for Promotion of Antibacterial Activity, Anti-Inflammation, and Type-H Vascularized Bone Regeneration.

The regeneration of oral tissues is a challenging clinical problem because of the complex microbial and biological stress environments. Electrospun fibrous scaffolds have attracted significant interest as effective barrier membranes for guided bone regeneration (GBR); however, no mature strategy yet exists for the surface modification of fibers to provide versatility to satisfy clinical requirements. This study demonstrated a practical biosafety strategy: the combined use of plant polyphenols and LL-37 peptides to modify the fiber surface to endow the fibrous scaffold with antimicrobial activity, immunoregulation, and vascularized bone regeneration. We confirmed that the LL-37 peptides interacted with tannic acid (TA) through noncovalent bonds through experiments and molecular docking simulation analysis. In vitro experiments showed that the TA coating imparted strong antibacterial properties to the fibrous scaffold, but it also caused cytotoxicity. The grafting of LL-37 peptide promoted the spreading, migration, and osteogenic differentiation of mesenchymal stem cells and was also conducive to the M2 polarization of RAW264.7 cells. In vivo experiments further verified that the LL-37 peptide-grafted fibrous scaffold significantly enhanced angiogenesis, anti-inflammatory effects, and type-H vascularized bone regeneration. Overall, the fibrous scaffold modified by the LL-37 peptide through TA grafting has significant potential for GBR applications.

Sequential Dual-Biofactor Release from the Scaffold of Mesoporous HA Microspheres and PLGA Matrix for Boosting Endogenous Bone Regeneration.

The combined design of scaffold structure and multi-biological factors is a prominent strategy to promote bone regeneration. Herein, a composite scaffold of mesoporous hydroxyapatite (HA) microspheres loaded with the bone morphogenetic protein-2 (BMP-2) and a poly(DL-lactic-co-glycolic acid) (PLGA) matrix is constructed by 3D printing. Furthermore, the chemokine stromal cell-derived factor-1α (SDF-1α) is adsorbed on a scaffold surface to achieve the sequential release of the dual-biofactors. The results indicate that the rapid release of SDF-1α chemokine on the scaffold surface effectively recruits bone marrow-derived mesenchymal stem cells (BMSCs) to the target defect area, whereas the long-term sustained release of BMP-2 from the HA microspheres in the degradable PLGA matrix successfully triggers the osteogenic differentiation in the recruited BMSCs, significantly promoting bone regeneration and reconstruction. In addition, these structures/biofactors specially combining scaffold exhibit significantly better biological performance than that of other combined scaffolds, including the bare HA/PLGA scaffold, the scaffold loaded with SDF-1α or BMP-2 biofactor alone, and the scaffold with surface SDF-1α and BMP-2 dual-biofactors. The utilization of mesoporous HA, the assembly method, and sequential release of the two biofactors in the 3D printed composite scaffold present a new method for future design of high-performance bone repairing scaffolds.

Chopped fibers and nano-hydroxyapatite enhanced silk fibroin porous hybrid scaffolds for bone augmentation.

Chopped fiber (CF)- and nano-hydroxyapatite (n-HA)-enhanced silk fibroin (SF) porous hybrid scaffolds (SHCF) were prepared by freeze-drying for bone augmentation. Compared with pristine SF scaffolds, the incorporation of CF and n-HA can significantly enhance the mechanical properties of the composite scaffold. The results of cell experiments and mouse subcutaneous implantation indicated that the SHCF could alleviate foreign body reactions (FBR) led by macrophages and neutrophils, promote the polarization of RAW264.7 cells to anti-inflammatory M2 macrophages, and inhibit the secretion of pro-inflammatory cytokine TNF-α. A rat femoral defect repair model and bulk-RNA-seq analysis indicated that the CF- and n-HA-enhanced SHCF promoted the proliferation and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) by the upregulation of Capns1 expression and regulated the calcium signaling pathway to mediate osteogenesis-related cell behavior, subsequently promoting bone regeneration.

A baicalin-loaded coaxial nanofiber scaffold regulated inflammation and osteoclast differentiation for vascularized bone regeneration.

