Effect of low-temperature ethylene oxide and electron beam sterilization on the in vitro and in vivo function of reconstituted extracellular matrix-derived scaffolds

Abstract

Reconstituted extracellular matrix (ECM) -derived scaffolds are commonly utilized in preclinical tissue engineering studies as delivery vehicles for cells and growth factors. Translation into clinical use requires identifying a sterilization method that effectively removes bacteria but doesn’t harm scaffold function. To determine effectiveness of sterilization and impact on ECM scaffold integrity and function low temperature ethylene oxide and 15kGy electron beam irradiation techniques were evaluated. Scaffold sterility was assessed in accordance to United States Pharmacopeia Chapter 71. Scaffold matrix degradation was determined in vitro using enzymatic resistance tests and gel electrophoresis. Scaffold mechanics including elastic modulus, yield stress and collapse modulus were tested. Lastly, 14 Yorkshire pigs underwent ACL transection and bio-enhanced ACL repair using sterilized scaffolds. Histologic response of ligament, synovium and lymph nodes was compared at 4, 6, and 8 weeks. Ethylene oxide as well as electron beam irradiation yielded sterile scaffolds. Scaffold resistance to enzymatic digestion and protein integrity slightly decreased after electron beam irradiation while ethylene oxide altered scaffold matrix. Scaffold elastic modulus and yield stress were increased after electron beam treatment, while collapse modulus was increased after ethylene oxide treatment. No significant changes in ACL dimensions, in vivo scaffold resorption rate, or histologic response of synovium, ligament and lymph nodes with either terminal sterilization technique were detectable. In conclusion, this study identifies two methods to terminally sterilize an ECM scaffold. In vitro scaffold properties were slightly changed without significantly influencing the biologic responses of the surrounding tissues in vivo. This is a critical step toward translating new tissue engineering strategies to clinical trials.

Biologic Scaffolds

“…Alternative methods of terminal sterilization include ethylene oxide 9 (ETO) and high-energy radiation (electron beam or g radiation), both of which can have a negative effect on the ECM bioscaffolds. ETO can react with the free amine groups of collagen, affecting the mechanical properties of ECM bioscaffolds, and high-energy radiation damages protein chains and promotes the formation of free radicals (Proffen et al 2015). Supercritical CO 2 sterilization 10 has been investigated for sterilization of collagen scaffolds and showed minimal compromise of mechanical properties (Bernhardt et al 2015).…”

Extracellular matrix‐blood composite injection reduces post‐traumatic osteoarthritis after anterior cruciate ligament injury in the rat

“…A hydrogel of extracellular matrix proteins was made by aseptically harvesting bovine connective tissue from the knee capsule, decellularization with a detergent solution and solubilization using pepsin in hydrochloric acid as previously described. 25 The resulting gel was neutralized using a buffer containing HEPES, sodium hydroxide, and calcium chloride to a pH of 7.4 and kept at 4˚C until use. At time of injection, animals were anaesthetized and 50 ml of blood was drawn from the tail vein of the rat and mixed with acidcitrate-dextrose (ACD) in a ratio of 10:1.…”

A Narrative Review of Four Different New Techniques in Primary Anterior Cruciate Ligament Repair: “Back to the Future” or Another Trend?

“…The authors found that this method of sterilization was effective and had no significant influence on in vivo function of the scaffolds. Moreover, Proffen et al after an animal study showed that low-temperature ethylene oxide and electron beam irradiation were acceptable methods of sterilization of reconstituted extracellular matrix-derived scaffolds [ 28 ]. The slight modification of in vitro scaffold properties did not significantly alter biologic reactions of the surrounding tissues in vivo.…”

“…The BEAR scaffold was manufactured at Boston Children’s Hospital and completed all biocompatibility and sterility testing prior to use in the clinical study [ 36 , 37 ]. The scaffold comprised extracellular matrix proteins, including collagen, that were obtained from bovine tissue [ 28 ].…”