The Experimental Study On Construction Of Tissue-Engineered Trachea With Allogenic Acellular Matrix

ObjectiveTracheal defects caused by wounds, tumors or congenital diseases are tough problems for clinic doctors. End-to-end suture can be used in repairing the short or partial defects of trachea, while once the length of crico-defect exceeds 5 tracheal rings or 3 cm, tracheal substitute became the only effective way to reconstruct the form and function of airway. The studies of tracheal substitute have been focused on artificial trachea, autologous grafts, allogenic grafts and tissue-engineered trachea since the end of 19^th century, and tissue-engineered trachea has become more and more hot due to its advantages in the area of immunologic rejection and material resource. Tissue-engineered trachea is made of seeds cells cultured in vitro and scaffolds in tracheal form, which has the form and function of real trachea and can be implant into the body to repair the defects of tissue and function. The goal of this study is to observe the adherence, proliferation and secretion of ECM of autogenic MSCs on allogenic tracheal acellular matrix, and then to evaluate the feasibility of constructing a new type of tissue-engineered trachea. We are looking forward to acquire the ideal seeds cells and scaffold materials. Methods1 Six 4-month Beagle canines were used in this study. 14-16ml bone marrow were isolated from both tibial tubercles of each canine, rabbit. The MSCS suspension was aspired after the bone marrow was centrifuged by 1.073g/ml percoll separating medium, cultured primarily, and then generated. The 3^rd passage MSCs were induced by the L-DMEM containing 15% FBS and 10ng/ml TGF-β1,10ng/ml IGF- I , 37.5μg/ml Vitamin C and 6.25μg/ml transferring, while no cytokines are added into the control group. The cells were viewed at different stages with phase contrast microscope every day. The growth curves were drawn by MTT assay. The content of GAG in the cell culture fluid were detected by Alcian blue. Toluidine blue O, Masson trichome stain and immunohistochemistry for collagen type II were used to measuring the secreting of cell matrix and collagen type II in monolayer plate of cells. The chondrogenic differentiation of induced MSCs were evaluated by above methods.2 Local healthy hybrid dogs, aged 4-6 month, were used as the trachea donors. The 5-ring tracheas (from the 6^th to the 10^th ring below thyroid cartilage) were cut and taken to the lab immediately after the execution of the donor dogs. The tracheas were immersed into the 1% Triton X-100 and solutions containing of DNAse and RNAse in sequence. According to the different length of time in Triton X-100 solution, 4 groups were divided: 24-hour group, 48-hour group, 72-hour group and 120-hour group. After all of the treatments, the samples were collected for HE staining and scanning electronic microscoping to observe the effect of removal of cells in the tracheal acellular matrixs and the effect of the four groups are compared.3 Adjust the cell density of 7-day induced MSCs to 3×10⁶/ml, then co-culture the autogenic MSCs and the allogenic tracheal acellular matrix for 14 days in vitro. The composites were harvested for scanning electronic microscoping at the 3^rd, 7^th and 14^th day to observe the adherence and proliferation of MSCs on the surface of matrixs. The 7-day co-cultured composites were implanted into the back of host Beagle canine subcutaneously, tracheal acellular matrixs without MSCs and fresh tracheal rings as control groups. The implants were harvested at 14 and 30 days for HE tissue staining to observe whether there's cell component generated.Results1 Adherence onto the bottom of culture dishes were observed in 24 hours after the isolation of primary passage MSCs under the phase contrast microscope. The number of adherent cells increased obviously in 72 hours and lots of cell-colony-units formed in 5 days. The colonies of MSCs increased in scalp constantly and the consecutive clonoes mix together in 7-8 days. The spread area of the primary passage reached 80% in 10 days. The subculture cells grew faster than the primary passage and the cell became bigger. The MTT growth curves showed that the proliferation ability of P1 cells is the best among P0, P1, P2 and P3 passages. The growth rate became steady in P3 cells.2 After adding induced cytokines, the rate of proliferation increased and the cells turned to be more circular and bigger. MTT assay showed that the growth curve of induced P3 MSCs is in the left side of control group. The content of GAG in cell culture fluid of induced MSCs (42.48±2.32) μg/ml is obviously higher than that of control group (19.62±1.30) μg/ml (p<0.01). In the histochemistry stanings, the cytoplasm of 14-day induced MSCs showed blue stained (Toluidine blue O staining) and green stained (Masson trichome staining) and yellow or brown-yellow stained (immunohistochemistry for collagen type II), while no obvious positively stained cell were detected in control group.3 It took at least 120 hours to remove all cells from the allogenic tracheas with 1% Triton X-100 solution. HE staining showed that all the ciliated epithelial cells, gland cells and chondrocytes of trachea samples in 120-hour group were removed entirely. The mucous membrane matrix separated with the cartilage acellular matrix partly, while there was no obvious evidence of destroys observed on the cartilage ring acellular matrix. The results of SEM also demonstrated that all the cells of trachea samples were removed in the 120-hour group. The cartilage lacunas, without chondrocytes, were clear on the surface of cartilage acellular matrixes, when the mucous membrane were removed away.4 There were adherent cells with pseudopodias observed under the SEM on the surface of 3-day co-cultured MSCs and allogenic trachea acellular matrixes in vitro. 2-3 layers of adherent cells close together and secreted so much ECM that the surface of composites looked like "frost and ice" in 7 days. While apoptosis of adherent cells were observed on the 14-day co-cultured composites in vitro and many cells turned to be vacuole-like. The HE staining after 14-day cultured subcutaneously in the host canine showed that there were many layers of cells on the surface of composites. The cells were flat and long, looked like spear, with bigger nucleus. But no cells were seen in the cartilage acellular matrix. The composites were packed up with granulation tissue and a small quantity of lymphocytes. Small amounts of cells grew into the superficial layer of cartilage matrix after 30-day cultured subcutaneously, but no newly-formed cartilage was detected. The cartilage acellular matrix was destroyed mostly and more lymphocytes were observed around the samples. Conclusion1 MSCs can be easily obtained, and percoll separation method can get huge MSCs, their characteristics were stable in vitro, and easy to generate. MSCs can be induced to chondrocytes in vitro by TGF-β1 and IGF-I . Therefore MSCs are the suitable seed cells for constructing tissue-engineered cartilage for trachea reconstruction.2 The detergent-nucleinase method can remove all the cells of trachea samples thoroughly by using 1% Triton X-100 solution, without damages to the ECM. It keeps the integrity of structure of trachea acellular matrix.3 The induced MSCs can adhere onto the surface of allogenic trachea acellular matrix and proliferate and secret mounts of ECM. But the cells can grow into the interior of matrixes neither cultured in vitro nor implanted subcutaneously. It showed that allogenic trachea acellular matrix has a good compatibility with induced MSCs, but before it was used for the scaffold material of tissue-engineered trachea, the features of interval porosity and feedthrough need to be improved better.