Junjie Guan

Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis

BackgroundHuman induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) have emerged as a promising alternative for stem cell transplantation therapy. Exosomes derived from mesenchymal stem cells (MSC-Exos) can play important roles in repairing injured tissues. However, to date, no reports have demonstrated the use of hiPSC-MSC-Exos in cutaneous wound healing, and little is known regarding their underlying mechanisms in tissue repair.MethodshiPSC-MSC-Exos were injected subcutaneously around wound sites in a rat model and the efficacy of hiPSC-MSC-Exos was assessed by measuring wound closure areas, by histological and immunofluorescence examinations. We also evaluated the in vitro effects of hiPSC-MSC-Exos on both the proliferation and migration of human dermal fibroblasts and human umbilical vein endothelial cells (HUVECs) by cell-counting and scratch assays, respectively. The effects of exosomes on fibroblast collagen and elastin secretion were studied in enzyme-linked immunosorbent assays and quantitative reverse-transcriptase–polymerase chain reaction (qRT-PCR). In vitro capillary network formation was determined in tube-formation assays.ResultsTransplanting hiPSC-MSC-Exos to wound sites resulted in accelerated re-epithelialization, reduced scar widths, and the promotion of collagen maturity. Moreover, hiPSC-MSC-Exos not only promoted the generation of newly formed vessels, but also accelerated their maturation in wound sites. We found that hiPSC-MSC-Exos stimulated the proliferation and migration of human dermal fibroblasts and HUVECs in a dose-dependent manner in vitro. Similarly, Type I, III collagen and elastin secretion and mRNA expression by fibroblasts and tube formation by HUVECs were also increased with increasing hiPSC-MSC-Exos concentrations.ConclusionsOur findings suggest that hiPSC-MSC-Exos can facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. These data provide the first evidence for the potential of hiPSC-MSC-Exos in treating cutaneous wounds.

Hybrid nanostructured hydroxyapatite–chitosan composite scaffold: bioinspired fabrication, mechanical properties and biological properties

The fabrication of bone scaffolds with interconnected porous structure, adequate mechanical properties, excellent biocompatibility and osteoinductivity presents a great challenge. Herein, a hybrid nanostructured hydroxyapatite-chitosan (HA-CS) composite scaffold has been fabricated according to the following steps: (i) the deposition of brushite-CS on a CS fibre porous scaffold by a dip-coating method; and (ii) the formation of a hybrid nanostructured HA-CS composite scaffold by the in situ conversion of brushite to HA using a bioinspired mineralization process. The hybrid HA-CS composite scaffold possesses three-dimensional (3D) interconnected pores with pore sizes of 30-80 μm. The HA rods with a length of ∼200 nm and width of ∼50 nm are perpendicularly oriented to the CS fibres. Interestingly, the abovementioned HA rods are composed of many smaller nanorods with a length of ∼40 nm and width of ∼10 nm oriented along the c-axis. The hybrid nanostructured HA-CS composite scaffold exhibits good mechanical properties with a compression strength of 9.41 ± 1.63 MPa and an elastic modulus of 0.17 ± 0.02 GPa, which are well-matched to those of trabecular bone. The influences of the hybrid HA-CS composite scaffold on cells have been investigated using human bone marrow stem cells (hBMSCs) as cell model and the CS fibre porous scaffold as the control sample. The hybrid HA-CS composite scaffold not only supports the adhesion and proliferation of hBMSCs, but also improves the osteoinductivity. The alkaline phosphatase activity and mineralization deposition on the hybrid HA-CS composite scaffold are higher than those on the CS fibre porous scaffold. Moreover, the hybrid HA-CS composite scaffold can promote the formation of new bone in rat calvarial defects as compared with the CS fibre porous scaffold. The excellent biocompatibility, osteoinductivity and mechanical properties suggest that the hybrid nanostructured HA-CS composite scaffold has great potential for bone tissue engineering.

