Tetratricopeptide repeat domain 36 deficiency mitigates renal tubular injury by inhibiting TGF-ß1-induced epithelial-mesenchymal transition in a mouse model of chronic kidney disease.

The damage of proximal tubular epithelial cells (PTECs) is considered a central event in the pathogenesis of chronic kidney disease (CKD) and deregulated repair processes of PTECs result in epithelial-mesenchymal transition (EMT), which in turn aggravates tubular injury and kidney fibrosis. In this study, we firstly revealed that the reduction of TTC36 is associated with unilateral ureteral obstruction (UUO)-induced CKD; besides, ablation of TTC36 attenuated tubular injury and subsequent EMT in UUO-treated mice kidneys. Consistently, TTC36 overexpression promoted EMT in TGF-ß1-induced HK2 cells. Moreover, TTC36 elevated the protein expression of CEBPB, which was involved in the regulation of TGF-ß/SMAD3 signaling, and augmented SMAD3 signaling and downstream genetic response were reduced by CEBPB silencing. Collectively, our results uncovered that TTC36 deficiency plays a protective role in tubular injury and renal fibrosis triggered by UUO; further, TTC36 overexpression exacerbated TGF-ß/SMAD3 signaling via elevating the stability of SMAD3 and CEBPB, suggesting that TTC36 inhibition may be a potential strategy in the therapy of obstructive nephropathy.

Biofabricated macrophage and fibroblast membranes synergistically promote skin wound healing.

Effective skin wound healing is a complex process involving anti-inflammation, fibrosis, matrix reconstruction, and angiogenesis. This work aimed to integrate the macrophage-mediated anti-inflammation and fibroblast-assisted matrix reconstruction for enhanced skin wound healing. Herein, we utilized the cytomembranes derived from repolarized M2 macrophages and fibroblasts to prepare the natural biologics. Results showed that the inflammatory M1 macrophages were repolarized to M2 phenotype by the M2 macrophage cytomembranes. As a consequence, the cytomembranes of M2 macrophage could facilitate the wound closure in mice. Furthermore, the addition of fibroblast membranes to the macrophage cytomembranes contributed to a better matrix reconstruction, neovascularization and angiogenesis. Next, we used a transforming growth factor-ß (TGF-ß) inhibitor to attenuate cutaneous scar formation. Therefore, our modality could promote skin wound healing and effectively suppress scar formation in the preclinical murine skin wounds. The cytomembrane biologics might provide a biocompatible and versatile tool for wound healing.

Macrophage-derived implantable vaccine prevents postsurgical tumor recurrence.

Immunotherapy emerges as a potential therapeutic strategy against tumor relapse. However, immunosuppressive tumor microenvironment poses an obstacle to immunotherapy. Of particular note is that macrophages are abundant in solid tumors and tumor-associated macrophages (TAMs) are mainly anti-inflammatory and protumoral. Therefore, re-educating TAMs will be critical for improving the antitumor efficacy of immunotherapy. Herein we engineered a macrophage-derived implantable vaccine for suppressing postsurgical tumor relapse. The vaccine comprised hybrid cytomembranes from macrophages/tumor cells and an immunoadjuvant, cytosinephosphate-guanosine oligodeoxynucleotides (CpG ODNs). The vaccine was further embedded into a calcium alginate hydrogel for tissue-localized delivery. Results show that the vaccine could induce the shift from anti-inflammatory M2-like TAMs to proinflammatory M1-like macrophage. Moreover, the vaccine stimulated systemic immunity by facilitating dendritic cells (DCs) maturation and memory T (T EM) cell activation, forming a self-replenishing circulation in tumor microenvironment. Consequently, the vaccine could prevent the postsurgical tumor relapse at both the primary and distant tumor sites. In addition, the lung metastasis was also reduced by the vaccine implantation in mice. The multifunctional vaccine prepared from biomacromolecule and nature-derived material provides a biocompatible and versatile tool for re-educating TAMs and preventing postsurgical tumor recurrence.

Role of Na+, K+-ATPase ion pump in osteoinduction.

