Characterization of Stem Cell-Natural Scaffold Construct for the Ligament and Tendon Reconstruction Cultured in Tissue Bioreactor

Abstract

Since many years, the injury of anterior cruciate ligament (ACL) has been cured by the biological grafting material such as autograft and allograft. These natural scaffolds derived from human tissues have many advantages, including eliminated donor site morbidity, shortened operation time, large-sized bone grafts, minimized surgical incisions, and reduced incidence of arthrofibrosis. However there are potential disadvantages such as some risk of viral and bacterial disease transmission, potential for immune rejection, and longer incorporation time, which can require longer protection from potentially injurious effects. However, donor screening, high pressure saline washing, antibiotics, and sterilization may safely preserve them from those disadvantages. Besides, it became known that natural graft can affect patient’s immune rejection by its own cellular components remaining in the tendon as antigens. Thus, the important of decellularization is increased for the safety of tendon graft. Decellularized tissues are successfully used in tissue engineering and regenerative medical field for the purpose of removing tissue antigens from a cellular component. However, chemical decellularization which is processed by ionic solution or enzymatic treatment that may decrease the mechanical property of tendon graft and deform the extracellular matrix (ECM). In addition, some cleaning detergents can be harmful and dangerous when it remains in the patient’s body. Even though the decellularization technique is considered as effective and safe method for tissue sterilization, most of soft tissues are processed restrictively by antibiotics or high pressure saline washing. While decellularization technique is used in tissue sterilization to remove immunoreactive material, the property of tendon graft must be preserved of mechanical strength decline by decellularization must be

recovered. For this study, we designed a tissue bioreactor to give biaxial cyclic loading to the tissue. The advanced bioreactor is expected to provide ideal conditions for the cultivation of functional tissue grafts. The system of tissue bioreactor is composed of motion part that gives mechanical stimulation such as translational and rotational strain, control part that regulates time, frequency, temperature, and gases such as CO2, and O2 and chamber part that receives physical stimulation conditionally. The purpose of this study is to confirm ECM synthesis and mechanical efficiency of cell-natural scaffold construct that is cultured under cyclic loading in the tissue bioreactor. During decellularization process, porcine anterior tibialis tendons were treated with enzymatic solution and ultrasonic cleaning. In the final step, hypertonic and hypotonic solutions were rinsed to remove residue during cell lysis. Decellularized tendons were recellularized by bone marrow derived stem cell-collagen mixture, and cultivated under cyclic loading in the bioreactor. In this study, the ultimate tensile load of the decellularized tendons was lowered by 20% than the fresh tendon. However, after cultivation in the tissue bioreactor with cyclic loading, which was conditioned as 10% of tension, 90° of torsion and 1Hz of frequency, the ultimate tensile load was further increased as compared with the fresh tendon. This study shows that the natural graft is decellularized as biologically safe and the cyclic loading in the bioreactor system increases the mechanical property of engineered cell-natural scaffold construct compared with the fresh natural graft. The recellularized natural scaffold composite, which is cultivated under cyclic loading in the bioreactor, will be essential for the ligament and tendon reconstruction.