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

BACKGROUND: Despite 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. METHODS: USCs 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. RESULTS: USCs 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-ß1, and USC-conditioned medium enhanced the migration, proliferation, and tube formation of endothelial cells. CONCLUSION: USCs 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.

[Influence of PGLA degradable products on main physiologic function].

The biological safety of degradable products is a key point in biological evaluation of biodegradable materials. This paper deals with an investigation of the influence of PGLA degradable products on physiologic function of liver and kidney by detecting the "before-after change" of some biochemical indexes with the use of self-control method. PGLA materials of different sizes were implanted into the subcutaneous tissue of Wistar rats and New Zealand rabbits respectively. Also for the purpose of group comparison, only surgical operation was performed on other animal at the same site (control group). The concentrations of urea nitrogen, uric creatinine, blood urea nitrogen (BUN), carbamide (Cr) and serum glutamio pyruvic transaminase (GPT) were measured before and after implantation (from 2 w to 10 w). The results showed the values of urea nitrogen and uric creatinine were significantly increased at 2-3 weeks after implantation (P < 0.01). After 2 weeks of implantation, the concentration of GPT was slightly decreased (P < 0.05). The BUN and Cr were remarkably increased (P < 0.01). But at the 4th week, all the values returned to normal and kept stable thenceforth. All the biochemical indexes in the control animal exhibited no abnormal change after operation. These results indicated: (1) There are no permanent harmful effects of PGLA degradable products on the function of liver and kidney; (2) By means of self-control and measurements of some biochemical indexes in the blood or urea, the method is suitable for evaluating the biological safety of degradable products; (3) The method has the advantages for evaluating the bioaccept ability of degradable products during the life period, it is objective and sensitive, the number of animal can be decreased, the process of dynamic change in vivo can be observed directly, and the effect of degradable products on physical function can be evaluated. Therefore, the method can provide a new approach for biological evaluation of biodegradable materials.

[Effect of degradable products on degradable property of PGLA in vitro].

In order to investigate the effect of degradable products on degradable property of PGLA in vitro, two kinds of media-PBS and artificial plasma were prepared for immersing PGLA under changing or non-changing media condition. The mass loss rate of PLGA was calculated and the pH value in the non-changing media was measured before and after immersing 2 w, 3 w, 4 w, 6 w, 8 w and 10 w respectively. The results showed that there was almost no statistically significant difference of mass loss rate of PGLA immersing in two kinds of media at 2 w (P > 0.05). But from 2 w to 6 w, the degradation of PGLA immersing in both media under non-changing media group was remarkably faster than those at the same period of changing media group (P < 0.01). During the whole degradable period, the pH value in PBS kept stable around 7.0-7.4, while the pH value in artificial plasma showed gradually decreased as the degradation of PGLA from 7.5 to 5.7. The change of pH values had statistically significant difference between two degradable media (P < 0.01). It was implied that the degradable products existed in immersing media had an effect on degradable speed of PGLA itself if the media was unchanged. It could accelerate the mass loss of material. The pH value also affected the degradable property of PGLA, the lower the pH value, the slower the degradable speed.

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 BG's 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 ß-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 ß-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.

Human urine-derived stem cells can be induced into osteogenic lineage by silicate bioceramics via activation of the Wnt/ß-catenin signaling pathway.

Human urine-derived stem cells (USCs) have great application potential for cytotherapy as they can be obtained by non-invasive and simple methods. Silicate bioceramics, including calcium silicate (CS), can stimulate osteogenic differentiation of stem cells. However, the effects of silicate bioceramics on osteogenic differentiation of USCs have not been reported. In this study, at first, we investigated the effects of CS ion extracts on proliferation and osteogenic differentiation of USCs, as well as the related mechanism. CS particles were incorporated into poly (lactic-co-glycolic acid) (PLGA) to obtain PLGA/CS composite scaffolds. USCs were then seeded onto these scaffolds, which were subsequently transplanted into nude mice to analyze the osteogenic differentiation of USCs and mineralization of extracellular matrix formed by USCs in vivo. The results showed that CS ion extracts significantly enhanced cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and expression of certain osteoblast-related genes and proteins. In addition, cardamonin, a Wnt/ß-catenin signaling inhibitor, reduced the stimulatory effects of CS ion extracts on osteogenic differentiation of USCs, indicating that the observed osteogenic differentiation of USCs induced by CS ion extracts involves Wnt/ß-catenin signaling pathway. Furthermore, histological analysis showed that PLGA/CS composite scaffolds significantly enhanced the osteogenic differentiation of USCs in vivo. Taken together, these results suggest the therapeutic potential of combining USCs and PLGA/CS scaffolds in bone tissue regeneration.

The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

The discovery of induced pluripotent stem cells (iPSCs) rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL)/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM), the Cell Counting Kit-8 (CCK-8), histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.