We demonstrate a simple, effective and feasible method to address the shrinkage of Poly (lactic-co-glycolic acid) (PLGA) through a core-shell structure fiber strategy. The results revealed that introducing size-stable poly-caprolactone (PCL) as the core fiber significantly improved the PLGA-based fibrous scaffold's dimensional maintenance. We further utilized fish collagen to modify the PLGA shell layer (PFC) of coaxial fibers and loaded baicalin (BA) into the PCL core layer (PCL-BA) to endow fibrous scaffold with more functional biological cues. The PFC/PCL-BA fibrous scaffold promoted the osteogenic differentiation of bone mesenchymal stem cells and stimulated the RAW264.7 cells to polarize into a pro-reparative phenotype. Importantly, the in vivo study demonstrated that the PFC/PCL-BA scaffold could regulate inflammation and osteoclast differentiation, favor neovascularization and bone formation. This work tactfully combined PLGA and PCL to establish a drug release platform based on the core-shell fibrous scaffold for vascularized bone regeneration.

Recent advances in PLGA-based biomaterials for bone tissue regeneration.

Bone regeneration is an interdisciplinary complex lesson, including but not limited to materials science, biomechanics, immunology, and biology. Having witnessed impressive progress in the past decades in the development of bone substitutes; however, it must be said that the most suitable biomaterial for bone regeneration remains an area of intense debate. Since its discovery, poly (lactic-co-glycolic acid) (PLGA) has been widely used in bone tissue engineering due to its good biocompatibility and adjustable biodegradability. This review systematically covers the past and the most recent advances in developing PLGA-based bone regeneration materials. Taking the different application forms of PLGA-based materials as the starting point, we describe each form's specific application and its corresponding advantages and disadvantages with many examples. We focus on the progress of electrospun nanofibrous scaffolds, three-dimensional (3D) printed scaffolds, microspheres/nanoparticles, hydrogels, multiphasic scaffolds, and stents prepared by other traditional and emerging methods. Finally, we briefly discuss the current limitations and future directions of PLGA-based bone repair materials. STATEMENT OF SIGNIFICANCE: As a key synthetic biopolymer in bone tissue engineering application, the progress of PLGA-based bone substitute is impressive. In this review, we summarized the past and the most recent advances in the development of PLGA-based bone regeneration materials. According to the typical application forms and corresponding crafts of PLGA-based substitutes, we described the development of electrospinning nanofibrous scaffolds, 3D printed scaffolds, microspheres/nanoparticles, hydrogels, multiphasic scaffolds and scaffolds fabricated by other manufacturing process. Finally, we briefly discussed the current limitations and proposed the newly strategy for the design and fabrication of PLGA-based bone materials or devices.

Dissecting the microenvironment around biosynthetic scaffolds in murine skin wound healing.

The structural properties of biomaterials play crucial roles in guiding cell behavior and influencing immune responses against the material. We fabricated electrospun membranes with three types of surface topography (random, aligned, and latticed), introduced them to dorsal skin excisional wounds in mice and rats, and evaluated their effects on wound healing and immunomodulatory properties. An overview of different immune cells in the microenvironment with the help of single-cell RNA sequencing revealed diverse cellular heterogeneity in vivo. The time course of immune response was advanced toward an adaptive immunity-dominant stage by the aligned scaffold. In mice without mature T lymphocytes, lack of wound-induced hair neogenesis indicated a regulatory role of T cells on hair follicle regeneration. The microenvironment around scaffolds involved an intricate interplay of immune and cutaneous cells.

Topological structure of electrospun membrane regulates immune response, angiogenesis and bone regeneration.

