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Objective To optimize the method of the directional differentiation of adipose-derived stem cells into keratinocytes.Methods Adipose-derived stem cells (ADSCs),separated by collagenase digestion method,were isolated and cultured.Then the expression of surface specific markers CD34,CD44 and CD90 were detected by flow cytometer.The effect of different induced mediums cultured for two weeks on the differentiation of ADSCs into KCs was demonstrated:Group 1,the DMEM supplied with 2％ FBS and 49％ supertant of KCs;group 2,KSFM medium;group 3,DMEM medium supplied with 10％ FBS and 5 μM ATRA;10％ FBS DMEM as the control group.Immunofluorescene staining was applied to detect the expression of keratin CK14 and F-actin.Results A flattened fibroblast-like morphology was observed in cells,the positive expression rate of CD34 was 0.08％,while those of CD44 and CD90 were 99％.The cells that could differentiate into osteoblasts and chondrocytes,indicated that the cells were ADSCs.There was no significant change in the cell morphology in the group 1 under the induction medium;about 10％ of the cells in group 2 were altered;the morphological changes were obvious in group 3,and approximately 20％ of the cells showed irregular polygon.The immunofluorescene staining of the cells in group 3 indicated that the cells showed cobblestone-like phenotype and an organized cytoskeletal network with dense actin fibers at the edges;some cells were positive for CK14.Conclusions ADSCs show higher induction rate under ATRA stimulation.
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Objective To evaluate the biomechanical effectiveness of hyaluronidase (HAase) on rabbit auricular cartilage in the early stage.Methods HAase in 3 different concentrations (75 U/ml,150 U/ml,300 U/ml) were injected subcutaneously to rabbit auricle in 3 groups,while normal saline were injected as control.For the comparison of biomechanism properties among different groups and different time,cartilages were harvested at 3rd and 7th day after injection,followed by stress-relaxation assay.Results Auricular cartilage displayed different levels in control group compared with other groups in elastic modulus (P＜0.05) and maxmum stress (P＜0.05) in 3th day as well as 7th day.While both in the same concentration group,there were also differences between the 3th and 7th day (P＜0.05).Conclusions HAase injection can cause changes in biomechanical properties of auricular cartilage.And 7 days would not be enough for the tissue recovery biomechanically.
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BACKGROUND:Inducing factor and chondrogenic microenvironment is a primary factor, which influences chondrogenic differentiation and chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). OBJECTIVE:To explore the feasibility of in vivo chondrogenesis by co-culture of bone marrow-derived MSCs and chondrocytes. DESIGN, TIME AND SETTING:A randomized controlled animal experiment was performed at Department of Pathology, Stomatological Hospital, Fourth Military Medical University of Chinese PLA between September 2004 and March 2005. MATERIALS:Fifteen New Zealand rabbits of clean grade were used for cell-scaffold construct transplantation. The rabbits were randomly divided into co-culture, chondrocyte, and bone marrow-derived MSC groups, with 5 rabbits in each group. Five neonatal New Zealand rabbits, aged 1-3 days, were used for isolation and culture of bone marrow-derived MSCs and chondrocytes. Polyglycolic acid (PGA) scaffold material (Shanghai Yikuo Company, China) has a fiber diameter of 15 μm, with an average interval of 150-200 μm, an interval porosity of 97% and 2-mm thickness. METHODS:In the co-culture group, bone marrow-derived MSCs and chondrocytes were mixed at a ratio of 3:1. The mixed cells were seeded onto a pre-wetted PGA scaffold (5 mm×5 mm )at the ultimate concentration of 6.0×1010 L-1. Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum was dropwise added to peripheral compound for 1 week of culture. In the chondrocyte, and bone marrow-derived MSC groups, chondrocytes and bone marrow-derived MSCs of the same ultimate concentration were seeded respectively onto the PGA scaffold. Then, the cell-scaffold constructs were transplanted into subcutaneous tissue of adult rabbits. MAIN OUTCOME MEASURES:Gross observation and hematoxylin-eosin & Masson staining of neo-cartilage were performed after in vivo culture for 8 weeks. RESULTS:Cell in all groups had a fine adhesion to the scaffold. In both co-culture and chondrocyte groups, the cell-scaffold constructs could maintain the original size and shape during in vivo culture and formed homogenous mature cartilage after 8 weeks of in vivo culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features. In the bone marrow-derived MSCs group, connective tissue rather than cartilage was found during in vivo culture. CONCLUSION:Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of bone marrow-derived MSCs and thus promote the chondrogenesis of bone marrow-derived MSCs in vivo.
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Nerve growth factor (NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound. We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells. NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate (APA) microencapsulation and cultivated in vitro. Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay (ELISA) method. These microencapsules were co-cultured with epidermic cells and fibroblast cells. Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method. NIH-3T3 microencapsules, empty microencapsules, normal culture media were control groups. After one week culture, the characteristics of the dermis were described by MTT test, the content of hydroxyproline (HP), HE staining and ultrastructure photograph. We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro, promot the proliferation of epidermic cells and secret collagen of fibroblast cells. These functions can maintaine in bioengineered dermis. So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis.