Yongyudh Vajaradul
Siriraj Hospital, Mahidol University, Bangkok, Thailand

Hendadura Piyanandana Jayathilaka
Medical Lab Technologist
Sri Lanka Model Human Tissue Bank

Biomechanical studies were carried out for porcine tracheal cartilage to determine the effectiveness of processing steps. This study shows that radiation reduces tensile strength by 30% in deep frozen trachea and by 38% in freeze dried samples. And also the structural changes such as length, thickness, inner diameter and outer diameter, due to freeze drying were studied. This study shows that thickness of the tracheal tube is more affected by freeze drying (15%) and the length was least affected (5%).

Trachea is a cartilage tube that brings air from the larynx to the bronchi that enter the lungs. It is lined with mucus membrane and ciliated epitheila. Cartilage is composed of cells, bound water and a proteoglycan matrix in which are contained fibers, the majority of which are type II collagen fibers. The cross-binding due to irradiation produces high degree of resistance to absorption. Grafting is required when primary reconstruction of trachea defect is not feasible. Surgical techniques of tracheal reconstruction for acquired or congenital tracheal stenosis and tracheal tumor have been developed for decades. But the limitation of maximal length of safe resection and reconstruction as well as frequency of recurrent stenosis was the obstacle of the surgical treatment for long segment of tracheal diseases. Recently tracheal allotransplantation as an ideal conduit for tracheal reconstruction have been investigated in various animal models, but free tracheal allograft, which were totally devascularized for several days after transplantation, have been used with little success because of strong immunologic reactions against the allograft and early immediate failure of revascularization.

Sung Bo Sim (2000) observed reliable allograft tracheal viability and better survival rate and lesser rejection reaction with trachea of deep frozen plus irradiation used in rats. Somyos(2000) had similar results using deep frozen irradiated tracheal allograft for subglottic tracheal stenosis in human for the first time.

The Bangkok Biomaterial Center usually preserved trachea by deep freezing and freeze drying in combination with gamma irradiation. For this study we used porcine trachea as an experimental model to prepare freeze dried irradiated and deep frozen irradiated tracheal grafts. The processing steps are expected to solve the problem of immunologic reaction. This study of mechanical properties with and without irradiation was carried out with the motive of finding techniques to improve the efficiency of grafts.

	To identify the tensile strength of trachea.
	To compare the tensile strength of trachea before and after irradiation.
	To identify the changes of length and diameter after freeze drying.
	To identify the techniques to improve the efficiency of grafts.

Procurement and Processing

Porcine trachea procured at slaughter house were transported to the center. After dissection to remove all soft tissues around, each trachea was cut into 5cm pieces. Pasteurization was done at 80⁰C for 10min. Several washing steps were done to clean the tissue using biofiltered water, betadine and 0.5% NaOCl. Divided the samples into two groups to prepare deep frozen irradiated and freeze dried irradiated trachea. To avoid structural changes during freeze drying and irradiation, a special precaution was taken. This was done by inserting a plastic test tube into tracheal duct. Both groups were sterilized by gamma irradiation at 40kGy using gamma irradiation. Measured the length, inner diameter, outer diameter and thickness before and after freeze drying.

Freeze dried tissues were immersed in normal saline for one hour.

The tensile strength test was carried out by universal testing machine (LLOYD instruments-LR10K). Both ends of the tracheal tube were properly fixed at the metal clamp and stretching was done until the maximum breakage of the tracheal tube was reached.

13. Results

Fig-7: Percentage decrease of tensile strength after Irradiation

Structural Changes due to freeze drying:

The decrease of dimensions such as thickness, length, inner diameter and outer diameter, due to freeze drying effect were compared in this experiment. Fig 1,2,3,4,: shows a marked decrease in every aspects of dimensions. Fig 5 shows the decrease of these parameters as a percentage. The thickness of the trachea shows a maximum decrease due to freeze drying to upto 15.77%. And also the inner and outer diameter shows higher decrease in their dimensions nearly 15%. But the decrease in length is only 5%. When compared to other dimensions, length shows lowest change of structure.

Tensile Strength of trachea

Fig 6 shows the tensile strength of deep frozen and freeze dried samples before and after irradiation. This reveals that the gamma irradiation clearly decrease the tensile strength of deep frozen upto 6.13 N/cm2 and freeze dried sample upto 7.14 N/cm2 from 20.32 and 18.67 subsequently. The decrease of tensile strength is denoted as a percentage in Fig 7. In the deep frozen sample, radiation decreases tensile strength by 30.17% and in freeze dried sample, radiation decreases tensile strength by 38.24%. This indicates that the tensile property is more affected by freeze drying and radiation.

Discussion

Cryopreserved tracheal transplantation has been done in animal models as a viable allograft (Heng Zhao). But this preservation method is an expensive and complex procedure. Less studies has been carried out on deep frozen and freeze dried trachea. These preservation methods give more economic grafts with less antigenicity, sterility, availability, elasticity and prolonged preservation. Hamaide and Arnoczky (1998) noted that biomechanical and biochemical properties of the canine tracheal ring cartilage are altered with age. However location of the ring along the trachea did not affect these properties. These results lend support to the theory that proteglycan content has some effect on tensile properties of tracheal rings. Many researchers report irradiated cartilage grafts absorption rates that differ widely. Donald PJ (1996) observed that a major factor governing this phenomenon might be radiation dose. Irradiation produces collagen cross-binding and increased resistance to absorption of such materials when implanted.

Guzman and Vajaradul (1996) noted that radiation and freeze drying decreases the tensile strength of fascia and duramater. We used porcine tracheal cartilage tubes for this study on mechanical strength. This study shows a marked decrease in tensile strength after irradiation in both deep frozen and freeze dried samples. Freeze drying also tends to decrease the tensile strength. This is because the cartilage tube become more fragile after freeze drying and also reconstitution is unable to bring the tensile strength to initial state. Clearly this experiment shows that radiation at 40kGy causes significant decrease in tensile strength of tracheal cartilage grafts. The loss of tensile strength in irradiated grafts may indeed, account for its susceptibility to absorption. The effect of radiation on the biomechanical properties of trachea are most evident when combined with lyophilization. Low doses of radiation and deep freezing are recommended in preparing tracheal grafts to avoid these problems.

References

	Hamaide A, Arnoczky SP, Ciarelli MJ, Gardner K, Effects of age and location on the biomechanical and biochemical properties of canine tracheal ring cartilage in dogs; American Journal of Veterinary Research; 59(1); 18-22, 1998 Jan
	Donald PJ, Deckrad JK, Sharkey N, Lagunas M,The effect of radiation dose on the cartilage grafts; Annals of plastic surgery; 36(3); 297-303, 1996 Mar.
	Somyos K, Yongyudh V: Cryopreserved irradiated tracheal homograft transplnatation for laryngotracheal reconstruction in human being; 2000, Vol; 22; 911-916
	Donald PJ: Cartilage grafting in tracheal reconstruction with special consideration of irradiated grafts. Laryngoscope: 1986:96:786-807
	Heng Zhao, Yun Zhong Zhou, Yi Yang: Transplantation of trachea with cryopreserved allograft in dogs.
	Zenida G, Yongyudh V; Mechanical properties of freeze dried and irradiated bone chips, fascia lata and dura mater; The Nucleus; 1996; 6-9.
	Sung Bo Sim MD, Seok WM, Young pil W, Moon SK; The effcet of cryopreservation and irradiation on tracheal allotransplantation in rats; Oct 2000; 90-91.

                                        Tensile strength test in progress  

       BACK TO HOMEPAGE