Porous biphasic calcium phosphate bioceramic (BCP) possesses osteoinductivity to induce the osteoblastic commitment of mesenchymal stem cells (MSCs) and ectopic bone formation. However, the underlying mechanism remains enigmatic. We performed a gene array analysis of MSCs cocultured with BCP to screen for candidate osteoinductive modulators. Na+, K+-ATPase (NKA), an ion transporter, therefore was identified as a crucial ion transporter in regulating the osteogenesis of the cells. NKA activator, a polyclonal antibody, enriched the cytomembrane abundance of NKA and lead to an enhanced osteogenic effect of BCP. As indicated in gene array analysis and suggested by co-immunoprecipitation assay, protein phosphatase 2A (PP2A) was elevated by BCP to dephosphorylate NKA and prevent its endocytosis. The inhibition of NKA by ouabain resulted in an adverse effect on osteoinductivity of BCP. We further altered NKA activity in mice implanted with BCP and found that the intensity and incidence of osteoinduction was increased by the NKA activator. We went one step further by investigating the potential of targeting NKA in osteoporotic bone regeneration. Activating NKA upregulated osteogenic gene expression and calcium deposition ability of osteoporotic osteoblasts. Furthermore, activation of NKA in mice ameliorated estrogen-deficiency induced bone loss, in terms of increased bone mass and improved bending strength. With this osteoinductive bioceramic derived ion transporter target, we demonstrate that the activation of NKA has significant potential to revolutionize the regeneration of bone. STATEMENT OF SIGNIFICANCE: In this study, we identified an important role of Na+, K+-ATPase (NKA) have played in osteoinductivity of biphasic calcium phosphate bioceramic (BCP). Furthermore, we demonstrated the therapeutic potential of targeting NKA in osteoporotic bone regeneration. Numerous gene and protein targets to treat osteoporosis were discovered every year, mainly obtained by genomic and proteomic screenings of a large population. In contrast, our study identified an unrevealed bone regenerating target from the upregulated genes induced by an osteoinductive biomaterial. The approach was cost-saving since it did not require a large sample pool. Furthermore, the target derived from this approach was proven to be anabolic. Identification of an anabolic agent holds significant value since most of the current anti-osteoporotic therapies are antiresorptive.

ID1 As a Prognostic Biomarker and Promising Drug Target Plays a Pivotal Role in Deterioration of Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common cancers in the world. Our aim is to identify prognostic biomarkers that contribute to the progression of early stage ccRCC and clarify the mechanism. Here, the mRNA microarray expression profile of ccRCC samples was obtained from Gene Expression Omnibus (GEO) (GSE68417). 62 differentially expressed genes (DEGs) were gained by R Studio, including 31 upregulated genes and 31 downregulated genes. Pathway enrichment analysis was performed in DAVID database. Then, the protein-protein interaction network was obtained through STRING database and visualized by Cytoscape. Subsequently, among the network, only inhibitor of DNA Binding 1 (ID1) was significant between low-grade and high-grade ccRCC patients in TCGA data set. After analysis of the corresponding clinical information in R Studio, it is shown that low ID1 expression correlated with poor survival, high probability of tumor metastasis, and relatively high serum calcium. Later, functional enrichment of ID1 in GeneMANIA uncovered that regulating DNA binding is a main characteristic of ID1 in ccRCC, which was validated by Kaplan-Meier curve of ID1 associated genes using KM plotter database and R Studio. Immune infiltration analysis performed by Tumor Immune Estimation Resource (TIMER) revealed that CD8+ T cells and macrophages were prognostic factors. Furthermore, Valproic acid was analyzed to be the most convinced target drug of ID1 identified by Comparative Toxicogenomics Database (CTD). Taken together, ID1, a biomarker of clinical outcome in early stage ccRCC patients, has the potential function of preventing deterioration in ccRCC progression and metastasis.

Fabrication and characterization of collagen-based injectable and self-crosslinkable hydrogels for cell encapsulation.

Injectable and self-crosslinkable hydrogels have drawn much attention for their potential application as cell delivery carriers to deliver cells to the injury site of arbitrary shape. In this study, injectable and self-crosslinkable hydrogels were designed and fabricated based on collagen type I (Col I) and activated chondroitin sulfate (CS-sNHS) by physical and chemical crosslinking without the addition of any catalysts. The physical properties of hydrogels, including mechanical properties, swelling and degradation properties, were investigated. The results demonstrated that the physical properties of hydrogels, especially the stiffness of hydrogels, were readily tuned by varying the degree of substitution (DS) of CS-sNHS without changing the concentration of collagen-based precursor. Chondrocytes were encapsulated into hydrogels to investigate the effects of hydrogels on the survival, proliferation and extracellular matrix (ECM) secretion of cells by FDA/PI staining, CCK-8 test and histological staining. The results suggested that all of these hydrogels supported the survival and ECM secretion of chondrocytes, while there was more ECM secretion around chondrocytes encapsulated in hydrogel Col I/CS-sNHS56% in which the DS of CS-sNHS was 56%. When the neutral precursor solution for hydrogel of Col I or Col I/CS-sNHS56% was subcutaneously injected into SD rats, hydrogels both displayed acceptable biocompatibility in vivo. These results imply that these injectable and self-crosslinkable hydrogels are suitable candidates for applications in the fields of cell delivery and tissue engineering.

Collagen structure regulates MSCs behavior by MMPs involved cell-matrix interactions.

