Gel microspheres enhance the stemness of ADSCs by regulating cell-ECM interaction.

The gel microsphere culture system (GMCS) showed various advantages for mesenchymal stem cell (MSC) expansion and delivery, such as high specific surface area, small and regular shape, extensive adjustability, and biomimetic properties. Although various technologies and materials have been developed to promote the development of gel microspheres, the differences in the biological status of MSCs between the GMCS and the traditional Petri dish culture system (PDCS) are still unknown, hindering gel microspheres from becoming a culture system as widely used as petri dishes. In the previous study, an excellent "all-in-one" GMCS has been established for the expansion of human adipose-derived MSCs (hADSCs), which showed convenient cell culture operation. Here, we performed transcriptome and proteome sequencing on hADSCs cultured on the "all-in-one" GMCS and the PDCS. We found that hADSCs cultured in the GMCS kept in an undifferentiation status with a high stemness index, whose transcriptome profile is closer to the adipose progenitor cells (APCs) in vivo than those cultured in the PDCS. Further, the high stemness status of hADSCs in the GMCS was maintained through regulating cell-ECM interaction. For application, bilayer scaffolds were constructed by osteo- and chondro-differentiation of hADSCs cultured in the GMCS and the PDCS. The effect of osteochondral regeneration of the bilayer scaffolds in the GMCS group was better than that in the PDCS group. This study revealed the high stemness and excellent functionality of MSCs cultured in the GMCS, which promoted the application of gel microspheres in cell culture and tissue regeneration.

A pathological joint-liver axis mediated by matrikine-activated CD4+ T cells.

The knee joint has long been considered a closed system. The pathological effects of joint diseases on distant organs have not been investigated. Herein, our clinical data showed that post-traumatic joint damage, combined with joint bleeding (hemarthrosis), exhibits a worse liver function compared with healthy control. With mouse model, hemarthrosis induces both cartilage degeneration and remote liver damage. Next, we found that hemarthrosis induces the upregulation in ratio and differentiation towards Th17 cells of CD4+ T cells in peripheral blood and spleen. Deletion of CD4+ T cells reverses hemarthrosis-induced liver damage. Degeneration of cartilage matrix induced by hemarthrosis upregulates serological type II collagen (COL II), which activates CD4+ T cells. Systemic application of a COL II antibody blocks the activation. Furthermore, bulk RNAseq and single-cell qPCR analysis revealed that the cartilage Akt pathway is inhibited by blood treatment. Intra-articular application of Akt activator blocks the cartilage degeneration and thus protects against the liver impairment in mouse and pig models. Taken together, our study revealed a pathological joint-liver axis mediated by matrikine-activated CD4+ T cells, which refreshes the organ-crosstalk axis and provides a new treatment target for hemarthrosis-related disease. Intra-articular bleeding induces cartilage degradation through down-reulation of cartilage Akt pathway. During this process, the soluble COL II released from the damaged cartilage can activate peripheral CD4+ T cells, differention into Th17 cells and secretion of IL-17, which consequently induces liver impairment. Intra-articular application of sc79 (inhibitor of Akt pathway) can prevent the cartilage damage as well as its peripheral influences.

Inhibition of PI3K/AKT signaling pathway prevents blood-induced heterotopic ossification of the injured tendon.

Objective: It is a common clinical phenomenon that blood infiltrates into the injured tendon caused by sports injuries, accidental injuries, and surgery. However, the role of blood infiltration into the injured tendon has not been investigated. Methods: A blood-induced rat model was established and the impact of blood infiltration on inflammation and HO of the injured tendon was assessed. Cell adhesion, viability, apoptosis, and gene expression were measured to evaluate the effect of blood treatment on tendon stem/progenitor cells (TSPCs). Then RNA-seq was used to assess transcriptomic changes in tendons in a blood infiltration environment. At last, the small molecule drug PI3K inhibitor LY294002 was used for in vivo and in vitro HO treatment. Results: Blood caused acute inflammation in the short term and more severe HO in the long term. Then we found that blood treatment increased cell apoptosis and decreased cell adhesion and tenonic gene expression of TSPCs. Furthermore, blood treatment promoted osteochondrogenic differentiation of TSPCs. Next, we used RNA-seq to find that the PI3K/AKT signaling pathway was activated in blood-treated tendon tissues. By inhibiting PI3K with a small molecule drug LY294002, the expression of osteochondrogenic genes was markedly downregulated while the expression of tenonic genes was significantly upregulated. At last, we also found that LY294002 treatment significantly reduced the tendon HO in the rat blood-induced model. Conclusion: Our findings indicate that the upregulated PI3K/AKT signaling pathway is implicated in the aggravation of tendon HO. Therefore, inhibitors targeting the PI3K/AKT pathway would be a promising approach to treat blood-induced tendon HO.

