Human umbilical cord mesenchymal stem cell-derived and dermal fibroblast-derived extracellular vesicles protect dermal fibroblasts from ultraviolet radiation-induced photoaging in vitro.

Ultraviolet B (UVB) radiation is a major cause of aging in dermal fibroblasts. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show antioxidant activity. In this study, the anti-aging effects of MSC-EVs on dermal fibroblast photoaging induced by UVB radiation were evaluated, and the effects of extracellular vesicles derived from dermal fibroblasts (Fb-EVs) were compared. Human umbilical cord mesenchymal stem cells and human dermal fibroblasts were cultured, and MSC-EVs and Fb-EVs were isolated and characterized. Human dermal fibroblasts were cultured in the absence or presence of different concentrations of EVs 24 hours prior to UVB radiation exposure. Cell proliferation and cell cycle were evaluated, and senescent cells and intracellular ROS were detected. The expressions of matrix metalloproteinase-1 (MMP-1), extracellular matrix protein collagen type 1 (Col-1), and antioxidant proteins such as glutathione peroxidase 1 (GPX-1), superoxide dismutase (SOD), and catalase were also analyzed. Pretreatment with MSC-EVs or Fb-EVs significantly inhibited the production of ROS induced by UVB radiation, increased dermal fibroblast proliferation, protected cells against UVB-induced cell death and cell cycle arrest, and remarkably decreased the percentage of aged cells. Pretreatment with MSC-EVs or Fb-EVs promoted the expressions of GPX-1 and Col-1 and decreased the expression of MMP-1. Both MSC-EVs and Fb-EVs protected dermal fibroblasts from UVB-induced photoaging, likely through their antioxidant activity.

Cartilage progenitor cells combined with PHBV in cartilage tissue engineering.

BACKGROUND: Bone marrow-derived stem cells (BMSCs) and chondrocytes have been reported to present "dedifferentiation" and "phenotypic loss" during the chondrogenic differentiation process in cartilage tissue engineering, and cartilage progenitor cells (CPCs) are novel seeding cells for cartilage tissue engineering. In our previous study, cartilage progenitor cells from different subtypes of cartilage tissue were isolated and identified in vitro, but the study on in vivo chondrogenic characteristics of cartilage progenitor cells remained rarely. In the current study, we explored the feasibility of combining cartilage progenitor cells with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to produce tissue-engineered cartilage and compared the proliferation ability and chondrogenic characteristics of cartilage progenitor cells with those of bone marrow-derived stem cells and chondrocytes. METHODS: These three cells combined with PHBV were cultured in vitro for 1 week without chondrogenic induction and then transplanted subcutaneously into nude mice for 6 weeks. The cell-PHBV constructs were evaluated by gross observation, histological staining, glycosaminoglycan content measurement, biomechanical analysis and RT-PCR. RESULTS: The chondrocyte-PHBV constructs and CPC-PHBV constructs became an ivory-whitish cartilage-like tissue, while the BMSC-PHBV constructs became vascularized 6 weeks after the subcutaneous implantation. Histological examination showed that many typical cartilage structures were present in the chondrocyte group, some typical cartilage structures were observed in the CPC group, while no typical cartilage structures were observed in the BMSC group. CONCLUSIONS: Cartilage progenitor cells may undergo chondrogenesis without chondrogenic induction and are better at chondrogenesis than BMSCs but worse than chondrocytes in the application of cartilage tissue engineering.

Isolation and identification of stem cells in different subtype of cartilage tissue.

