Experimental Studies On Biocompatibility Of Xenogenic Tendon Matrix Materials

Background:Treatment of soft tissue defects caused by trauma, tumour surgery or pressure sores is a challenge to the plastic and reconstructive surgeon. Soft tissue defects may not only cause aesthetic and functional problems if not properly repaired, but also affect their mental health and quality of life. Current treatment modalities for soft tissue defects due to various pathologies and trauma include autologous grafting and commercially available fillers. However, these treatment methods present a number of challenges and limitations, such as donor site morbidity and volume loss over time. As such, improved therapeutic modalities need to be developed. In recent years, tissue engineering and regenerative medicine have developed rapidly, especially in the theory of regenerative medicine. Regenerative medicine is the process of creating living, functional tissues to repair or replace tissue or organ function lost due to age, disease, damage, or congenital defects. This field holds the promise of regenerating damaged tissues and organs in the body by stimulating previously irreparable tissues to heal themselves. Xenogenic materials has been used as therapeutic scaffolds for cell attachment and proliferation and as templates for tissue repair. It was gradually applied to the field of regenerative medicine, because it can provide the ECM environments which regulate the migration, proliferation, and differentiation of progenitor cells in and around a scaffold during tissue repair. It has been previously reported that acellular xenogeneic extracellular matrix facilitate the constructive, tissue-specific replacement of diverse tissue structure such as urinary bladder, urethra, bone, dura mater, bioprosthetic heart valve (BPHVs), and acellular dermal materials (ADM) in experimental animals and in human patients. However, there were few reports about xenogenic materials used in the field of plastic surgery, except for the skin defects with ADM.Many attempts have been made to produce long-lasting, biocompatible implants. To overcome the mechanical and biological limitations of synthetic implants, various researchers have begun to focus on the development of a naturally derived biomaterial. In order for materials to be transplanted to a patient from a donor, especially an animal donor, the tissue must be modified to increase resistance to degradation and to decrease immunogenicity, while maintaining natural mechanical properties. Glutaraldehyde (GA) is the first and most prevalent fixative of Fixation with glutaraldehyde was once thought to largely mitigate the immune response to connective tissue xenografts by irreversibly cross-linking graft matrix proteins. It is now clear that both humoral and cell-mediated immune responses to glutaraldehyde-fixed xenografts occur. Moreover, this treatment causes altered mechanical properties, increased calcification, and cytotoxicity. In this study a novel polyepoxy compounds was used as an alternative fixative (Patent number: US6106555, US6231614B1). In some previous reports, xenogenic dura fixed with this polyepoxy compounds represents an effort to overcome some of the drawbacks that are typically encountered with GA. The xenogenic dura was approved by SFDA and widely used in clinical practices. In this study, xenogenic tendon matrix materials were prepared by means of treating porcine tendon with epoxy cross-linking fixation, diversified antigen minimization process, mechanic enhancement modification and surface activating process. The purpose of this study was to evaluate the application potential of the recently developed xenogenic tendon matrix materials as a novel soft-tissue implant by investigating its properties in vivo and in vitro.Methods1. Preparation of xenogenic tendon matrix materialsTaking fresh pig tendon→bio-burden→eliminating impurities and making pretreatments→epoxy cross-linking fixation→protein molecules modification raising the mechanical strength of the material→diversified eliminations of antigen→surface activity modification making the material capable for adhesion to enriched growing factors→packaging→terminal sterilization usingγ-ray→finished.2. Morphologic Observation of xenogenic tendon matrix materialsGross examination; HE stained and observed under the optical microscope; Sample preparation for SEM (scanning electron microscope). Parallel and cross sections were taken, fixed 24 hours at 4℃with 2.5%(v/v) glutaraldehyde, via gradient dehydration with 30%-100% alcohol, soaked in isoamyl acetate 30 minutes, and dried at isoelectric point. The dried surface of the sample was sprayed with platinum. Then, the morphology and structure was observed under SEM.3. Mechanical and chemical testing on of xenogenic tendon matrix materialsTensile properties were determined by using a testing machine (Micro-controlled electron tension-testing device). The protein content of xenogenic tendon matrix materials was tested by Kjeldahl method. Heavy metal content of materials and epoxide residue were examined. The chemical stability of materials was tested by measuring the PH value of the material in PBS solution.4. Biocompatibility of xenogenic tendon matrix materialsAccording to the standard for the biological evaluation of the medical devices, the acute toxicity test, sensitization test, skin stimulating test, cellular toxicity test, and subcutaneous embedded test were carried out to evaluate the biocompatibility of the prepared implants.5. Evaluation of xenogenic tendon matrix materialsTo observe evolution of the xenogenic tendon matrix materials in different implanting periods by means of a canine model.24 mongrel dogs were divided into 6 groups (4 in each group). The animals in each group were sacrificed at 3,6,9,12 months after implantation, respectively.6. StatisticsAll values are mean±SEM. The significance of differences among mean values was determined by t-test. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS 13.0. The accepted level of significance was P<0.05.Results1. Morphologic Observation of xenogenic tendon matrix materialsHistology observation suggested that the xenogenic implant material consisted primarily of collagen without cell fragments. Scanning electron micrograph demonstrated that collagenous fibers were arranged uniformly.2. Mechanical and chemical testing on of xenogenic tendon matrix materialsThe tensile strength was at 11～16MPa.The breaking elongation rate was at 52～67%. The contents of protein was 94%.The residual of polyepoxy compound was lower than the standard line. The pH value in PBS kept stable around 7.24-7.32.3. Biocompatibility of xenogenic tendon matrix materialsNo animal died and no toxicity symptom or adverse effects were shown within 72 hours. No obvious sensitivity were observed. Skin stimulating reactions were not found in the experimental groups and negative control group by intradermal stimulation test. The toxicity of materials leaching liquor was graded from 0 to 1, which means the material has no cytotoxicity. All dogs survived well during the embedded test. There was no tissue necrosis, effusion or inflammation at all implantation sites. The xenogenic implant materials promoted slight to moderate inflammation process at 14 days, however, at 30 days, there was a regression of inflammation. After 60 days, it was observed the presence of well-organized connective tissue, and few inflammatory cells. Score evaluation of inflammation response at different time after operation of two groups showed no statistically significant difference (P> 0.05).4. Histological evaluation of xenogenic tendon matrix materials in animalsThe xenogenic tendon matrix materials indicated that regenerated collagen fibers had gradually encroached the implant and became a "composite"biomaterial with both original implanting tissue and host living tissue. After a year of remodeling the remaining materials, although reverse substituted half way through, was still kept its microscopic structure. The process of evaluation can be described as "creeping substitution", which is similar to the process of bone remodeling. ConclusionsThe xenogenic tendon matrix materials consisted primarily of collagen without cell fragments, and it has acceptable biomechanical properties and superior biocompatibility. It may be served as a promising implant material.