Document Type Master's Dissertation Author Wessels, Quenton Bester URN etd-08262008-181356 Document Title Engineering of a Collagen-glycosaminoglycan copolymer dermal regeneration matrix Degree MSc Department Anatomy Supervisor
Advisor Name Title Prof E Pretorius Supervisor Keywords
- full thickness burn wounds
- tissue engineering
- collagen-glycosaminoglycan copolymer; regeneration
- extracellular matrix analogue
- regenerative medicine
Date 2008-04-24 Availability unrestricted Abstract
Background:Tissue engineering and its contribution to regenerative medicine has advanced through the years. It has proven its efficacy especially in the treatment of advanced full thickness burn wounds. Tissue engineering is the synergy between biology and engineering. This fairly young science has one common goal and that is to regenerate new tissue. Various commercially available products have appeared on the market and this due to the ground-breaking work of many. One such well known product is Integraź which is the brain child of Yannas and Burke. This is a collagen-glycosaminoglycan copolymer which serves as a bioactive regeneration template or extracellular matrix analogue. Advanced wound healing is promoted along with the prevention of scar tissue formation and consequent contractures.
Aims:This study provides an extensive review on the development of this dermal regeneration matrix and also aims to develop an equivalent product. Attention will be paid to: the biological building blocks and the motivation for their use; the essential production steps; and the final processing required in order to deliver a sterile product.
Materials and Methods:
A collagen and chondroitin 6-sulphate coprecipitate was prepared and subjected to either controlled or uncontrolled freezing. The frozen slurry was dried under vacuum for 17 hours after which each sample was coated with a thin silicone film. Glutaraldehyde crosslinking followed after which the product was thoroughly rinsed. The packaged products were then subjected to terminal sterilisation via gamma irradiation under various conditions. Various tests were conducted to evaluate the newly formed regeneration matrices and included scanning electron microscopy, enzymatic degradation by collagenase, and a cytotoxicity assay. Scanning electron microscopic analysis was done in order to reveal the adequacy of the scaffold architecture. Collagenase degradation of the scaffolds was used to project the rate of degradation of each template. Integraź served as the gold standard for each test. Quantifiable data was statistically analysed and any comparison made included the calculation of means, standard deviations and p-values (confidence interval of 95%).
Results indicated that highly porous bioactive tissue engineering matrices were obtained by either controlled freezing or uncontrolled freezing. The average pore diameter of the most homogenous scaffolds ranged between 52.47 and 136.44 ”m with a mean of 87.34 ”m. These templates were formed by using a 0.5% collagen concentration and a controlled freeze rate of 0.92 °C/min. Uncontrolled freezing (1.3 °C/min) of a 0.5% collagen concentration resulted in the formation of an irregular scaffold with an average pore diameter of 174.08 ”m. It was found that the architecture of the most equivalent scaffold compared well with that of Integraź with p = 0.424. Scaffolds prepared using higher collagen concentrations (1.0%) and controlled freezing resulted in dense sponges with average pore diameters of 56.51 ”m. Statistical analysis upon comparison indicated a significant difference p = 0.000 in the micro architecture. The rate of degradation of the most equivalent scaffold was 1.9 times that of Integraź. This implicates that the crosslinking was insufficient and due to one of the following: poor collagen quality; method of crosslinking; and degradation due to terminal sterilization. The rate of scaffold degradation can be extended, either by additional crosslinking or the prevention of degradation induced by irradiation. Temperature vacuum dehydration crosslinking through esterification or amide formation can be used as an initial crosslinking method in further studies. This form of crosslinking will complete the conventional glutaraldehyde crosslinking that reacts with the free amine groups of lysine or hydroxylysine of the protein backbone of collagen. It should be stressed that the determination of an in vivo degradation rate, in the form of an animal study, will aid to confirm the efficacy of the biologically active regeneration matrix.
© University of Pretoria 2007E1092/gm
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