Lithium stimulates human bone marrow derived mesenchymal stem cell proliferation through GSK‐3β‐dependent β‐catenin/Wnt pathway activation

Mesenchymal stem cells (MSCs) are multipotent cells that have been widely used in cell based transplantation therapy. The use of MSCs requires in vitro expansion in order to fulfill their regenerative capacity. Therefore the proliferative ability of MSCs is one of the key factors which determine MSC therapeutic efficacy. In the present study, we showed for the first time that lithium, a well‐known antidepressant, reversibly promoted the proliferation of human bone marrow derived MSCs in vitro. MSCs treated with 5 mm lithium proliferated more rapidly than untreated cells without undergoing apoptosis. Lithium increased the proportion of cells in S phase as well as cyclin D1 expression. Mechanistic studies revealed that these effects were dependent upon the activation of the glycogen synthase kinase 3β (GSK‐3β) mediated canonical Wnt pathway. Lithium induced Ser9 phosphorylation, which results in the inhibition of GSK‐3β activity, β‐catenin accumulation and Wnt pathway activation. Utilizing a specific GSK‐3β inhibitor SB216763 or siRNA‐mediated inhibition of GSK‐3β produced effects similar to those induced by lithium. In contrast, either quercetin, an inhibitor of the β‐catenin/TCF pathway, or siRNA‐mediated knockdown of β‐catenin abolished the proliferative effect of lithium, suggesting that lithium stimulates MSC proliferation via the GSK‐3β‐dependent β‐catenin/Wnt pathway. Collectively, these studies elucidate a novel role of lithium, which may not only provide a simple and effective way to strengthen MSC transplantation therapy efficacy but also shed light on lithiums clinical application for the treatment of certain disorders resulting from β‐catenin/Wnt pathway suppression.

Human urine-derived stem cells in combination with polycaprolactone/gelatin nanofibrous membranes enhance wound healing by promoting angiogenesis

BackgroundDespite advancements in wound healing techniques and devices, new treatments are needed to improve therapeutic outcomes. This study aimed to evaluate the potential use of a new biomaterial engineered from human urine-derived stem cells (USCs) and polycaprolactone/gelatin (PCL/GT) for wound healing.MethodsUSCs were isolated from healthy individuals. To fabricate PCL/GT composite meshes, twin-nozzle electrospinning were used to spin the PCL and gelatin solutions in normal organic solvents. The morphologies and hydrophilicity properties of PCL/GT membranes were investigated. After USCs were seeded onto a PCL/GT, cell adhesion, morphology, proliferation, and cytotoxicity were examined. Then, USCs were seeded on a PCL/GT blend nanofibrous membrane and transplanted into rabbit full-thickness skin defects for wound repair. Finally, the effect of USCs condition medium on proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were performed in vitro.ResultsUSCs were successfully isolated from urine samples and expressed specific mesenchymal stem cells markers and could differentiate into osteoblasts, adipocytes, and chondrocytes. PCL/GT membrane has suitable mechanical properties similar with skin tissue and has good biocompatibility. USCs-PCL/GT significantly enhanced the healing of full-thickness skin wounds in rabbits compared to wounds treated with PCL/GT membrane alone or untreated wounds. USCs-PCL/GT-treated wounds closed much faster, with increased re-epithelialization, collagen formation, and angiogenesis. Moreover, USCs could secrete VEGF and TGF-β1, and USC-conditioned medium enhanced the migration, proliferation, and tube formation of endothelial cells.ConclusionUSCs in combination with PCL/GT significantly prompted the healing of full-thickness skin wounds in rabbits. USCs based therapy provides a novel strategy for accelerating wound closure and promoting angiogenesis.

