[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.
Sun
An-ke
AK
Centre of Orthopaedics and Traumatology of PLA, Nan Fang Hospital, The First Military Medical University, Guangzhou 510515, China. sak00161@hotmail.com
Pei
Guo-xian
GX
Hu
Ping
P
Chen
Jiang-rui
JR
Ren
Gao-hong
GH
Zhang
Yu
Y
Hu
Ba-sheng
BS
Qin
Yu
Y
D000818:Animals; D001672:Biocompatible Materials; D002356:Cartilage / Q000166:cytology; D002358:Cartilage, Articular / Q000166:cytology*; D018929:Cell Culture Techniques; D019902:Chondrocytes / Q000166:cytology; D007830:Larynx; D011817:Rabbits; D012272:Ribs / Q000166:cytology; D046509:Tissue Culture Techniques; D023822:Tissue Engineering / Q000379:methods*
D001672:Biocompatible Materials