The fate of biomaterials is orchestrated by biocompatibility and bioregulation characteristics, reported to be closely related to topographical structures. For the purpose to investigate the topography of fibrous membranes on the guided bone regeneration performance, we successfully fabricated poly (lactate-co-glycolate)/fish collagen/nano-hydroxyapatite (PFCH) fibrous membranes with random, aligned and latticed topography by electrospinning. The physical, chemical and biological properties of the three topographical PFCH membranes were systematically investigated by in vitro and in vivo experiments. The subcutaneous implantation of C57BL6 mice showed an acceptable mild foreign body reaction of all three topological membranes. Interestingly, the latticed PFCH membrane exhibited superior abilities to recruit macrophage/monocyte and induce angiogenesis. We further investigated the osteogenesis of the three topographical PFCH membranes via the critical-size calvarial bone defect model of rats and mice and the results suggested that latticed PFCH membrane manifested promising performance to promote angiogenesis through upregulation of the HIF-1α signaling pathway; thereby enhancing bone regeneration. Our research illustrated that the topological structure of fibrous membranes, as one of the characteristics of biomaterials, could regulate its biological functions, and the fibrous structure of latticed topography could serve as a favorable surface design of biomaterials for bone regeneration. STATEMENT OF SIGNIFICANCE: In material-mediated regeneration medicine, the interaction between the biomaterial and the host is key to successful tissue regeneration. The micro-and nano-structure becomes one of the most critical physical clues for designing biomaterials. In this study, we fabricated three topological electrospun membranes (Random, Aligned and Latticed) to understand how topological structural clues mediate bone tissue regeneration. Interestingly, we found that the Latticed topographical PFCH membrane promotes macrophage recruitment, angiogenesis, and osteogenesis in vivo, indicating the fibrous structure of latticed topography could serve as a favorable surface design of biomaterials for bone regeneration.

The shrinking behavior, mechanism and anti-shrinkage resolution of an electrospun PLGA membrane.

The deformation shrinkage of a poly(lactide-co-glycolide) (PLGA) fibrous material seriously affects its biomedical application. To demonstrate the underlying shrinking mechanism and to find a method to prevent the shrinkage of an electrospun PLGA membrane, we investigated the shrinking behavior of PLGA electrospun membranes under various test conditions and discussed the underlying shrinking mechanism. The results indicated that the shrinkage of the electrospun PLGA membrane was mainly regulated by the glass transition of its polymer fiber; the temperature and liquid environment were found to be the two main factors leading to the shrinkage of the electrospun PLGA membrane through affecting its glass transition. Then a heat stretching (HS) technique was proposed by us to stabilize the electrospun PLGA membrane. After HS treatment, the glass transition temperature (Tg) of the electrospun PLGA membrane could increase from 48.38 °C to 54.55 °C. Our results indicated that the HS-treated membranes could maintain a high area percentage of 90.89 ± 2.27% and 84.78 ± 3.36% after immersion respectively in PBS and blood at 37 °C for 2 hours. Further experiments confirmed that the HS technique could also stabilize the dimensional structure of the electrospun PDLLA membrane in PBS and blood at 37 °C. This study provides an effective strategy for preventing the shrinkage of electrospun polyester biomaterials in a physiological environment that may benefit both the material structural stability and the in vivo biological performance.

Physicochemical and In Vitro Degradation Behaviors of Fibrous Membranes with Different Polycaprolactone and Gelatin Proportions.

Mechanical and degradation properties are crucial factors of guided tissue/bone regeneration (GTR/GBR) membranes. In this work, a series of fibrous membranes with different ratios of polycaprolactone (PCL) and gelatin (Gel) were prepared (PCL:Gel = 1:9 (P1G9), 3:7 (P3G7), 5:5 (P5G5), 7:3 (P7G3), and 9:1 (P9G1)) by electrospinning, and their physicochemical properties and In Vitro degradation behaviors were systematically investigated. Mechanical tests showed that tensile strength was enhanced with the presence of Gel, and the tensile strength of the P9G1 membrane reached nearly three times that of the pure PCL membrane. The degradation rate of the composite membranes could be adjusted by controlling the ratio of PCL and Gel; the higher the Gel content was, the faster the degradation of the PCL/Gel membrane. The higher PCL content favored maintaining the fibrous structure of the electrospun membranes. These findings will be beneficial for designing PCL/Gel composite materials for biomedical applications.

The ultralong-term comparison of osteogenic behavior of three scaffolds with different matrices and degradability between one and two years.

