Matrix alteration and not residual sodium dodecyl sulfate cytotoxicity affects the cellular repopulation of a decellularized matrix.

It has been suggested that residual cytotoxic sodium dodecyl sulfate (SDS) is responsible for the low levels of cell in-growth observed in SDS decellularized tissues. To determine whether this is the case, we used 2 washing methods to remove residual SDS and extensive biochemical, mechanical, and structural analyses to determine the effects of SDS-based decellularization on porcine anterior cruciate ligament (ACL) tissue and its propensity for cellular repopulation. The level of residual SDS in decellularized tissue was reduced using 2 different washing techniques (pH = 9 buffer, 75% ethanol). After washing in pH = 9 or 75% ethanol, residual SDS concentrations in decellularized tissues were found to be approximately 8 and 23 times less than reported SDS cytotoxic levels, respectively. It was found that SDS treatment significantly reduced glycosaminoglycan levels, increased collagen crimp amplitude and periodicity, and increased susceptibility of collagen to degradation by the gelatinase enzyme trypsin. The level of repopulation and viability of autologous ACL fibroblasts in the decellularized tissue after 28 days of culture were found to be the same regardless of the washing technique and resulting level of residual SDS in the tissue. This strongly indicates that alterations in tissue matrix biochemistry or structure from SDS treatment and not residual SDS cytotoxicity are responsible for the low cell re-population observed in SDS decellularized tissues.

Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft.

In this study, porcine bone-anterior cruciate ligament-bone (B-ACL-B) grafts were decellularized using one of three protocols incorporating surfactants lauryl sulfate (SDS), Triton X-100, and/or an organic solvent (tributyl phosphate (TnBP)). The effectiveness of Triton-SDS, Triton-Triton or Triton-TnBP treatments in removing cellular materials was determined and possible changes in biochemical composition and mechanical properties due to each treatment were investigated. Treatment with Triton-SDS was most effective at removing cell nuclei and intracellular protein (vimentin) from the ACL but affected both the collagen and glycosaminoglycan (GAG) components of the extracellular matrix while increasing the tensile stiffness of the ligament. Triton-Triton was the least effective of the three treatments in terms of cellular extraction, but did not significantly change the mechanical and biochemical properties of the ACL. Triton-TnBP matched the level of decellularization achieved by Triton-SDS in terms of visible cell nuclei; however, the extraction of intracellular vimentin was less consistent. TnBP treatment also slightly decreased the collagen content of the ACL but did not alter its mechanical properties. Overall, all three decellularization treatments maintained adequate mechanical and biochemical properties of B-ACL-B grafts to justify the further investigation of all three decellularization protocols. The selection of a superior treatment will depend on future studies of the propensity of treated tissues for repopulation by host ACL fibroblasts and, ultimately, on any immunogenic and/or remodeling host response induced in vivo.