Construction of tissue-engineered laryngeal cartilage with a hollow, semi-flared shape using poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) as a scaffold.

The aim of the present study was to construct tissue-engineered laryngeal cartilage with a hollow, semi-flared shape using a poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHH) scaffold. Porous PHBHH was prepared and formed into a hollow, semi-flared shape, and the cell-material composites were cultured for one week in vitro prior to implantation in vivo. Cells of the nine rabbits of the experimental group were filled and encapsulated in the myofascial flap-shaping material composite for in situ implantation. The three rabbits in the control group were treated with the shaping material without the chondrocytes. Cartilage regeneration was assessed at six, 12 and 18 weeks after surgery. In the experimental group, the shape and porosity of the material were ideal, the cells exhibited good adhesion with the material and the myofascial flap blood supply was rich. The engineered laryngeal cartilage with the hollow, semi-flared shape was ideally formed, and the cartilage formed at six weeks after the surgery. Further maturation of the cartilage was observed at 12 and 18 weeks after the surgery. PHBHH was a suitable material for the formation of a hollow, semi-flared shape with good cellular compatibility. Myofascial flap filling and wrapping can be used to build tissue-engineered laryngeal cartilage with a hollow, semi-flared shape.

A comparative study on inhibition of total astragalus saponins and astragaloside IV on TNFR1-mediated signaling pathways in arterial endothelial cells.

BACKGROUND: Both total astragalus saponins (AST) and it's main component astragaloside IV (ASIV) have been used in China as cardiovascular protective medicines. However, the anti-inflammatory activities that are beneficial for cardiovascular health have never been compared directly and the molecular mechanisms remain unresolved. This study was conducted to compare the inhibitory effects of these drugs on TNFα-induced cell responses, related signaling pathways, and the underlying mechanisms in mouse arterial endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Real-time qRT-PCR was performed to determine the expression of cell adhesion molecule (CAM) genes. Immunofluorescent staining was used to detect the nuclear translocation of transcription factor NF-κB-p65. Western Blot analysis was used to identify TNFα-induced NF-κB-p65 phosphorylation, IκBα degradation, and caspase-3 cleavage. Cell surface proteins were isolated and TNFα receptor-1(TNFR1) expression was determined. The results suggest that both AST and ASIV attenuate TNFα-induced up-regulation of CAMs mRNA and upstream nuclear translocation and phosphorylation of NF-κB-p65. However, TNFR1-mediated IκBα degradation, cleavage of caspase-3 and apoptosis were inhibited only by AST. These differences in the actions of AST and ASIV could be explained by the presence of other components in AST, such as ASII and ASIII, which also had an inhibitory effect on TNFR1-induced IκBα degradation. Moreover, AST, but not ASIV, was able to reduce TNFR1 protein level on the cell surface. Furthermore, mechanistic investigation demonstrated that TNFR1-mediated IκBα degradation was reversed by the use of TAPI-0, an inhibitor of TNFα converting enzyme (TACE), suggesting the involvement of TACE in the modulation of surface TNFR1 level by AST. CONCLUSION: ASIV was not a better inhibitor than AST, at least on the inhibition of TNFα-induced inflammatory responses and TNFR1-mediated signaling pathways in AECs. The inhibitory effect of AST was caused by the reduction of cell surface TNFR1 level, and TACE could be involved in this action.

[Causes of vocal cord dyscinesia and its original factors after endotracheal intubation].

OBJECTIVE: To research the causes of postintubation vocal cord dyskinesia and its contributing factors. METHOD: The causes of vocal cord dyskinesia were confirmed by laryngoscope, three-dimensional spiral CT, stroboscope, and the analysis of therapy. The factors relevant to the causes of vocal cord dyskinesia were analysed based on the following elements: (1) the anatomic or pathological condition of patients or the technical skills of anesthetists. (2) emaciated or obese body and neck. (3) the age of patients. (4) the duration of endotracheal tube retention. (5) the types of operations. (6) anesthesia procedure. RESULT: Among 135 patients, 128 cases (94.81%) manifested arytenoid dislocation, 7 cases (5.19%) vocal cord paralysis. The study showed that the vocal cord dyskinesia associated with anatomic or pathological condition of patients and technical skills of anesthetists (with intubation difficulty) accounted for 76.30%. The patients with relative emaciated body or neck accounted for 90.62% in cases without intubation difficulty. Age had no significant analytical relationship with vocal cord dyskinesia. Prolonged intubation (endotracheal tube retention over 12 hours) was accounted for only 17.64%. The incidence of vocal cord dyskinesia was nearly 0.5% in patients underwent cardio-thoracic surgery, accounting for 59.26% of all the patients. CONCLUSION: There are two major causes of vocal cord dyskinesia: arytenoid dislocation and vocal cord paralysis, and the rate of vocal cord dyskinesia could be reduced by the improvement of technical skill of anesthetists and/or sufficient attention to the intubation condition of patients.

[Fabrication of larynx-shape tissue engineered cartilage by means of filling together with wrapping with pedicle myofascial flap].

