Promoted Viability and Differentiated Phenotype of Cultured Chondrocytes With Low Level Laser Irradiation Potentiate Efficacious Cells for Therapeutics.

Effective clinical treatments of cartilage lesions in affected joints require large numbers of viable chondrogenic cells generated through in vivo stimulation or ex vivo expansion of chondrocytes isolated from small biopsy specimens. Conventional passaging of chondrocytes in culture provides sufficient cells for treatments but these cells usually lose their differentiated phenotype. This leads to the formation of fibrocartilaginous tissue due to a malfunctioning repair process. Biostimulation of passaging chondrocytes with low level laser irradiation (LLLI) may theoretically produce more functional chondrocytes for cell-based repair of cartilage defects. Molecular and cellular analyses, cytochemistry, cell cultivation, and microscopy showed that LLLI treatments were found to (1) increase chondrocyte viability, (2) promote secretion of matrix proteins, (3) upregulate expression of chondrogenic genes, and (4) downregulate gene expression of cell destructive proteases and genes coding for mediators involved in the extrinsic apoptosis signaling pathway. Furthermore, LLLI attenuated induction of genes associated with cell death and matrix breakdown induced by IL-1ß, some of which was seen at the protein level, with verification of effects on gene expression in the C28/I2 human chondrocyte line. LLLI treatments during culture generated larger numbers of viable chondrocytes compared to untreated cultures. Moreover, LLLI-treated chondrocytes in culture also rectified and simultaneously maintained their differentiated phenotype. Cultured chondrocytes treated with LLLI are a promising cell source for repairing cartilage lesions in vivo and restoration of articular function using tissue engineering strategies.

Fibrochondrogenic differentiation potential of tendon-derived stem/progenitor cells from human patellar tendon.

BACKGROUND: Bone-tendon junction (BTJ) is a unique structure connecting tendon and bone through a fibrocartilage zone. Owing to its unique structure, the regeneration of BTJ remains a challenge. Here, we study the fibrochondrogenic differentiation of human tendon-derived stem/progenitor cells (TSPCs) both in vitro and in vivo. METHODS: TSPCs were isolated from human patellar tendon tissues and investigated for their multidifferentiation potential. TSPCs were cultured in chondrogenic medium with transforming growth factor beta 3 (TGF-ß3) and BMP-2 in vitro â€‹and examined for the expression of fibrochondrogenic marker genes by quantitative real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence. TSPCs pretreated were also seeded in collage II sponge and then transplanted in immunocompromised nude mice to examine if the fibrochondrogenic characteristics were conserved in vivo. RESULTS: We found that TSPCs were differentiated towards fibrochondrogenic lineage, accompanied by the expression of collagen I, collagen II, SRY-box transcription factor 9 (Sox 9), and tenascin C. Furthermore, after TSPCs were seeded in collagen II sponge and transplanted in immunocompromised nude mice, they expressed fibrochondrogenic genes, including proteoglycan, collagen I, and collagen II. CONCLUSION: Taken together, this study showed that TSPCs are capable of differentiating towards fibrocartilage-like cells, and the fibrochondrogenic characteristics were conserved even in vivo, and thus might have the potential application for fibrocartilage regeneration in BTJ repair. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: TSPCs are able to differentiate into fibrocartilage-like cells and thus might well be one potential cell source for fibrocartilage regeneration in a damaged BTJ repair.

Knockdown of FUSE binding protein 1 enhances the sensitivity of epithelial ovarian cancer cells to carboplatin.

Epithelial ovarian cancer (EOC) affects almost 25,000 women annually and is the fifth most common malignancy in women in North America. A combination of surgery and cytotoxic chemotherapy may produce a favorable clinical response. The platinum-paclitaxel combination regimen is the chemotherapy gold-standard for advanced ovarian cancer, and carboplatin is one of the agents in this combination therapy. However, the majority of patients eventually experience a relapse due to the development of platinum resistance. FUSE binding protein 1 (FBP1) has been identified as an anti-apoptotic and pro-proliferative oncoprotein that is overexpressed in hepatocellular carcinoma. Its high expression is also associated with carboplatin resistance. In the present study, it was identified that the expression of FBP1 was significantly higher in EOC tissues than in normal epithelial ovarian or in epithelial ovarian adenoma tissue. FBP1 expression was significantly correlated with the grade of epithelial ovarian cancer. Carboplatin inhibited the expression of FBP1 in epithelial ovarian cancer cells and the knockdown of FBP1 enhanced the inhibition of cell viability and migration by carboplatin. In addition to FBP1, carboplatin also inhibited the expression of ß-catenin and matrix metalloproteinase (MMP)-9. Furthermore, the expression of ß-catenin and MMP-9 were lower in FBP1 knockdown cells compared with control EOC cells. FBP1 may thus serve a role in the regulation of the expression of ß-catenin and MMP-9; the inhibition of ß-catenin and MMP-9 by carboplatin may be mediated through the inhibition of FBP1. The inhibition of FBP1 expression by carboplatin may be a mechanism in the treatment of EOC by carboplatin.