3D-printed biomimetic scaffolds with precisely controlled and tunable structures guide cell migration and promote regeneration of osteochondral defect.

Untreated osteochondral defects will develop into osteoarthritis, affecting patients' quality of life. Since articular cartilage and subchondral bone exhibit distinct biological characteristics, repairing osteochondral defects remains a major challenge. Previous studies have tried to fabricate multilayer scaffolds with traditional methods or 3D printing technology. However, the efficacy is unsatisfactory because of poor control over internal structures or a lack of integrity between adjacent layers, severely compromising repair outcomes. Therefore, there is a need for a biomimetic scaffold that can simultaneously boost osteochondral defect regeneration in both structure and function. Herein, an integrated bilayer scaffold with precisely controlled structures is successfully 3D-printed in one step via digital light processing (DLP) technology. The upper layer has both 'lotus- and radial-' distribution pores, and the bottom layer has 'lotus-' pores to guide and facilitate the migration of chondrocytes and bone marrow mesenchymal stem cells, respectively, to the defect area. Tuning pore sizes could modulate the mechanical properties of scaffolds easily. Results show that 3D-printed porous structures allow significantly more cells to infiltrate into the area of 'lotus- and radial-' distribution pores during cell migration assay, subcutaneous implantation, andin situtransplantation, which are essential for osteochondral repair. Transplantation of this 3D-printed bilayer scaffold exhibits a promising osteochondral repair effect in rabbits. Incorporation of Kartogenin into the upper layer of scaffolds further induces better cartilage formation. Combining small molecules/drugs and precisely size-controlled and layer-specific porous structure via DLP technology, this 3D-printed bilayer scaffold is expected to be a potential strategy for osteochondral regeneration.

A Strong Adhesive Biological Hydrogel for Colon Leakage Repair and Abdominal Adhesion Prevention.

Colon leakage is one of the most severe complications in abdominal trauma or surgery cases. It can lead to severe abdominal infection and abdominal adhesions, resulting in prolonged hospital stays and increased mortality. In this study, a photosensitive hydrogel is proposed, which can swiftly form a strong adhesion coating on the damaged colon after UV irradiation, to realize quick cure and suture-free repair of colon leakage. The newly developed biological gel consists of hyaluronic acid methacryloyl (HAMA) and hyaluronic acid o-nitroso benzaldehyde (HANB) in the optimal ratio of 3: 1, which exerts both the rapid photocuring properties of HAMA and the strong tissue adhesion properties of HANB. HAMA/HANB shows excellent adhesion stability on wet surfaces, presenting controllable mechanical properties, ductility, adhesion stability, and chemical stability; it also evades foreign body response, which relieves the degree of abdominal adhesion. The underlying mechanism for HAMA/HANB promoting wound healing in colon leakage involves the reconstruction of the colon barrier, as well as the regulation of the immune reaction and neovascularization. In all, HAMA/HANB is a promising alternative suture-free approach for repairing colon leakage; it has a reliable healing effect and is expected to be extended to clinical application for other organ injuries.

Biomaterial based implants caused remote liver fatty deposition through activated blood-derived macrophages.

Understanding the biocompatibility of biomaterials is a prerequisite for the prediction of its clinical application, and the present assessments mainly rely on in vitro cell culture and in situ histopathology. However, remote organs responses after biomaterials implantation is unclear. Here, by leveraging body-wide-transcriptomics data, we performed in-depth systems analysis of biomaterials - remote organs crosstalk after abdominal implantation of polypropylene and silk fibroin using a rodent model, demonstrating local implantation caused remote organs responses dominated by acute-phase responses, immune system responses and lipid metabolism disorders. Of note, liver function was specially disturbed, defined as hepatic lipid deposition. Combining flow cytometry analyses and liver monocyte recruitment inhibition experiments, we proved that blood derived monocyte-derived macrophages in the liver underlying the mechanism of abnormal lipid deposition induced by local biomaterials implantation. Moreover, from the perspective of temporality, the remote organs responses and liver lipid deposition of silk fibroin group faded away with biomaterial degradation and restored to normal at end, which highlighted its superiority of degradability. These findings were further indirectly evidenced by human blood biochemical ALT and AST examination from 141 clinical cases of hernia repair using silk fibroin mesh and polypropylene mesh. In conclusion, this study provided new insights on the crosstalk between local biomaterial implants and remote organs, which is of help for future selecting and evaluating biomaterial implants with the consideration of whole-body response.

Stage-specific and location-specific cartilage calcification in osteoarthritis development.

OBJECTIVES: This study investigated the stage-specific and location-specific deposition and characteristics of minerals in human osteoarthritis (OA) cartilages via multiple nano-analytical technologies. METHODS: Normal and OA cartilages were serially sectioned for micro-CT, scanning electron microscopy with energy dispersive X-ray spectroscopy, micro-Raman spectroscopy, focused ion beam scanning electron microscopy, high-resolution electron energy loss spectrometry with transmission electron microscopy, nanoindentation and atomic force microscopy to analyse the structural, compositional and mechanical properties of cartilage in OA progression. RESULTS: We found that OA progressed by both top-down calcification at the joint surface and bottom-up calcification at the osteochondral interface. The top-down calcification process started with spherical mineral particle formation in the joint surface during early-stage OA (OA-E), followed by fibre formation and densely packed material transformation deep into the cartilage during advanced-stage OA (OA-A). The bottom-up calcification in OA-E started when an excessive layer of calcified tissue formed above the original calcified cartilage, exhibiting a calcified sandwich structure. Over time, the original and upper layers of calcified cartilage fused, which thickened the calcified cartilage region and disrupted the cartilage structure. During OA-E, the calcified cartilage was hypermineralised, containing stiffer carbonated hydroxyapatite (HAp). During OA-A, it was hypomineralised and contained softer HAp. This discrepancy may be attributed to matrix vesicle nucleation during OA-E and carbonate cores during OA-A. CONCLUSIONS: This work refines our current understanding of the mechanism underlying OA progression and provides the foothold for potential therapeutic targeting strategies once the location-specific cartilage calcification features in OA are established.

Seamless and early gap healing of osteochondral defects by autologous mosaicplasty combined with bioactive supramolecular nanofiber-enabled gelatin methacryloyl (BSN-GelMA) hydrogel.

Autologous mosaicplasty is a common approach used to treat osteochondral defects in clinical practice. Gap integration between host and transplanted plugs requires bone tissue reservation and hyaline cartilage regeneration without uneven surface, graft necrosis and sclerosis. However, poor gap integration is a serious concern, which eventually leads to deterioration of joint function. To deal with such complications, this study has developed a strategy to effectively enhance integration of the gap region following mosaicplasty by applying injectable bioactive supramolecular nanofiber-enabled gelatin methacryloyl (GelMA) hydrogel (BSN-GelMA). A rabbit osteochondral defect model demonstrated that BSN-GelMA achieved seamless osteochondral healing in the gap region between plugs of osteochondral defects following mosaicplasty, as early as six weeks. Moreover, the International Cartilage Repair Society score, histology score, glycosaminoglycan content, subchondral bone volume, and collagen II expression were observed to be the highest in the gap region of BSN-GelMA treated group. This improved outcome was due to bio-interactive materials, which acted as tissue fillers to bridge the gap, prevent cartilage degeneration, and promote graft survival and migration of bone marrow mesenchymal stem cells by releasing bioactive supramolecular nanofibers from the GelMA hydrogel. This study provides a powerful and applicable approach to improve gap integration after autologous mosaicplasty. It is also a promising off-the-shelf bioactive material for cell-free in situ tissue regeneration.

HECTD1-Mediated Ubiquitination and Degradation of Rubicon Regulates Autophagy and Osteoarthritis Pathogenesis.