Bone morphogenetic protein 2 gene transduction enhances the osteogenic potential of human urine-derived stem cells

IntroductionUrine-derived stem cells (USCs) have the ability to differentiate into osteogenic lineage. Previous studies have raised the possibility that USCs could be used for bone repair. To harness the power of USCs in promoting bone regeneration, methods must be developed to induce USCs to osteogenic lineage efficiently. The present study investigates the effect of lentivirus-encoded bone morphogenetic protein 2 (BMP2) gene transduction on the osteogenic potential of USCs.MethodsUSCs were isolated from voided urine and transduced with Lentiviral vector encoding BMP2. An in vitro study was performed to detect Lentiviral-BMP2 transduced USCs differentiated towards osteogenic lineage. Furthermore, Lentiviral-BMP2 transduced USCs were transplanted in vivo to examine the ectopic bone formation ability. After six weeks, retrieval samples were obtained for immunostaining and histological analysis.ResultsThe results showed that the transduction efficiencies were over 90%, and transduced USCs had high expression levels of the BMP2 gene and secreted BMP2 protein. Alkaline activity and mineral deposition staining demonstrated that transduced USCs differentiate into osteogenic lineages without the addition of osteogenic supplements. Transduced USCs also showed high expression of bone-related markers, including runt-related protein-2 (Runx2) and osteocalcin (OCN), confirming this lentiviral-BMP2 construct provides sufficient stimuli for osteogenic differentiation. Histological analysis indicated that the transduced USCs induced robust new bone formation in nude mice. Six weeks after transplantation, human derived cells were observed to participate in bone formation.ConclusionsThese results demonstrate that BMP2 gene transduction provides an effective method to enhance the osteogenic potential of USCs.

Bioinspired nanostructured hydroxyapatite/collagen three-dimensional porous scaffolds for bone tissue engineering

During the biomineralization process of bone minerals, amorphous calcium phosphate (ACP) is converted to apatite crystals by using octacalcium phosphate (OCP) and brushite (DCPD) as transitory precursors, resulting in the formation of hybrid nanostructured collagen/apatite composites. Herein, we report, for the first time, the bioinspired synthesis of a collagen/hydroxyapatite (HA) porous scaffold (CHPS) according to the following stages: (i) fabrication of collagen fibre porous scaffold (CFPS) by a needle-punching process; (ii) deposition of brushite/chitosan (DCPD/CS) on CHPS by a dip-coating method; and (iii) formation of CHPS by in situ conversion of DCPD to HA. The CHPS exhibits three-dimensional (3D) interconnected porous structures with pore sizes of around 60 μm. HA crystals distribute homogeneously on the CHPS, and display wheat-like shapes with a length of approximately 200 nm and a width of approximately 80 nm. The in vitro cell tests by using human bone marrow stromal cells (hBMSCs) indicate that the HA crystals in the CHPS not only promote the cell adhesion and proliferation of the hBMSCs, but also stimulate osteogenic differentiation. The in vivo results reveal that the CHPS exhibits better osteoinductivity than the CFPS because of its similar chemical components, crystallinity and crystallographic texture to natural bone. Moreover, the CHPS can stimulate new bone formation in rat critical-sized calvarial defects within 8 weeks. The CHPS possesses a favourable pore structure, and excellent biocompatibility and osteoinductivity, and thus it has great potential applications for bone tissue engineering.

Dimethyloxaloylglycine Promotes the Angiogenic Activity of Mesenchymal Stem Cells Derived from iPSCs via Activation of the PI3K/Akt Pathway for Bone Regeneration

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1α (HIF-1α) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1α in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study, we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1α and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1α. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats, DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone, leading to bone regeneration. Collectively, the results indicate that DMOG, via activation of the PI3K/Akt pathway, promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration.

Human Urine Derived Stem Cells in Combination with β-TCP Can Be Applied for Bone Regeneration