OBJECTIVE: To explore the method of fabricating larynx-shape tissue engineered cartilage by means of filling together with wrapping with pedicle myofascial flap. METHODS: Serial steps of solution casting, extrusion molding and particulate leaching were used to make larynx-shape [poly (3-hydroxybutyrate-co-3-hydroxyhexanoate), PHBHH] biomaterial models. The chondrocytes were seeded onto PHBHH models to form cell-PHBHH composites for culture in vitro for one week and then to fill and wrap larynx-shape composites with pedicle myofascial flap. After that to implant larynx-shape composites in situ on the back of adult New Zealand white rabbits (experimental groups n = 9). Control groups (n = 3) were the same measure as experimental groups but without chondrocytes on PHBHH models. Finally, morphological observation, HE & special staining and immunohistochemical test were conducted to evaluate the cartilage regeneration and its shape at 6, 8 and 12 weeks following implantation. RESULTS: The PHBHH models appeared to be hollow half-trumpet with edges and corners of larynx-shape and its porosity > 90%. Pedicle myofascial flap using fascia as lining presented rich blood supply and had enough to fill and wrap larynx-shape composites. Tissue engineered larynx-shape cartilage specimens could be harvested at different period. It was demonstrated that the cartilaginous tissue formed in 6 weeks after implantation through histological and immunohistochemical examination and further maturity in 12 weeks and 18 weeks. But no cartilaginous tissue showed without chondrocytes on PHBHH as control groups to implant at the same time. CONCLUSION: It seems that pedicled myofascial flap showed sufficient blood supply and that the filling together with wrapping method with pedicled myofascial flap is appropriate for fabricating larynx-shape tissue engineered cartilage.

[Fabrication of laryngeal cartilage by means of tissue engineering technique].

OBJECTIVE: To explore the method of fabricating tissue engineered laryngeal cartilage. METHODS: The rib and articular cartilage of infant New Zealand white rabbits were harvested in sterile condition. The chondrocytes were separated by collagenase digestion and cultured in vitro for 3 passage. Serial steps of solution casting, extrusion molding and particulate leaching were used to make larynx-shaped biomaterial models with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHH). The chondrocytes were seeded onto PHBHH scaffolds to form cell-PHBHH composites, which were subsequently in vitro for one week. After that, the measure of filling inner space of cell-PHBHH composites together with wrapping total composites using either greater omentum (n = 9) or fascia flap and muscle (also n = 9) in experimental groups was taken to implant the larynx-shaped biomaterial models seeded with chondrocytes into the belly and the back of adult New Zealand white rabbits. Control groups (every group n = 3) were the same measure as experimental groups but without chondrocyte on PHBHH scaffolds. Finally, morphological observation, HE staining & special staining and immunohistochemical test were conducted to assess cartilage regeneration and its shape at different period following implantation. RESULTS: The rate of viable cell in the final cell suspension was (93 +/- 2)% after well-controlled prolongation of digestion trypsin. Similar to that by traditional procedures (94 +/- 2)% (P > 0.05). The larynx-shaped PHBHH models with edges and corners of laryngeal cartilage made by us appeared to be hollow half-trumpet shape and its porosity was more than 90%. It showed that chondrocytes equally attached to the surface of porous PHBHH and filled within porousness with scanning electron microscopic examination. Tissue engineered larynx-shaped specimens could alternatively be harvested with the above mentioned two different implantation measures. The specimens presented to be similar to that before implantation in gross shape. It was demonstrated to be cartilaginous tissue through histological and immunohistochemical examination. Furthermore, There was nearly no difference between two kinds of tissue engineered laryngeal cartilage with two measures of implantation in morphology and histology. CONCLUSIONS: The regeneration of tissue engineered cartilage in vivo is not influenced by the chondrocytes harvested by improvement of well-controlled prolonged digestion with trypsin during in vitro cell culture. It seems that PHBHH may be used as scaffold in cartilage tissue engineering and wrapping together with filling method with either greater omentum or fascia flap and muscle is appropriate for fabricating tissue engineered laryngeal cartilage.

[Improvement upon construction of tissue-engineered allogeneic cartilage molded with polyglycolic acid as the scaffold].

OBJECTIVE: To improve the method for constructing allogeneic molded cartilage by means of tissue engineering techniques. METHODS: The chondrocytes from the rib and articular cartilage of infant rabbits were harvested by type II collagenase digestion, followed by in vitro cell culture for 3 to 4 passages. The chondrocytes were then prepared into cell suspension and seeded onto C -and O -shaped pre-molded polyglycolic acid (PGA) scaffolds form chondrocyte-PGA composites, which were subsequently cultured in vitro for 7 to 10 d before implanted subcutaneously into adult rabbits. Improvement was made upon conventional shaping and implantation procedures. Morphological observation and cartilage regeneration assessment were conducted at different time points following the implantation, in comparison with the observation by conventional shaping and implantation methods. RESULTS: During in vitro cell culture, the rate of viable chondrocytes in the final cell suspension was (92+/-2)% after well-controlled prolongation of digestion trypsin, similar to the viable cell rate (93+/-2) % by traditional procedures (P>0.05). Gross observation found milk-white, newly generated cartilage which had good flexibility 4 weeks after implantation, and after 8 weeks and later, the cartilage took on the color of porcelain-white. Histological examination showed a few inflammatory cells around the newly generated immature cartilage 4 weeks after implantation, and the inflammation abated when the newly generated cartilage acquired similar histological properties to that of the original cartilage 8 weeks postoperatively and later. After 16 weeks, no blood vessel or capillaries were visible within the new cartilage. CONCLUSION: The chondrocyte viability is not affected when the cells are treated with well-controlled prolonged digestion with trypsin during in vitro cell culture. Improved PGA scaffolds shaping and the implantation procedure facilitate the regeneration of the cartilage after the implantation of the composites.