OBJECTIVE: Osteoarthritis (OA) is one of the most common degenerative joint diseases and is associated with autophagy suppression. However, the molecular mechanism of autophagy regulation in the context of OA is not fully understood. In this study, we sought to determine the role that HECTD1 plays in the pathogenesis of OA. METHODS: We used RNA sequencing analysis to explore the differential expression of E3 ubiquitin ligase genes in healthy human cartilage and human cartilage affected by OA. Using surgery- and aging-induced OA mouse models, we comprehensively analyzed the function of the screened gene Hectd1 in the development of OA; furthermore, we dissected the mechanism by which HECTD1 regulates autophagy and OA progression using a combination of molecular biologic, cell biologic, and biochemical approaches. RESULTS: HECTD1 was significantly down-regulated in human OA cartilage samples compared to healthy cartilage samples. Overexpression of HECTD1 in mouse joints alleviated OA pathogenesis, whereas conditional depletion of Hectd1 in cartilage samples aggravated surgery- and aging-induced OA pathogenesis. Mechanistically, HECTD1 bound to Rubicon and ubiquitinated Rubicon at lysine residue 534, which targets Rubicon for proteasomal degradation. More importantly, HECTD1-mediated Rubicon degradation regulated chondrocyte autophagy, leading to mitigation of stress-induced chondrocyte death and the subsequent progression of OA. CONCLUSION: HECTD1 plays a crucial role in the pathogenesis of OA, in that HECTD1 regulates chondrocyte autophagy by ubiquitinating and targeting Rubicon for proteasomal degradation.

Msx1+ stem cells recruited by bioactive tissue engineering graft for bone regeneration.

Critical-sized bone defects often lead to non-union and full-thickness defects of the calvarium specifically still present reconstructive challenges. In this study, we show that neurotrophic supplements induce robust in vitro expansion of mesenchymal stromal cells, and in situ transplantation of neurotrophic supplements-incorporated 3D-printed hydrogel grafts promote full-thickness regeneration of critical-sized bone defects. Single-cell RNA sequencing analysis reveals that a unique atlas of in situ stem/progenitor cells is generated during the calvarial bone healing in vivo. Notably, we find a local expansion of resident Msx1+ skeletal stem cells after transplantation of the in situ cell culture system. Moreover, the enhanced calvarial bone regeneration is accompanied by an increased endochondral ossification that closely correlates to the Msx1+ skeletal stem cells. Our findings illustrate the time-saving and regenerative efficacy of in situ cell culture systems targeting major cell subpopulations in vivo for rapid bone tissue regeneration.

High-efficient engineering of osteo-callus organoids for rapid bone regeneration within one month.

Large bone defects that cannot form a callus tissue are often faced with long-time recovery. Developmental engineering-based strategies with mesenchymal stem cell (MSC) aggregates have shown enhanced potential for bone regeneration. However, MSC aggregates are different from the physiological callus tissues, which limited the further endogenous osteogenesis. This study aims to achieve engineering of osteo-callus organoids for rapid bone regeneration in cooperation with bone marrow-derived stem cell (BMSC)-loaded hydrogel microspheres (MSs) by digital light-processing (DLP) printing technology and stepwise-induction. The printed MSC-loaded MSs aggregated into osteo-callus organoids after chondrogenic induction and showed much higher chondrogenic efficiency than that of traditional MSC pellets. Moreover, the osteo-callus organoids exhibited stage-specific gene expression pattern that recapitulated endochondral ossification process, as well as a synchronized state of cell proliferation and differentiation, which highly resembled the diverse cell compositions and behaviors of developmentally endochondral ossification. Lastly, the osteo-callus organoids efficiently led to rapid bone regeneration within only 4 weeks in a large bone defect in rabbits which need 2-3 months in previous tissue engineering studies. The findings suggested that in vitro engineering of osteo-callus organoids with developmentally osteogenic properties is a promising strategy for rapid bone defect regeneration and recovery.

Chemical-Empowered Human Adipose-Derived Stem Cells with Lower Immunogenicity and Enhanced Pro-angiogenic Ability Promote Fast Tissue Regeneration.