Bone tissue engineering requires highly proliferative stem cells that are easy to isolate. Human urine stem cells (USCs) are abundant and can be easily harvested without using an invasive procedure. In addition, in our previous studies, USCs have been proved to be able to differentiate into osteoblasts, chondrocytes, and adipocytes. Therefore, USCs may have great potential and advantages to be applied as a cell source for tissue engineering. However, there are no published studies that describe the interactions between USCs and biomaterials and applications of USCs for bone tissue engineering. Therefore, the objective of the present study was to evaluate the interactions between USCs with a typical bone tissue engineering scaffold, beta-Tricalcium Phosphate (β-TCP), and to determine whether the USCs seeded onto β-TCP scaffold can promote bone regeneration in a segmental femoral defect of rats. Primary USCs were isolated from urine and seeded on β-TCP scaffolds. Results showed that USCs remained viable and proliferated within β-TCP. The osteogenic differentiation of USCs within the scaffolds was demonstrated by increased alkaline phosphatase activity and calcium content. Furthermore, β-TCP with adherent USCs (USCs/β-TCP) were implanted in a 6-mm critical size femoral defect of rats for 12 weeks. Bone regeneration was determined using X-ray, micro-CT, and histologic analyses. Results further demonstrated that USCs in the scaffolds could enhance new bone formation, which spanned bone defects in 5 out of 11 rats while β-TCP scaffold alone induced modest bone formation. The current study indicated that the USCs can be used as a cell source for bone tissue engineering as they are compatible with bone tissue engineering scaffolds and can stimulate the regeneration of bone in a critical size bone defect.

Dimethyloxaloylglycine Improves Angiogenic Activity of Bone Marrow Stromal Cells in the Tissue-Engineered Bone

One of the big challenges in tissue engineering for treating large bone defects is to promote the angiogenesis of the tissue-engineered bone. Hypoxia inducible factor-1α (HIF-1α) plays an important role in angiogenesis-osteogenesis coupling during bone regeneration, and can activate a broad array of angiogenic factors. Dimethyloxaloylglycine (DMOG) can activate HIF-1α expression in cells at normal oxygen tension. In this study, we explored the effect of DMOG on the angiogenic activity of bone mesenchymal stem cells (BMSCs) in the tissue-engineered bone. The effect of different concentrations of DMOG on HIF-1a expression in BMSCs was detected with western blotting, and the mRNA expression and secretion of related angiogenic factors in DMOG-treated BMSCs were respectively analyzed using qRT-PCR and enzyme linked immunosorbent assay. The tissue-engineered bone constructed with β-tricalcium phosphate (β-TCP) and DMOG-treated BMSCs were implanted into the critical-sized calvarial defects to test the effectiveness of DMOG in improving the angiogenic activity of BMSCs in the tissue-engineered bone. The results showed DMOG significantly enhanced the mRNA expression and secretion of related angiogenic factors in BMSCs by activating the expression of HIF-1α. More newly formed blood vessels were observed in the group treated with β-TCP and DMOG-treated BMSCs than in other groups. And there were also more bone regeneration in the group treated with β-TCP and DMOG-treated BMSCs. Therefore, we believed DMOG could enhance the angiogenic activity of BMSCs by activating the expression of HIF-1α, thereby improve the angiogenesis of the tissue-engineered bone and its bone healing capacity.

Bioactive borate glass promotes the repair of radius segmental bone defects by enhancing the osteogenic differentiation of BMSCs.

Bioactive borate glass (BG) has emerged as a promising alternative for bone regeneration due to its high osteoinductivity, osteoconductivity, compressive strength, and biocompatibility. However, the role of BG in large segmental bone repair is unclear and little is known about the underlying mechanism of BGs osteoinductivity. In this study, we demonstrated that BG possessed pro-osteogenic effects in an experimental model of critical-sized radius defects. Transplanting BG to radius defects resulted in better repair of bone defects as compared to widely used β-TCP. Histological and morphological analysis indicated that BG significantly enhanced new bone formation. Furthermore, the degradation rate of the BG was faster than that of β-TCP, which matched the higher bone regeneration rate. In addition, ions from BG enhanced cell viability, ALP activity, and osteogenic-related genes expression. Mechanistically, the critical genes Smad1/5 and Dlx5 in the BMP pathway and p-Smad1/5 proteins were significantly elevated after BG transplantation, and these effects could be blocked by the BMP/Smad specific inhibitor. Taken together, our findings suggest that BG could repair large segmental bone defects through activating the BMP/Smad pathway and osteogenic differentiation in BMSCs.