Various scaffolds have been studied in the formation of cell niches and regulation of mesenchymal stem cells (MSCs) behaviors. Collagen serves as one of the most promising materials for tissue engineering, but the cell-matrix interactions between MSCs and collagen are still poorly understood. In this study, we prepared methacrylated collagen (CMA) and gelatin (GMA) to form photo cross-linking hydrogels. The structure, morphology, mechanical properties and degradation behaviors of the derivatives and hydrogels were characterized and it was found that the advanced structure was the major difference between collagen and gelatin hydrogels. MSCs were encapsulated in the hydrogels and cultured for 14 days in vitro, with or without the tissue inhibitor of metalloproteinase (TIMP). The CCK-8 and CLSM demonstrated that the cells in the CMA hydrogels showed better spreading and proliferation than those in GMA hydrogels. The qRT-PCR and quantitative protein assay verified the inhibition effect of TIMP on metalloproteinases (MMPs). Since the inhibited MMPs led to inferior MSCs adhesion and proliferation, we considered that the appropriate degradation by MMPs would generate more bioactive domains and improve the cell microenvironment. Immunofluorescence staining further proved that the distribution of vitronectin was significantly related to MMP-1 and MMP-2. It was concluded that the differences in the advanced structures of the scaffold materials were amplified to significant differences in multiple biological cell-matrix interactions, and finally led to different cellular fates.

Comparison of ectopic bone formation process induced by four calcium phosphate ceramics in mice.

Phase composition played a key role in the biodegradation of calcium phosphate ceramics (CaP), which in turn influences the osteoinductive ability. The in vivo biological mechanism is still poorly understood. In this study, four types of porous CaP ceramics were investigated, namely, hydroxyapatite (HA), ß-tricalcium phosphate (TCP), and biphasic calcium phosphates BCP1 and BCP2, with HA to ß-TCP ratios of 70/30 and 30/70, respectively. The four types of ceramics were implanted into thigh muscle of mice for 16weeks. Longitudinal ectopic bone formation process at gene, protein, and tissue level induced by the material was assessed. Histological analysis revealed that BCP2 was the only group that had promoted new bone formation after 16weeks. In micro-CT analysis of biodegradation, the BCP2 group had the least increment of porosity due to the new bone formation, resulting in a significant elevation in material density. Instead of a steady increase, multiple peaks were observed in most of the temporal gene expression patterns. The gene expression results were further confirmed by immunohistochemical staining of the corresponding proteins. Among the target genes, Osterix and type I collagen were activated successively. The osteoinductive BCP2 group showed earlier and significantly higher peaks in BMP2, BMPR1A, and OPG expressions than non-bone forming groups. These findings revealed that the occurrence time and magnitude of these osteogenetic gene expression peaks can be crucial in the osteoinduction process.

Effects of Composition and Mechanical Property of Injectable Collagen I/II Composite Hydrogels on Chondrocyte Behaviors.

Satisfactory repair of damaged articular cartilage is still a challenge, while tissue engineering provides a promising strategy. Collagen-based hydrogels have been widely applied in cartilage tissue engineering due to their biocompatibility. In this study, type I collagen and type II collagen were selected to prepare physically crosslinked composite hydrogels by self-assembly of collagen, and the effects of their physicochemical properties on chondrocyte phenotype maintenance and extracellular matrix (ECM) secretion were investigated. First, the microstructure of hydrogels was observed by a scanning electron microscope, and the compressive modulus was measured by a dynamic mechanical analyzer. Then, chondrocytes were encapsulated in hydrogels and detected by Live/Dead staining. The secretion of ECM was qualitatively estimated by histological staining and quantitatively analyzed by sulfated glycosaminoglycans and DNA content detection. Finally, cartilage-specific gene expression was analyzed by quantitative real-time polymerase chain reaction analysis. The results showed that the microstructure and mechanical property of hydrogels were relevant to the composition of composite hydrogels. The compressive modulus of hydrogels improved with the increase of type I collagen content in the hydrogels. Chondrocytes could maintain their round or oval morphology and secrete cartilage-specific ECM in the four groups of hydrogels, but higher the compressive modulus of composite hydrogels, the more ECM secretion of chondrocytes.

Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway.

Tough and elastic hydrogel of hyaluronic acid and chondroitin sulfate as potential cell scaffold materials.

Natural polysaccharides are extensively investigated as cell scaffold materials for cellular adhesion, proliferation, and differentiation due to their excellent biocompatibility, biodegradability, and biofunctions. However, their application is often severely limited by their mechanical behavior. In this study, a tough and elastic hydrogel scaffold was prepared with hyaluronic acid (HA) and chondroitin sulfate (CS). HA and CS were conjugated with tyramine (TA) and the degree of substitution (DS) was 10.7% and 11.3%, respectively, as calculated by (1)H NMR spectra. The hydrogel was prepared by mixing HA-TA and CS-TA in presence of H2O2 and HRP. The sectional morphology of hydrogels was observed by SEM, static and dynamic mechanical properties were analyzed by Shimadzu electromechanical testing machine and dynamic mechanical thermal analyzer Q800. All samples showed good ability to recover their appearances after deformation, the storage modulus (E') of hydrogels became higher as the testing frequency went up. Hydrogels also showed fatigue resistance to cyclic compression. Mesenchymal stem cells encapsulated in hydrogels showed good cell viability as detected by CLSM. This study suggests that the hydrogels have both good mechanical properties and biocompatibility, and may serve as model systems to explore mechanisms of deformation and energy dissipation or find some applications in tissue engineering.