Mesenchymal stem cells (MSCs) have been widely used as functional components in tissue engineering. However, the immunogenicity and limited pro-angiogenic efficacy of MSCs greatly limited their pro-regenerative ability in allogenic treatment. Herein, utilizing a chemically defined cocktail in the culture system, including cytokines, small molecules, structural protein, and other essential components, we generated the immunoprivileged and pro-angiogenic cells (IACs) derived from human adipose tissues. Conventional adipose-derived MSCs (cADSCs) were used as a control in all the experiments. IACs show typical MSC properties with enhanced stemness capacity and a robust safety profile. IACs induce a significantly milder immune response of allogenic peripheral blood mononuclear cells in an H3K27me3-HLA axis-dependent manner. IACs, through superior paracrine effects, further promote nitric oxide production, anti-apoptotic ability, and the tube formation of human vein endothelial cells. Embedded in a photo-reactive hydrogel (Gel) termed as GelMA/HA-NB/LAP for tissue engineering treatment, IACs promote faster tissue regeneration in a xenogeneic full-thickness skin defect model, eliciting a milder immune response and enhanced blood vessel formation in IACs-treated defect areas. Together with its excellent pro-regenerative potential and robust safety, our findings suggest that IACs may be a promising candidate for clinically relevant stem cell and tissue engineering therapeutics.

Free or fixed state of nHAP differentially regulates hBMSC morphology and osteogenesis through the valve role of ITGA7.

Nano-hydroxyapatite (nHAP) has been widely used in bone repair as an osteo-inductive and naturally-occurring material. However, the optimal applied form of nHAP and the underlying mechanisms involved remain unclear. Herein, to investigate into these, a range of corresponding models were designed, including three applied forms of nHAP (Free, Coating and 3D) that belong to two states (Free or fixed). The results indicate that when fixed nHAP was applied in the 3D form, optimal osteogenesis was induced in human bone marrow stem cells (hBMSCs) with increased bone volume via integrin α7 (ITGA7)-mediated upregulation of the PI3K-AKT signaling pathway, while contrary results were observed with free nHAP. Ectopic osteogenesis experiments in mice subcutaneous transplantation model further confirmed the different tendencies of ITGA7 expression and osteogenesis of hBMSCs in free and fixed states of nHAP. Our results revealed that the two states of nHAP play a different regulatory role in cell morphology and osteogenesis through the valve role of ITGA7, providing cues for better application of nanoparticles and a potential new molecular target in bone tissue engineering.

Polyglutamic Acid-Based Elastic and Tough Adhesive Patch Promotes Tissue Regeneration through In Situ Macrophage Modulation.

Adhesive patches are advanced but challenging alternatives to suture, especially in treating fragile internal organs. So far there is no suture-free adhesive patch based on metabolizable poly(amino acid) materials with excellent mechanical strength as well as immunomodulation functionality. Here, a polyglutamic acid-based elastic and tough adhesive patch modified by photosensitive groups on the surface to achieve robust light-activated adhesion and sealing of flexible internal organs is explored. With the porous internal morphology and excellent biodegradability, the patches promote regeneration through a macrophage-regulating microenvironment. Treated rabbits achieve rapid full-thickness gastric regeneration with complete functional structure within 14 d, suggesting its robust tissue adhesion and repair-promoting ability.

Modular protein engineering-based biomaterials for skeletal tissue engineering.

Biomaterials are indispensable for tissue engineering, which plays a pivotal role in the skeletal tissue repair. However, biomaterials currently used such as animal extracts and chemically synthesized polymers display unsatisfactory bioactivity and safety. In recent years, modular protein engineering-based (MPE) biomaterials composed of polypeptides produced by molecular cloning and protein synthesis have greatly developed due to their lower batch-to-batch variation, avoidance of possible pathogens and, most importantly, sequence-tunable property. In this review, we first briefly describe the properties of different MPE biomaterials classified by the structural domains of polypeptides, and techniques to engineer the polypeptide sequence and synthesize MPE biomaterials at will. Then, we focus on the application of bio-designed MPE biomaterials in skeletal tissue engineering. Different structural domains of polypeptides are used individually or covalently fused with different bioactive motifs to generate a variety of MPE biomaterials. The sequence (protein modules) of MPE biomaterials would determine and guide their cytocompatibility, their effects on cell fate and ECM formation, the mechanical properties and functions during the in vivo skeletal tissue repair. Moreover, we propose several bio-design strategies and potential directions to develop MPE biomaterials for better performing skeletal tissue engineering and to achieve fast skeletal tissue regeneration. Combinations of material science and protein engineering would provide solutions to the obstacles in regenerative medicine. This article provides a board review of skeletal tissue engineering in a polypeptide sequence-guided way by using MPE biomaterials.