BACKGROUND: Cartilage tissue engineering provided a promising therapy for the repair of cartilage defects, and seeding cells play a vital role in cartilage regeneration. Chondrocytes and bone marrow-derived mesenchymal stem cells (BMSCs) were reported to be the ideal seeding cells, but 'dedifferentiation' and 'unstable phenotype' of tissue-engineered cartilage constructed by the two cell type hamper their clinical application. Recently, cartilage tissue was reported to possess a stem cell population, which may be a more superior cell source in cartilage tissue engineering. METHODS: In current study, we isolated a cell population from different subtype of cartilage tissue via a differential adhesion assay to fibronectin. RESULTS: Flow cytometry analysis demonstrates the cell lines expressed mesenchyme stem cell positive surface marker such as CD29 and CD90. Meanwhile, the cells are highly proliferative and multipotent. Reverse transcription-PCR detection showed the cell population expressed osteogenic and adipogenic differentiation under different induction conditions. More interesting, monolayer cells underwent chondrogenic differentiation in the presence of dexamethasone and insulin-like growth factor 1. In addition, the expression of chondrogenic genes in cartilage-derived stem cells (CSCs) was higher than those in BMSCs. CONCLUSION: CSC may become an ideal seeding cell in cartilage tissue engineering, owing to its stemness and chondrogenic characteristics.

Adenoviral transduction of hTGF-beta1 enhances the chondrogenesis of bone marrow derived stromal cells.

TGF-beta1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells (BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the hTGF-beta1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-beta1 expressed and secreted more hTGF-beta1 protein in vitro than those of the control group. Histological and immunohistological examination of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-beta1 exhibited neocartilage formation after 3 weeks in vivo. The neocartilage occupied 42 +/- 5% of the total tissue volume which was significantly greater than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen in the AdhTGF-beta1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG in experimental groups. The results demonstrate that transfer of hTGF-beta1 into BMSCs via adenoviral transduction can induce chondrogenic differentiation in vitro and enhance chondrogenesis in vivo.

[Creation of tissue engineered cartilage with internal support].

OBJECTIVE: To test the hypothesis that tissue-engineered cartilage can be bioincorporated with a nonreactive, permanent endoskeletal scaffold. METHODS: Chondrocytes obtained from swine articular were seeded onto polyglycolic acids(PGA) scaffold which was incorporated with high-density polyethylene (Medpor). After cultured in vitro for two weeks,the cell-scaffold construct was implanted into subcutaneous pockets on the back of nude mice. Six weeks later,the newly formed cartilage prosthesis was harvested, and a small part of sample was evaluated by gross view, histology, type II collagen immunohistochemistry and biochemistry. PGA scaffold seeded with cells as the control group. RESULTS: The newly formed cartilage was very similar to normal cartilage in both gross view and histology, and jointed Medpor tightly. The center of control group was hollow. CONCLUSION: This pilot technique combining tissue engineering with a permanent success in creating cartilage without "hollow" phenomenon. biocompatible endoskeleton demonstrated

[Regeneration of autologous tissue-engineered cartilage by using basic-fibroblast growth factor in vitro culture].

OBJECTIVE: To investigate the effect of the basic fibroblast growth factor (b-FGF) to regenerate an autologous tissue-engineered cartilage in vitro. METHODS: The Cells were harvested from the elastic auricular cartilage of swine,and were plated at the concentration of 1 x 10(4) cells/cm2 , studied in vitro at two different media enviroments: Group I contained Ham's F-12 with supplements and b-FGF, Group II contained Ham's F-12 only with supplements. The passage 2 cells (after 12.75 +/- 1.26 days) were harvested and mixed with 30% pluronic F-127/Ham's F-12 at the concentration of 50 x 10(6) cells/ml. It was injected subcutaneously at 0.5 ml per implant. The implants were harvested 8 weeks after the vivo culture and examined with the histological stains. RESULTS: The chondrocytes displayed morphologically similar to the fibroblasts in the media containing basic-FGF. The number of cell doublings (after 12.75 +/- 1.26 days) in vitro culture was as the following: Group I, 70; Group II, 5.4. Eight 8 weeks after the vivo autologous implantation, the average weight (g) and volume (cm3) in each group was as the following: Group I, 0.371 g/0.370 cm3 Group II, 0.179 g/0.173 cm3 (P < 0.01). With the b-FGF in vitro culture, the cells were expanded by 70 times after 2 weeks. Histologically, all of the engineered cartilage in the two groups were similar to the native elastic cartilage. CONCLUSION: These results indicate that the basic-FGF could be used positively to enhance the quality and quantity of the seeding cells for the generation of the well-engineered cartilage.

Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds.

Chitosan has been shown to be a promising scaffold for various applications in tissue engineering. In this study, a chitosan-gelatin complex was fabricated as a scaffold by a freezing and lyophilizing technique. Chitosan's structure and characteristics are similar to those of glycosaminoglycan (GAG) and its analogs, and possesses various biological activities, whereas gelatin can serve as a substrate for cell adhesion, differentiation, and proliferation. With the use of autologous chondrocytes isolated from pig's auricular cartilage and seeded onto the chitosan-gelatin scaffold, elastic cartilages have been successfully engineered at the porcine abdomen subcutaneous tissue. After 16 weeks of implantation, the engineered elastic cartilages have acquired not only normal histological and biochemical, but also mechanical properties. The tissue sections of the engineered elastic cartilages showed that the chondrocytes were enclosed in the lacuna, similar to that of native cartilage. The presence of elastic fibers in the engineered cartilages was also demonstrated by Vehoeff's staining, and immunohistochemical staining confirmed the presence of type II collagen in the engineered cartilages. Quantitatively, the GAG in the engineered cartilages reached 90% of the concentration in native auricular cartilage. Furthermore, biomechanical analysis demonstrated that the extrinsic stiffness of the engineered cartilages reached 85% of the level in native auricular cartilage when it was harvested at 16 weeks. Thus, this study demonstrated that the chitosan-gelatin complex may serve as a suitable scaffold for cartilage tissue engineering.

[The experimental study of tissue engineered autologous cartilage using chitosan-gelatin complex scaffolds].

OBJECTIVE: To investigate whether man-made porous chitosan-gelatin complex scaffold was a appropriate scaffold for tissue engineering cartilage. METHODS: Chondrocytes isolated from Changfeng crossbred swines' auricular cartilage were seeded onto chitosan- gelatin scaffolds to be cultured in a three dimensional environment. The chondrocyte- polymer constructs were implanted into the subcutaneous tissue of the swines' abdomenal wall. Specimens were harvested and analyzed by gross observation, histology, type II collagen immunohistochemistry and biochemistry after 10 and 16 weeks in vivo respectively. RESULTS: H.E staining showed cartilage was formed, and chondrocytes were enclosed in lacuna with histological characteristics similar to natural cartilage. Some clusters of neocartilage surrounded by fibrous tissues were observed. Elastic fibres were observed in the mesenchyma of cartilage 16 weeks after by Vehoeff's staining. Immunohistochemical staining of the neocartilage with anti- type II collagen showed the presence of type II collagen in the ECM of tissue engineered cartilage. The proteoglycans content in tissue engineered cartilage was close to that of natural swine's auricular cartilage. CONCLUSION: The experiments demonstrated that using chitosan-gelatin complex scaffold we can generate autologous cartilage on animals with normal immune system. Porous chitosan- gelatin complex scaffolds may be a suitable scaffolds for tissue engineered cartilage.

[In vitro chondrogenic phenotype differentiation of bone marrow stem cells].

OBJECTIVE: To investigate the feasibility of chondrogenic phenotype differentiation of adult swine bone marrow stem cells(MSCs) in a defined medium as seeding cells in cartilage tissue engineering. METHODS: A volume of 5 ml bone marrow was aspirated from swine iliac crest and cultured in the complete medium of DMEM-LG for two weeks. The growth and ultrastructure of the cultured MSCs were observed. Immunohistochemistry and in situ hybridization were applied to detect the expression of collagen type II. RESULTS: The MSCs changed from a spindle-like fibroblastic appearance to a polygonal shape when transferred from the complete medium of DMEM-LG to a defined medium. A large amount of endoplasmic reticulum was observed in large Golgi ccmplex and mitochondria. The differentiation of MSCs toward chondrogenic phenotype was verified by the positive result of collagen type II through immunohistochemistry and in situ hybridization respectively. CONCLUSIONS: Bone marrow stem cells obtained from adult swine can differentiate to be chondrogenic phenotype when cultured in vitro. MSCs can likely be served as optimal autogenous cell source for cartilage tissue engineering.