Attributed to their structure and composition manipulated to mimic natural bone tissue, porous scaffolds composed of inorganic nano-hydroxyapatite (n-HA) and organic polymers with different degrees of degradability have been proven to be a promising bone regeneration strategy. However, long-term and in-depth comparative research on the effects of scaffolds with different matrices and degrees of degradability on bone reconstruction is still lacking. In this study, the ultralong-term osteogenic performance of three polymeric composite scaffolds based on non-degradable polyamide 66 (PA66), slowly degradable polycaprolactone (PCL) and fast degradable poly (lactic-co-glycolic acid) (PLGA) were investigated comparatively after implanting the scaffolds into rabbit femoral defects for 12, 15, 18 and 21 months. The results demonstrated that the structural integrity of the scaffolds played a positive role in long-term bone reconstruction. Thus the n-HA/PA66 and n-HA/PCL scaffolds have a higher relative bone volume and bone density than the n-HA/PLGA scaffolds from 12 to 21 months. In addition, the favorable surface wettability and collagen-like molecular structure should endow the n-HA/PA66 scaffold with the best long-term osteogenic property among the three scaffolds. The ultralong-term comparative study reveals that a relatively stable scaffold integrity, together with favorable matrix molecular characteristics and hydrophilicity, may be more important for long-term osteogenesis besides the effect of scaffold pore structure, rather than the pursuit of fast scaffold degradation. The results also show that the space left by scaffold degradation is not easily occupied by new bone tissue, especially after bone tissue has formed a stable structure or the bone interface has become inert.

Enhanced osteogenesis and angiogenesis by PCL/chitosan/Sr-doped calcium phosphate electrospun nanocomposite membrane for guided bone regeneration.

Membranes play pivotal role in guided bone regeneration (GBR) technique for reconstruction alveolar bone. GBR membrane that is able to stimulate both osteogenic and angiogenic differentiation of cells may be more effective in clinic practice. Herein, we fabricated the Sr-doped calcium phosphate/polycaprolactone/chitosan (Sr-CaP/PCL/CS) nanohybrid fibrous membrane by incorporating 20 wt% bioactive Sr-CaP nanoparticles into PCL/CS matrix via one-step electrospinning method, in order to endow the membrane with stimulation of osteogenesis and angiogenesis. The physicochemical properties, mechanical properties, Sr2+ release behavior, and the membrane stimulate bone mesenchymal stem cell (BMSCs) differentiation were evaluated in comparison with PCL/CS and CaP/PCL/CS membranes. The SEM images revealed that the nanocomposite membrane mimicked the extracellular matrix structure. The release curve presented a 28-day long continuous release of Sr2+ and concentration which was certified in an optimal range for positive biological effects at each timepoint. The in vitro cell culture experiments certified that the Sr-CaP/PCL/CS membrane enjoyed excellent biocompatibility and remarkably promoted rat bone mesenchymal stem cell (BMSCs) adhesion and proliferation. In terms of osteogenic differentiation, BMSCs seeded on the Sr-CaP/PCL/CS membrane showed a higher ALP activity level and a better matrix mineralization. What's more, the synergism of the Sr2+ and CaP from the Sr-CaP/PCL/CS membrane enhanced BMSCs angiogenic differentiation, herein resulting in the largest VEGF secretion amount. Consequently, the Sr-CaP/PCL/CS nanohybrid electrospun membrane has promising applications in GBR.

The long-term behaviors and differences in bone reconstruction of three polymer-based scaffolds with different degradability.

Scaffolds composed of polymers and nano-hydroxyapatite (n-HA) have received extensive attention in bone reconstructive repair; however there is a lack of in-depth and long-term comparative study on the effect of scaffold degradability on bone reconstruction. In this study, the osteogenic behaviors of three polymeric composite scaffolds based on fast degradable poly(lactic-co-glycolic acid) (PLGA), slowly degradable polycaprolactone (PCL) and non-degradable polyamide 66 (PA66) were investigated and compared via implanting the scaffolds into rabbit femoral defects for 1, 3, 6 and 12 months. The in vivo results demonstrated that although the n-HA/PLGA scaffold could obtain higher new bone volume at 3 months, its fast degradation caused the loss of scaffold structural integrity and led to reduction of bone volume after 3 months. The n-HA/PCL scaffold displayed slow degradation mainly after 6 months (∼20% degradation) and the n-HA/PA66 scaffold showed no degradation during the entire 12 months; these two scaffolds could maintain their structural integrity and exhibited a constant increase in bone volume with the implantation time, and even achieved higher bone volume than the n-HA/PLGA scaffold at 12 months. The year-long in vivo research revealed the following important aspects: (1) bone reconstruction is strongly related to scaffold degradability, and the scaffold structural integrity should be maintained at least for one year before complete degradation in vivo; (2) the in vivo experiment of a bone scaffold must take more time than the conventional 3 or 6 months, which is normally neglected. The study suggests a principle for future design and application of bone scaffolds that must have a relatively stable osteogenic space and scaffold interface, or have a scaffold degradation speed slower than the time of bone reconstruction completion.