In-cytoplasm mitochondrial transplantation for mesenchymal stem cells engineering and tissue regeneration.

Stem cell therapies are unsatisfactory due to poor cell survival and engraftment. Stem cell used for therapy must be properly "tuned" for a harsh in vivo environment. Herein, we report that transfer of exogenous mitochondria (mito) to adipose-derived mesenchymal stem cells (ADSCs) can effectively boost their energy levels, enabling efficient cell engraftment. Importantly, the entire process of exogeneous mitochondrial endocytosis is captured by high-content live-cell imaging. Mitochondrial transfer leads to acutely enhanced bioenergetics, with nearly 17% of higher adenosine 5'-triphosphate (ATP) levels in ADSCs treated with high mitochondrial dosage and further results in altered secretome profiles of ADSCs. Mitochondrial transfer also induced the expression of 334 mRNAs in ADSCs, which are mainly linked to signaling pathways associated with DNA replication and cell division. We hypothesize that increase in ATP and cyclin-dependent kinase 1 and 2 expression might be responsible for promoting enhanced proliferation, migration, and differentiation of ADSCs in vitro. More importantly, mito-transferred ADSCs display prolonged cell survival, engraftment and horizontal transfer of exogenous mitochondria to surrounding cells in a full-thickness skin defect rat model with improved skin repair compared with nontreated ADSCs. These results demonstrate that intracellular mitochondrial transplantation is a promising strategy to engineer stem cells for tissue regeneration.

Engineered adipose-derived stem cells with IGF-1-modified mRNA ameliorates osteoarthritis development.

BACKGROUND: Osteoarthritis (OA), a prevalent degenerative disease characterized by degradation of extracellular matrix (ECM), still lacks effective disease-modifying therapy. Mesenchymal stem cells (MSCs) transplantation has been regarded as the most promising approach for OA treatment while engrafting cells alone might not be adequate for effective regeneration. Genetic modification has been used to optimize MSC-based therapy; however, there are still significant limitations that prevent the clinical translation of this therapy including low efficacy and safety concerns. Recently, chemically modified mRNA (modRNA) represents a promising alternative for the gene-enhanced MSC therapy. In this regard, we hypothesized that adipose derived stem cells (ADSCs) engineered with modRNA encoding insulin-like growth factor 1 (IGF-1) were superior to native ADSCs on ameliorating OA development. METHODS: Mouse ADSCs were acquired from adipose tissue and transfected with modRNAs. First, the kinetics and efficacy of modRNA-mediated gene transfer in mouse ADSCs were analyzed in vitro. Next, we applied an indirect co-culture system to analyze the pro-anabolic potential of IGF-1 modRNA engineered ADSCs (named as IGF-1-ADSCs) on chondrocytes. Finally, we evaluated the cell retention and chondroprotective effect of IGF-1-ADSCs in vivo using fluorescent labeling, histology and immunohistochemistry. RESULTS: modRNA transfected mouse ADSCs with high efficiency (85 ± 5%) and the IGF-1 modRNA-transfected ADSCs facilitated burst-like production of bio-functional IGF-1 protein. In vitro, IGF-1-ADSCs induced increased anabolic markers expression of chondrocytes in inflammation environment compared to untreated ADSCs. In a murine OA model, histological and immunohistochemical analysis of knee joints harvested at 4 weeks and 8 weeks after OA induction suggested IGF-1-ADSCs had superior therapeutic effect over native ADSCs demonstrated by lower histological OARSI score and decreased loss of cartilage ECM. CONCLUSIONS: These findings collectively supported the therapeutic potential of IGF-1-ADSCs for clinical OA management and cartilage repair.

Macrophage membrane-functionalized nanofibrous mats and their immunomodulatory effects on macrophage polarization.