Protective effect of p53 on the viability of intervertebral disc nucleus pulposus cells under low glucose condition.

P53 is a famous cancer suppressor and plays key roles in metabolism. Intervertebral disc (IVD) is the largest avascular cartilaginous structure in humans and its degeneration is a common cause of spine diseases initiated from damaged nucleus pulposus (NP) cells. The potential cause of disc degeneration has been attributed to aging, genetic factors, mechanical factors and nutrition. In this study, we found that p53 decreased and leaked to the cytoplasm in NP cells as the glucose level decreases, in contrast to cancer cells in which p53 increases and concentrates to the nuclei. Comparing with in p53 knockdown NP cells, relative high p53 expression in normal control NP cells inhibited autophagy and the pentose phosphate pathway. Furthermore, the expression of Sox 9 and type II collagen were higher in p53 normal control than p53 knockdown NP cells. Based on these results, we believe that relative high p53 facilitates NP cell viability and integrity.

Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes.

Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule ß-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

New bone formation and microstructure assessed by combination of confocal laser scanning microscopy and differential interference contrast microscopy.

Bone is a mineralized connective tissue that is continuously and microstructurally remodeled. Altered bone formation and microstructure arise in pathological bone conditions such as osteoporosis, osteonecrosis, fracture repair, and Paget disease of bone. A proper and objective assessment of bone formation and microstructure will provide insight into the understanding of bone pathogenesis and remodeling. Here, new bone formation ex vitro and its microstructure were evaluated in in vivo multiple sequential polychrome-labeled samples using confocal laser scanning microscopy (CLSM), which generated clearer and more reliable images of thick bone sections than conventional fluorescence microscopy (CFM). Intriguingly, fine details of the bone microstructural features, including the mineralization fronts, quiescent versus active osteons, and Volkmann's channel, were elucidated using CLSM, which defines the relationship between morphological changes and function, when combined with differential interference contrast microscopy. Furthermore, CLSM provided objective evaluations of bone formation, such as the ratio of labeled areas of new bone formation in a rabbit model when compared with CFM. Altogether, new bone formation and its microstructure can be evaluated more adequately using a combination of CLSM and DIC microscopies.

MRI and histologic analysis of collagen type II sponge on repairing the cartilage defects of rabbit knee joints.

There are limited treatment options for cartilage defects in clinical practice because of the lack of suitable biomaterials. Here, we evaluated the effects of collagen type II sponge on the articular cartilage repairing process using a cartilage injury of a rabbit knee joint model. We showed that the home-made collagen type II sponges appeared to have a suitable pore size of 93.26 ± 38.4 µm for chondrocyte growth. MRI with H&E staining results demonstrated that the effusion absorption in the collagen type II sponge treated group was quicker than that of the control group. Moreover, sporadic cartilage signals first appeared at 6 weeks in the collagen type II sponge treated group. Safranin O staining and immunohistochemical analysis confirmed that the newly formed cartilage expresses glycosaminoglycan and type II collagen matrix. Using Sirius red polarized light staining, we showed that the newly formed cartilage-like areas from the collagen type II treated group are significantly greater than those of the control group. Taken together, our data demonstrated that the home-made collagen type II sponge is able to promote cartilage repair in the cartilage injury of a rabbit knee joint model.

[An experimental study of using chitinous membrane as the culture scaffold for epidermal stem cells].

OBJECTIVE: To investigate the feasibility of constructing a skin tissue engineering covering on chitinous membrane using rat epidermal stem cells (ESCs). METHODS: Rat ESCs were isolated and cultured by cold digestive method and collagen type IV adherent method. Cell colonies were observed with inverted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal microscope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of surface markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expressions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chitinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the vehicle was observed. RESULTS: Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive; CD71 and CD34 were negative; CK19, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P > 0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chitinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. CONCLUSIONS: Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.

[Detection of heterogeneous type II collagen transplantation mediated cytotoxic response in vitro].

To detect the cellular immunity state of New Zealand white rabbit immunized by pig type II collagen. The New Zealand white rabbit was immunized by type II collagen for sixty days. The plasma was collected at a regular interval and the anti-type II collagen antibodies were examined. At the sixtieth day, the peripheral circular lymphocytes and the lymphocytes separated from spleen cells of rabbit and lymph nodes were collected and were stimulated by type II collagen in vivo again. The regulation of reactive cellular proliferation caused by the stimulation was detected. The experiment samples were divided into two groups. The first group was the positive control group by adding different concentrations of PHA and the non-specific immunity was assayed. The different concentrations of type II collagen were added to the second group and the specific immunity was assayed. The lymphocytes of normal rabbits showed proliferation by PHA stimulation but no proliferation by the first stimulation of type II collagen. Obvious proliferation due to the stimulation of both PHA and type II collagen in the immunized rabbit were observed. It shows that certain concentration of heterogeneous collagen may cause an increase of anti-type II collagen antibody in immunized rabbit and may cause a proliferation of lymphocytes in rabbit spleen and peripheral blood. The heterogeneous type II collagen causes cellular immunity in vivo.

[The effect of fibroblast growth factors and epidermal growth factors on the proliferation of rabbit anterior cruciate and medial collateral ligament cell in vitro].

OBJECTIVE: To investigate the effect of acid, basic fibroblast, growth factor (aFGF, bFGF) and epidermal growth factor (EGF), and their combination on the proliferation of rabbit anterior cruciate ligament (ACL) and medial collateral ligament (MCL) in vitro. METHODS: The cells of ACL and MCL were isolated and subcultured from the knee joints of ten-week-old New Zealand white rabbits. The cells were seeded into 96-well corning cluster plates. Three growth factors of different concentration alone or in combination were added into the culture medium respectively, which were 0, 1, 5, 10, 50 and 100 ng/ml for aFGF, bFGF and 0, 1.56, 3.13, 6.25, 12.5, 25 and 50 ng/ml for EGF. The proliferation of the fibroblasts was measured for 48 h with XTT method. RESULTS: All of the three growth factors alone promoted the cell proliferation of ACL and MCL fibroblasts. The concentration of aFGF had a significant effect on the proliferation of both ACL and MCL fibroblasts. The concentration of 1 ng/ml bFGF and 5 ng/ ml EGF was most effective in promoting the proliferation of ACL, and both bFGF and EGF had a significant effect on MCL. 5 ng/ml aFGF with 50 ng/ml EGF had effect on ACL. 1 ng/ml aFGF with 3.13 ng/ml EGF had effect on MCL. CONCLUSION: The three growth factors may promote the cell proliferation of ACL and MCL. These findings suggest that topical application of aFGF, either alone or in combination with EGF may have the potential to promote the proliferation of rabbit ACL and MCL,and aFGF of low concentration in combination with EGF is more effective than single growth factor.

Effect of basic fibroblast growth factor and hyaluronic acid on proliferation of rabbit chondrocytes in vitro.

OBJECTIVE: To investigate the effect of basic fibroblast growth factor (bFGF) and hyaluronic acid (HA) on the proliferation of rabbit chondrocytes in vitro. METHODS: Chondrocytes from the knee joints of New Zealand white rabbits were cultured. bFGF or HA or both were added into the culture medium respectively, and the proliferation of the chondrocytes was measured with MTT 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetra-zolium bromide. (MTT, Sigma, M2128). RESULTS: Basic fibroblast growth factor (10 ng/ml) with low concentration of fetal bovine serum in the culture medium promoted the proliferation of chondrocytes significantly, and this effect reached its maximum when concentration of bFGF reached 50 ng/ml. HA itself had no effect on the proliferation of chondrocytes. However, when bFGF was used in combination with HA, especially when the concentration of bFGF was 50-500 ng/ml and that of HA was 10-50 ng/ml, the effect on the proliferation of chondrocytes was much more than when bFGF or HA was used alone. CONCLUSIONS: bFGF can promote the proliferation of chondrocytes. HA, which has no effect on the proliferation of the cells, can maintain a normal growth of chondrocytes. When bFGF is used in combination with HA, more proliferation is obtained.

[Study on preparation of a wound healing agent: fibronectin].

In search of the optimal preparation method for large-scale purification of human plasma fibronectin, we adopted affinity chromatography with gelatin and the Sepharose 4B activated with cyanogen bromide to purify fibronectin from type "C" plasma of healthy males, and scanned the best method under the conditions of different amount of plasma loading and different residence time in column. In a given column volume of gelatin, the absorbent was related with the plasma residence time in column and the total amount of plasma loaded. As a result, the optimal loading amount of plasma is 150 ml, and the residence time is 20 minutes. The preparation method, herein, has been proved to require small amount of plasma and yield large amount of fibronectin.