Comparison between xCELLigence biosensor technology and conventional cell culture system for real-time monitoring human tenocytes proliferation and drugs cytotoxicity screening.

BACKGROUND: Local injections of anesthetics, NSAIDs, and corticosteroids for tendinopathies are empirically used. They are believed to have some cytotoxicity toward tenocytes. The maximal efficacy dosages of local injections should be determined. A commercial 2D microfluidic xCELLigence system had been developed to detect real-time cellular proliferation and their responses to different stimuli and had been used in several biomedical applications. The purpose of this study is to determine if human tenocytes can successfully proliferate inside xCELLigence system and the result has high correlation with conventional cell culture methods in the same condition. METHODS: First passage of human tenocytes was seeded in xCELLigence and conventional 24-well plates. Ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone with different concentrations (100, 50, and 10% diluted of clinical usage) were exposed in both systems. Gene expression of type I collagen, type III collagen, tenascin-C, decorin, and scleraxis were compared between two systems. RESULTS: Human tenocytes could proliferate both in xCELLigence and conventional cell culture systems. Cytotoxicity of each drug revealed dose-dependency when exposed to tenocytes in both systems. Significance was found between groups. All the four drugs had comparable cytotoxicity in their 100% concentration. When 50% concentration was used, betamethasone had a relatively decreased cytotoxicity among them in xCELLigence but not in conventional culture. When 10% concentration was used, betamethasone had the least cytotoxicity. Strong and positive correlation was found between cell index of xCELLigence and result of WST-1 assay (Pearson's correlation [r] = 0.914). Positive correlation of gene expression between tenocytes in xCELLigence and conventional culture was also observed. Type I collagen: [r] = 0.823; type III collagen: [r] = 0.899; tenascin-C: [r] = 0.917; decorin: [r] = 0.874; and scleraxis: [r] = 0.965. CONCLUSIONS: Human tenocytes could proliferate inside xCELLigence system. These responses varied when tenocytes were exposed to different concentrations of ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone. The result of cell proliferation and gene expression of tenocytes in both xCELLigence and conventional culture system is strongly correlated. CLINICAL RELEVANCE: xCELLigence culture system may replace conventional cell culture, which made real-time tenocyte proliferation monitoring possible.

Effects of hyperbaric oxygen treatment on tendon graft and tendon-bone integration in bone tunnel: biochemical and histological analysis in rabbits.

Despite moderate success in clinical applications, outcome of tendon grafts employed for anterior cruciate ligament (ACL) reconstruction remains unsatisfactory. This study investigated the effects of hyperbaric oxygen (HBO) on neovascularization at the tendon-bone junction, collagen fibers of the tendon graft, and the tendon graft-bony interface incorporated into the osseous tunnel in rabbits. Forty rabbits were assigned to two groups. The HBO group was exposed to 100% oxygen at 2.5 atmospheres pressure for 2 h daily, 5 consecutive days in a week. The control group was maintained in cages exposed to normal air. Histological studies of 12 rabbits were performed postoperatively at 6, 12, and 18 weeks. Biomechanical studies of 24 rabbits were conducted postoperatively at 12 and 18 weeks. Electron microscopy (EM) analyses of four rabbits were performed postoperatively at 18 weeks. Experimental results demonstrated that a higher number of Sharpey's fibers bridged the newly formed fibrocartilage and graft in the HBO group than in the control group. In addition, HBO treatment increased neovascularization and enhanced the incorporation of the progressive interface between tendon graft and bone. Biomechanical analysis showed that the HBO group achieved higher maximal pullout strength than the control group. Examination by EM showed that HBO treatment resulted in regenerated collagen fibers with increased compaction and regularity. Based on experimental results, HBO treatment is a treatment modality that potentially improves outcome following ACL reconstruction.

Attenuation of apoptosis and enhancement of proteoglycan synthesis in rabbit cartilage defects by hyperbaric oxygen treatment are related to the suppression of nitric oxide production.

Proinflammatory cytokine, nitric oxide (NO) and localized hypoxia-induced apoptosis and proteoglycan (PG) degradation are thought to be correlated to the degree of cartilage injury. This study evaluated hyperbaric oxygen (HBO)-induced changes in joint cavity oxygen tension, antigenickeratan sulfate (KS) content, inducible nitric oxide synthase (iNOS) expression, PG synthesis, and cell apoptosis in full-thickness defects of rabbit cartilage. The HBO group was exposed to 100% oxygen at 2.5 atm for 2 h daily, 5 days per week. Meanwhile, the control group was kept in housing cages with normal air. The joint cavity oxygen tension was determined with an oxygen sensor. Blood serum KS was quantified by competitive indirect enzyme-linked immunosorbent assay (ELISA). After sacrifice, specimen sections were sent for histological and histochemical examination with a standardized scoring system. In situ analysis of iNOs expression and apoptosis detection were performed using immunostaining and TUNEL staining, respectively and quantified by a computerized imagine analysis system. This study demonstrated that HBO treatment increased joint cavity oxygen tension but decreased blood KS content. Histological and histochemical score results showed that HBO treatment significantly increased the cartilage repair. Moreover, immunostaining and TUNEL staining showed that HBO treatment suppressed the iNOs expression and apoptosis of chondrocytes, respectively. Accordingly, HBO offers a potential treatment method for cartilage injury.