Nanoscale delivery based on polyethylene glycol (PEG)ylated graphene oxide (GO-PEG) merits attention for biomedical applications owing to its functional surface modification, superior solubility/biocompatibility and controllable drug release capability. However, impaired skin regeneration in applications of these fascinating nanomaterials in diabetes is still limited, and critical issues need to be addressed regarding insufficient collagen hyperplasia and inadequate blood supply. Therefore, a high-performance tissue engineering scaffold with biocompatible and biodegradable properties is essential for diabetic wound healing. Natural and artificial acellular dermal matrix (ADM) scaffolds with spatially organized collagen fibers can provide a suitable architecture and environment for cell attachment and proliferation. Here, a novel collagen-nanomaterial-drug hybrid scaffold was constructed from GO-PEG-mediated quercetin (GO-PEG/Que)-modified ADM (ADM-GO-PEG/Que). The resulting unique and versatile hybrid scaffold exhibited multiple advantages, including the following: a biocompatible, cell-adhesive surface for accelerating mesenchymal stem cell (MSC) attachment and proliferation; superior stability and adjustability of the conduction potential of quercetin for inducing the differentiation of MSCs into adipocytes and osteoblasts; and a biodegradable nanofiber interface for promoting collagen deposition and angiogenesis in diabetic wound repair. This study provides new prospects for the design of innovative GO-PEG-based collagen hybrid scaffolds for application in efficient therapeutic drug delivery, stem cell-based therapies, tissue engineering and regenerative medicine.

Human embryonic stem cells offer a scalable and renewable source of all somatic cell types. Human embryonic progenitor (hEP) cells are partially differentiated endodermal, mesodermal and ectodermal cell types that have not undergone terminal differentiation and express an embryonic pattern of gene expression. Here, we describe a large-scale and reproducible method of isolating a diverse library of clonally purified hEP cell lines, many of which are capable of extended propagation in vitro. Initial microarray and non-negative matrix factorization gene-expression profiling suggests that the library consists of at least 140 distinct clones and contains many previously uncharacterized cell types derived from all germ layers that display diverse embryo- and site-specific homeobox gene expression. Despite the expression of many oncofetal genes, none of the hEP cell lines tested led to tumor formation when transplanted into immunocompromised mice. All hEP lines studied appear to have a finite replicative lifespan but have longer telomeres than most fetal- or adult-derived cells, thereby facilitating their use in the manufacture of purified lineages for research and human therapy.

Chronic skin wounds caused by diabetes mellitus (DM) have been acknowledged as one of the most intractable complications. Local transplantation of mesenchymal stem cells (MSCs) is a promising method, but strategies for stabilizing and efficiently delivering active MSCs according to the wound circumstance with high proteolysis remain the main barrier. Hereon, the study demonstrates the feasibility of incorporating reduced graphene oxide (RGO) nanoparticles with an acellular dermal matrix (ADM) to improve physicochemical characteristics of natural scaffold material and fabricate a highly efficient local transplantation system for MSCs in diabetic wound healing. Under the influence of RGO nanoparticles, the ADM-RGO composite scaffolds achieved high stability and strong mechanical behaviors. In vitro, conductive ADM-RGO scaffolds demonstrated an admirable milieu for stem cells adhesion and proliferation. After having been cocultured with MSCs, the ADM-RGO-MSC composite scaffolds were transplanted into the full-thickness wound of a diabetic model that was induced by streptozotocin (STZ) to evaluate its effects. As a result, the ADM-RGO composite scaffold delivered with MSCs supported robust vascularization and collagen deposition as well as rapid re-epithelialization during diabetic wound healing. Overall, the versatile nature of the ADM-RGO composite scaffold makes it an efficient transplanting mediator for pluripotent stem cells in tissue engineering applications. The composite scaffold delivered with MSCs presents a promising approach for nonhealing diabetic wounds.

BackgroundAdult full-thickness cutaneous wound repair suffers from an imbalanced immune response, leading to nonfunctional reconstructed tissue and fibrosis. Although various treatments have been reported, the immune-mediated tissue regeneration driven by biomaterial offers an attractive regenerative strategy for damaged tissue repair.MethodsIn this research, we investigated a specific bone marrow-derived mesenchymal stem cell (BMSC) sheet that was induced by the Traditional Chinese Medicine curcumin (CS-C) and its immunomodulatory effects on wound repair. Comparisons were made with the BMSC sheet induced without curcumin (CS-N) and control (saline).ResultsIn vitro cultured BMSC sheets (CS-C) showed that curcumin promoted the proliferation of BMSCs and modified the features of produced extracellular matrix (ECM) secreted by BMSCs, especially the contents of ECM structural proteins such as fibronectin (FN) and collagen I and III, as well as the ratio of collagen III/I. Two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging of mouse implantation revealed superior engraftment of BMSCs, maintained for 35 days in the CS-C group. Most importantly, CS-C created a favorable immune microenvironment. The chemokine stromal cell-derived factor 1 (SDF1) was abundantly produced by CS-C, thus facilitating a mass migration of leukocytes from which significantly increased expression of signature TH1 cells (interferon gamma) and M1 macrophages (tumor necrosis factor alpha) genes were confirmed at 7 days post-operation. The number of TH1 cells and associated pro-inflammatory M1 macrophages subsequently decreased sharply after 14 days post-operation, suggesting a rapid type I immune regression. Furthermore, the CS-C group showed an increased trend towards M2 macrophage polarization in the early phase. CS-C led to an epidermal thickness and collagen deposition that was closer to that of normal skin.ConclusionsCurcumin has a good regulatory effect on BMSCs and this promising CS-C biomaterial creates a pro-regenerative immune microenvironment for cutaneous wound healing.

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP-labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full-thickness cutaneous wound site in streptozotocin-induced diabetic mice. Wounds treated with MSC-ADM demonstrated an increased percentage of wound closure. The treatment of MSC-ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col-I) fibers synthesis via second harmonic generation imaging. The synthesis of Col-I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP-labeled MSCs during wound healing was simultaneously traced via two-photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo-multiplier tube.

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin-induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha-smooth muscle actin and transforming growth factor-beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing.

Mesenchymal stem cells (MSCs) had been reported as a novel therapeutic strategy for non-healing diabetic cutaneous wound mainly by promoting the formation of extracellular matrix (ECM) and neovasculature. Collagen regeneration is one of the key processes of ECM remodeling in wound healing. Accordingly, rapid assessment of the collagen content in a noninvasive manner can promptly provide objective evaluation for MSC therapy of cutaneous wound healing and strength evidence to adjust therapeutic regimen. In the present study, noninvasive Raman microspectroscopy was used for tracing the regeneration status of collagen during diabetic wound healing with MSCs. Wound tissues of normal mice, diabetic mice, and MSC-treated diabetic mice were subjected to Masson trichrome staining assay and submitted to spectroscopic analysis by Raman microspectroscopy after wounding 7, 14, and 21 days. Masson trichrome staining demonstrated that there was more collagen deposition in diabetic + MSCs group relative to diabetic group. The relative intensity of Raman collagen peak positions at 937, 1004, 1321, 1452, and 1662 cm increased in MSC-treated diabetic group compared to diabetic group, although normal mice group had the highest relative intensity of collagen peak bands. Correlation analysis suggested that the spectral bands had a high positive correlation with the collagen intensity detected by Masson trichrome staining in wound tissues of three groups. Our results demonstrate that Raman microspectroscopy has potential application in rapidly and quantitatively assessing diabetic wound healing with MSCs by monitoring collagen variation, which may provide a novel method for the study of skin regeneration.

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