Decellularization of cartilage microparticles: Effects of temperature, supercritical carbon dioxide and ultrasound on biochemical, mechanical, and biological properties
V. I. Sevastianov, Yu. B. Basok, Alexey M. Grigoriev, E. A. Nemets, A. D. Kirillova, Liudmila A. Kirsanova, A. E. Lazhko, А.М. Суббот, M V Kravchik, Yusef D. Khesuani, Elizaveta V. Koudan, S. V. Gautier
Abstract
One of the approaches to restoring the structure of damaged cartilage tissue is an intra-articular injection of tissue-engineered medical products (TEMPs) consisting of biocompatible matrices loaded with cells. The most interesting are the absorbable matrices from decellularized tissues, provided that the cellular material is completely removed from them with the maximum possible preservation of the structure and composition of the natural extracellular matrix. The present study investigated the mechanical, biochemical, and biological properties of decellularized porcine cartilage microparticles (DCMps) obtained by techniques, differing only in physical treatments, such as freeze-thaw cycling (Protocol 1), supercritical carbon dioxide fluid (Protocol 2) and ultrasound (Protocol 3). Full tissue decellularization was achieved, as confirmed by the histological analysis and DNA quantification, though all the resultant DCMps had reduced glycosaminoglycans (GAGs) and collagen. The elastic modulus of all DCMp samples was also significantly reduced. Most notably, DCMps prepared with Protocol 3 significantly outperformed other samples in viability and the chondroinduction of the human adipose-derived stem cells (hADSCs), with a higher GAG production per DNA content. A positive ECM staining for type II collagen was also detected only in cartilage-like structures based on ultrasound-treated DCMps. The biocompatibility of a xenogenic DCMps obtained with Protocol 3 has been confirmed for a 6-month implantation in the thigh muscle tissue of mature rats (n = 18). Overall, the results showed that the porcine cartilage microparticles decellularized by a combination of detergents, ultrasound and DNase could be a promising source of scaffolds for TEMPs for cartilage reconstruction.