[Experimental study of repairing full-thickness articular cartilage defect with chondrocyte-sodium alginate hydrogel-SIS complex].

OBJECTIVE: To explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects. METHODS: SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articular cartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3 weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multiplication method were diluted by sodium alginate to (5-7) x 10(7) cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n = 20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was established in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation. RESULTS: Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the interface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in the implant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly and active cell metabolism and proliferation were evident 9 months after operation. While in the control group, no repair of the defect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9 months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P < 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P < 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P < 0.05). CONCLUSION: Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in line with physiological repair process of articular cartilage.

Variations in the ratios of co-cultured mesenchymal stem cells and chondrocytes regulate the expression of cartilaginous and osseous phenotype in alginate constructs.

As mesenchymal stem cells (MSCs) are capable of self-renewal and multilineage differentiation, the feasibility and efficacy of co-culturing human MSCs (hMSCs) with rabbit articular chondrocytes (rACs) to promote chondrogenic and osteogenic differentiation of hMSCs for clinical osteoarthritic therapy were investigated in the present study. The two distinct cell types were encapsulated in alginate hydrogels singly or in one of three ratios (2:1, 1:1, 1:2 of hMSCs to rACs) and cultured under chondrogenic conditions for 28 days. The results demonstrated that newly synthesized cartilaginous extracellular matrix (ECM) and type II collagen (col-2) gene signal were upregulated with greater hMSC ratios and longer culture periods. However, a specific col-2 gene probe for human was found only in single hMSC group but absent in all co-culture groups, which indicate that the enhanced cartilaginous phenotype originated from the co-cultured rACs. Osseous phenotype was histologically detected only in the 2:1 group on day 28; and xenogenic osteocalcin assay showed that it originated from hMSCs. This suggests that variations in the ratios of co-cultured hMSC and rAC regulated the cartilaginous and osseous phenotype as well as the differentiation of hMSCs in alginate constructs. The study provides new insights into the role of cell-cell interactions in regulating both cell differentiation and cell function and highlights the importance of developing appropriate differentiation protocols for tissue engineering therapies.

[Manufacture and biocompatibility study of poly-D,L-lactic acid plate containing rhBMP-2].

OBJECTIVE: To investigate the manufacture and biocompatibility of a bioabsorbable poly-D,L-lactic acid (PDLLA) plate containing rhBMP-2. METHODS: rhBMP-2 was composited with PDLLA (0.05 mg/plate) through vacuum to prepare PDLLA plate containing rhBMP-2. Thirty-two New Zealand rabbits (male or female) weighted (3.0 +/- 0.5) kg were used in the study. A 2.5 mm middle ulna osteotomy was made bilaterally. The bones as well as periosteum were removed. The right side of all the animals was experimental side (n=32), was fixed internally by PDLLA plate containing rhBMP-2.The left side of all the animals was control side (n=32), was fixed by common PDLLA plate. After a follow-up of 2, 4, 8 and 12 weeks, the ulnas were examined visually, radiographically, histologically, and by computer graph analysis to compare the biocompatibility. RESULTS: Porosity of PDLLA plate containing rhBMP-2 was 90%, aperture was 150 microm, tensile strength was higher than 50 MPa, three point flexural strength was higher than 90 MPa and intrinsic viscosity was 1.6 dL/g (chloroform solvent). All animals had a good healing 1 or 2 weeks after operation. All the animal's diet and movement were normal. All the fractures were stable. The plates in the experimental group degraded faster than those in the control group. Relative values of callus density evaluated by X-ray at 2, 4, 8 and 12 weeks after operation in the experimental group were 39.22 +/- 2.48, 48.79 +/- 1.26, 63.78 +/- 1.78 and 78.60 +/- 1.25 respectively. Those in the control defects were 33.83 +/- 1.13, 41.28 +/- 1.25, 55.23 +/- 0.68 and 66.54 +/- 1.33. At each time point, the experimental side produced better and more trabeculae than the control side did (P < 0.01). Histological examination showed that the new bone formation in the experimental side at 2, 4, 8 and 12 weeks after operation was 0.106% +/- 0.015%, 0.292% +/- 0.019%, 0.457% +/- 0.048% and 0.601% +/- 0.037%, while those in the control side was 0, 0.193% +/- 0.019%, 0.339% +/- 0.029% and 0.574% +/- 0.047% respectively. At each time point, the experimental side produced better new bone formation than the control side did (P < 0.05). The experimental side showed better compatibility between plates and surrounding tissue, faster bone formation, more bone regeneration mass and better medullary canal structure than the control side. CONCLUSION: PDLLA plate containing rhBMP-2 has good biocompatibility and osteoinducibility to enhance fracture healing.