Expressions Of Alpha-smooth Muscle Actin In Goat Temporomandibular Joint Disc And Self-assembled Engineered Constructs

Objective:Purpose of this study was to investigate the expression of smooth muscle actin-alpha (α-SMA) in native goat temporomandibular joint (TMJ) disc and in self-assembled construct of TMJ disc.Methods:1.Isolation and culture of goat TMJ disc cellsTMJ disc cells (fibrochondrocytes) were aseptically harvested from the TMJ disc tissues approximately1-month old goats less than24hours after slaughter in local abattoir.The disctissues were minced with ophthalmic scissors about1mm3size of small pieces, then moved to triangle flask containing fifteen times volume of DMEM and0.5%collagenase1was used to isolate and passage when80%cells of monolayer attained confluence. P2cells were used for self-assembling engineered constructs.2.Self-assembling constructs of goat TMJ discA diameter of5mm×10mm cylindrical stainless steel rod dies were processed which made it fit the dimensions of the24-well plate.One of dieends was pasted to the lid inside of24-well plate and faced to the corresponding well at the middle of24-well plate. Then2%agarose were perfused into wells between24-well plate and stainless steel rod with1.5mL in each well and formed agarose molds at room temperature for30minutes. After the stainless steel rod dies were separated from each agarose mold,500uL complete medium with10μg/L IGF-Ⅰ were added into each and changed the medium everyday until completely saturated to the time when the cells will be inoculated. A number of4.6×106cells containing150μl DMEM medium were added to each agarose mold and carefully added400μL culture mediumcontaining10μg/L IGF-Ⅰ into agarose well four hours later.500μL/well medium was changed everyday.4. Immunohistochemistry of α-SMA in native goat TMJ discTMJ disc tissues were embedded in paraffin and serially sectioned at4μm. Tissue sections were stained with hematoxylin-eosin and picrosirius red to examine type Ⅰ collagen distributions according to standard histological protocols. The localization of a-SMA in disc tissues were evaluated in the same way.5. Expression and distribution of a-SMA inconstructs of self-assembled constructsAt t=2w,4w,6w,8w, self-assembled constructs were frozen and fixed in4%paraformaldehyde and cut into8μm sections. Picrosirius red staining was used to examine collagentype I distribution and safranin-O/fast green for GAG distribution observations. The IHC staining steps for a-SMA are the same mentionedas above.RESULTS:1. Construct was small spherically at first about4hours and started to contract after24hours. Significantly shrinkage in shape were taken place after two weeks and changed to be stable since then.2. Gross morphology of self-assembled constructs of the goat TMJ disc cells at2,4,6,8weeks, we found that after2weeks dry weight and wet weight of self-assembled constructs were significantly increased overtime.3. Picrosirius red staining shows that there arelarge amount of collagens formed inside the constructs. Safranin-O/fast green staining indicates that a weakly positive distribution in the constructs.4. Immunohistochemistry of a-SMA in native goat TMJ articulardiscStaining for a-SMA in the goat TMJ disc was weakly positivein which expression was distributed in extracellular matrix.5. Immunohistochemical staining for a-SMA in self-assembled constructs of the goat TMJ disc cells at differentweeks showed the strong positive expression of the a-SMA all in four self-assembled construct groups.ConclusionThe experiment result showed self-assembled construct starts to contract obviously in shape within two weeks after cell inoculation, and then gradually grows stable. IHC staining of α-SMA revealed highly positive expressionin self-assembled constructs than that in normal native TMJ disc. We suggested that a-SMA may be contributed to volume shrinkage of engineered TMJ disc construct, but its specific mechanism needs further elucidation.