Immunomodulation is an important phenomenon in the normal mammalian host response toward an injury, and plays a critical role in tissue regeneration and regenerative medicine. Different phenotypes of macrophages show an array of activation states compassing pro-inflammatory to pro-alleviating cells, which are the critical players to modulate immune response and tissue regeneration. In this study, macrophage membranes of different phenotypes (macrophages (M0), classically activated macrophages (M1) and alternatively activated macrophages (M2)) were coated onto poly-ε-caprolactone (PCL) nanofibers to acquire exterior surface proteins and similar functions of the natural membranes. In vitro results unveiled that these nanofibers, especially the M2-PCL nanofibers, can suppress the activities of inflammatory markers such as TNF-α and IL-1ß, and stimulate anti-inflammatory markers such as Arg-1, IL-10 and TGF-ß. In a C57BL/6 mouse model, the macrophage membrane-coated nanofibers, especially the M2-PCL nanofibers, displayed minimal cellular infiltration and low collagen deposition, increased anti-inflammatory CD206 and decreased inflammatory CD86 levels. The M2-PCL nanofibers most effectively neutralized inflammatory chemokines, regulated the expression of inflammation-associated genes as well as anti-inflammatory genes, and showed strong immunomodulatory effects than the PCL, M0-PCL and M1-PCL nanofibers. STATEMENT OF SIGNIFICANCE: Different types of macrophage membrane-functionalized PCL nanofibers were successfully prepared and well characterized. They inherited the surface proteins imitating the source macrophages, and played an important role in limiting cellular infiltration and collagen deposition. These different macrophages and their membrane-coated nanofibers (M0-PCL, M1-PCL and M2-PCL) behaved like their respective source cells. The M2 mimicking M2-PCL nanofibers effectively polarized macrophages to M2 phenotype and decreased the expression of inflammation-associated chemokines and promoted the anti-inflammation in vitro and in vivo, which is critical for tissue regeneration. The mice implanted with the bio-mimicking M2-PCL nanofibers effectively inhibited toll like receptors signaling induced NF-kB and IRF-5 and their target genes such as Edn-1, IL-6, iNOS, TNF-α, etc. compared to the PCL, and M0-PCL and M1-PCL macrophage membrane-coated nanofibers.

Single-cell RNA-seq reveals functionally distinct biomaterial degradation-related macrophage populations.

Macrophages play crucial roles in host tissue reaction to biomaterials upon implantation in vivo. However, the complexity of biomaterial degradation-related macrophage subpopulations that accumulate around the implanted biomaterials in situ is not fully understood. Here, using single cell RNA-seq, we analyze the transcriptome profiles of the various cell types around the scaffold to map the scaffold-induced reaction, in an unbiased approach. This enables mapping of all biomaterial degradation-associated cells at high resolution, revealing distinct subpopulations of tissue-resident macrophages as the major cellular sources of biomaterial degradation in situ. We also find that scaffold architecture can affect the mechanotransduction and catabolic activity of specific material degradation-related macrophage subpopulations in an Itgav-Mapk1-Stat3 dependent manner, eventually leading to differences in scaffold degradation rate in vivo. Our work dissects unanticipated aspects of the cellular and molecular basis of biomaterial degradation at the single-cell level, and provides a conceptual framework for developing functional tissue engineering scaffolds in future.

Light-induced osteogenic differentiation of BMSCs with graphene/TiO2 composite coating on Ti implant.

Light-induced surface potential have been demonstrated as an effective bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation regulator. However, traditional bone repair implants almost were weak or no light-responsive. Fortunately, surface modification was a feasible strategy to realize its light functionalization for bone implants. Herein, a graphene oxide (GO)/titanium dioxide (TiO2) nanodots composite coating on the surface of titanium (Ti) implant was constructed, and GO was reduced to reduced graphene oxide (rGO) with the method of UV-assisted photocatalytic reduction. After rGO deposited on the surface of TiO2, a heterojunction formed at the interface of rGO and TiO2. With visible light illumination, positive charges accumulated on the surface of rGO/TiO2 film, and performed as a positive surface potential change. The light-induced surface potential which was generated under proper light intensity is harmless to the cell adhesion and proliferation behavior, but presented a good BMSCs osteogenic differentiation promoting effect, and the activation of the voltage-gated calcium channels through surface potential and the promotion of the adsorption of osteogenic growth factors could be the reason. This work given a new insight of the modification for Ti implant with a light-induced surface potential, and shows potential application for bone regeneration on the clinical practice through light stimulation.