Development of biomimetic trilayer fibrous membranes for guided bone regeneration.

in order to build fibrous bone tissue scaffolds for guided bone regeneration and to mimic the trilayer structure and the multifunctional properties of the natural periosteum, we fabricated two fibrous trilayer membranes by conjugate electrospinning technology, in which poly(ε-caprolactone) (PCL) fiber was designed as an outer layer, the mixed fibers of PCL and polyurethane (co-PUPCL) as the interlayer, and degradable polyurethane fibers with or without nano-hydroxyapatite (n-HA) as the inner layer (PUHA or PU). The microstructure and characteristics of the trilayer membranes were evaluated and different monolayer fibers were fabricated as the contrast samples. The tensile strength values of each layer increased from the inner layer to the outer layer in the designed structure, while the step-by-step electrospinning method produced good adhesion of different layers. Furthermore, the degradable properties and hydrophilicity of the layers changed with dissymmetric fibrous structures. Cell proliferation assay and cell morphology observation indicated that the PUHA inner fibrous layer exhibited better cell attachment and proliferation than PU. In addition, the osteogenicity of the PUHA fibrous layer has been attested through protein expression by the differentiation of rat mesenchymal stem cells (rMSCs) into the osteogenic lineage. Cell infiltration testing on the two sides of the trilayer membranes in vitro and in vivo showed that the inner layer had good cellular penetration deep into the scaffolds, whereas the cells were barred by the outer layer. We have developed a trilayer structured membrane with different polymer fibers to replicate the natural periosteum by improving functional outcomes, which is a promising fibrous scaffold for clinical use in the repair of destroyed bone.

Electrospun silver ion-loaded calcium phosphate/chitosan antibacterial composite fibrous membranes for guided bone regeneration.

PURPOSE: The purpose of this study is to construct a guided bone regeneration membrane that is similar to bone components and structurally resembles the native extracellular matrix with sufficient antibacterial properties. MATERIALS AND METHODS: A novel type of biomimetic and bioactive silver ion-loaded calcium phosphate/chitosan (Ag-CaP/CS) membrane with antibacterial ability was successfully developed by incorporation of silver ion-loaded CaP via a one-step electrospinning method and subsequently crosslinked with vanillin. RESULTS: Evaluation of the physicochemical properties revealed that the fabricated fibrous membranes mimicked the extracellular matrix structure and the addition of CaP significantly increased the mineralization ability of the membranes. Importantly, the Ag-CaP/CS membranes exhibited a sustainable release of Ag+, which in turn inhibited the adhesion and growth of Staphylococcus mutans. The results of cell adhesion and MTT assay revealed that the Ag-CaP/CS membranes were cytocompatible with bone marrow stromal cells. CONCLUSION: The fabricated electrospinning Ag-CaP/CS nanofiber membranes with excellent biocompatibility and strong antimicrobial properties have great potential to be used for guided bone regeneration.

High incidence of JC viruria in JC-seropositive older individuals.

The prevalence of the human JC virus (JCV) in the general population at various ages was investigated. Polymerase chain reaction was employed to detect viral DNA in the urine. The results showed that the incidence of JC viruria was low in the young population, but it was high in the elderly. Hemagglutination inhibition assay was performed for JCV seroprevalence study. The results showed that the seropositive rate of JCV was lower in children than that in adults. The ratio of viruria to seropositive for JCV increased with age and reached 79.7% for those older than 70 years. The results indicated that aging immunity may correlate with JCV reactivation.