Abstract
Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC) culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS), trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS) are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.
Highlights
Extracellular matrix (ECM) has emerged as a fundamental tool for developing regenerative tissue prostheses [1]
Engineered scaffolds including decellularized tendon scaffolds (DTS) are geared to provide a tunable microenvironment in which we may study fundamental development as well as cell-mediated mechanisms of tissue regeneration. Advanced scaffolds such as DTS may prove valuable in extending the phenotypic stability of tenocytes [39] and tendon stem cells [40], which experience drift over extended culture periods [41]
Our novel protocol resulted in formation of biocompatible, acellular constructs that will prove valuable in this pursuit
Summary
Extracellular matrix (ECM) has emerged as a fundamental tool for developing regenerative tissue prostheses [1]. Biomaterials based on stem cells seeded on decellularized tissue scaffolds are emerging as exciting options for clinical therapy, by obviating the need for traditional organ transplant or autologous donation techniques which are associated with significant morbidity [4,5,6,7]. Equine athletes routinely function close to the mechanical threshold for tendon damage [12] in a mildly hyperthermic environment [13], resulting in cumulative cellular and extracellular breakdown as well as changes in tissue biochemistry [14]. Tendinopathy results when this deterioration exceeds the capacity for restorative remodeling [15,16]. Due to the high in vivo tensile load experienced by the equine flexor digitorum superficialis tendon (5% average strain at a speed of 7 m/s) [18], a maximal load on the order of 10 kN [12], and the low cellularity and vascularity of the tissue, it is a strong and homogeneous starting material as a source of xenogeneic scaffold material
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