Exploring the mechanical properties of ex vivo human dermis in vitro and in silico

Human acellular dermal matrices (HADMs) are used in reconstructive surgery as scaffolds promoting autologous tissue regeneration. Despite their primary usage was in burn surgery, HADMs have recently been employed in reconstructive surgeries, which involve the high mechanical resistance prerequisite (e.g. rotator cuff tears repair, Achilles tendon augmentation, breast reconstruction procedure, hernia repair). Critical to the HADM ability to remodel and integrate into the host tissue is the removal of cells while maintaining an intact extracellular architecture. First objective of this research is to develop a methodology to analyze the mechanical properties of HADMs after decellularization to identify its ideal form of treatment and its duration. Two different decellularization techniques were used as a benchmark: the first is a well-established technique (incubation in NaOH for 1 to 7 weeks), and the second is an innovative technique developed by the Turin Skin Bank (AOU Città della Salute e della Scienza) research group (incubation in DMEM (Dulbecco's modified Eagle medium) for 1 to 7 weeks). After decellularization, the specimens underwent uniaxial tensile tests, and experimental data were represented with stress strain curves, calculating both engineering and true values. Mechanical tests, coupled to the immunohistochemical evaluation and the surgeons’ macroscopic analysis, have led to the identification of the optimal method (DMEM) and duration (5 weeks) for the decellularization treatment. Moreover, despite differences have been found between engineering and true values, which can reach 84%, the engineering values remain useful to make comparisons, providing reliable indications with a simpler experimental set up and data processing. Once identified the most suitable decellularization treatment, the preservation process has been inquired analysing its effects on the ECM mechanical properties. The standardized glycerolization procedure for the preservation of skin allograft is considered a simple and cost-effective method resulting in non-viable but intact skin that can be used as biological dressing on scalds, temporary coverage on excised burns and as a means of wound bed preparation. This same procedure can be used for the conservation of the decellularized dermis, whose application is in the reconstruction of tissues subjected to high mechanical in vivo solicitations. The preservation of the ECM’s integrity during the storage period is mandatory. Therefore, dermis tissue harvested from four donors was subjected to glycerolization and uniaxial mechanical tests were carried out on paired samples composed by de-glycerolized allograft and freshly excised human dermis collected from the same donor. Mechanical tests have led to the identification of the treatment influence, showing post-treatment increases in ultimate stress and elastic modulus of up to 191% and 212% respectively. In addition, donor and orientation factors were investigated, confirming the higher anisotropy of skin in older donors. Fundamental in the clinical uses of HADM is its suturability during the surgery procedure and the sutured allograft behavior when physiologically solicited. The first feature can be evaluated through a macroscopic analysis carried out by an experienced clinical examination. For the second one a mechanical characterization is mandatory, and it was here performed investigating the sutured HADM in various condition (one single suture at one end of the specimen (I), one or two stitches between two pieces of dermis in quasi-static (II) and dynamic conditions (III)). Standard uniaxial tensile tests were performed, coupling tensile machine sensor outputs to image analysis, with the aim of providing additional information other than just the “suture retention strength” value. Characteristic curves of the sutured dermis behaviour were obtained and were compared with control intact specimens. This analysis showed an increase in the elasticity of the sutured specimens, which appears to have beneficial effects in the immediate post-operative period, when the dermis allograft has not yet been incorporated and colonized by the host tissue. Uniaxial engineering stress-strain curves obtained in the first phase of this work were then exploited for the implementation of four different computational model (both linear and non-linear), and an evaluation procedure was carried out based on a quantitative comparison of the simulation results with parameters extracted from the true stress-strain curves. It was found that the results obtained from the hyperelastic models well represent the experimental situation, and in particular, the incompressible Odgen model shows less than 7% percentage variations for all the considered parameters. It was then confirmed the inadequacy of the linear models to describe the behaviour of a biological material like dermis. The last aspect investigated in this research was the biaxial characterization of the engineered HADMs. In fact, planar biaxial testing allows for a two-dimensional stress-state that can be used to fully characterize the dermis properties. The high cost related to this type of analysis has lead to the development of a new low cost biaxial conversion mechanism for a uniaxial testing machine. This mechanism was realized through rapid prototyping considerably reducing the implementation costs and facilitating the design phase, assisted by the immediate printing and testing of the components. Before use, the device was validated through both experimental and computational tests which involved the response of the mechanism when subjected to unbalanced loads and the evaluation of the uniformity of the strain distributions in a small central region of the specimen. This latter was performed through different procedures (the finite element method, the DIC method and the rosette gage theory) all employing the optical measure of strains. The biaxial mechanical behaviour of untreated and decellularized dermis was measured exploiting the purpose-made biaxial fixture. Stress-strain curves were evaluated from loads acquired by two load cells positioned along two orthogonal axis and the optical measure of the deformations of four markers located in the central area of the specimen. The specimens resulted on average less extensible in the medio-lateral direction (namely, along the Langer lines) then in the cranio-caudal direction, confirming the correlation of dermis mechanical response with collagen fibres disposition with respect to the loading direction