Ex Vivo Dermis Mechanical Behavior in Relation to Decellularization Treatment Length

1. BACKGROUND

So far, artificial scaffold-based constructs are unable to closely mimic the properties of the respective native tissues in terms of biocompatibility, function, structure, and so on. On the other hand, human-derived biomaterials preserve the natural architecture from the native tissue; however, allogenic biomaterials would solicit an adverse immune response [1, 2]. The solution is making use of decellularized allogenic material, where cells have been removed to produce a tissue as free as possible of potentially immunogenic antigens. The optimal decellularization treatment should minimize cellular and nuclear materials in the matrix, without affecting the integrity of the tissue structure, and consequently its mechanical properties [2, 3]. Generally speaking, the duration of the decellularization treatment has to counterbalance two benefits: on the one hand, a longer duration gives more chance to eliminate all cellular and nuclear components; on the other hand, it could adversely influence collagen, elastin, and extracellular components.

This work focuses on acellular dermal allografts; these grafts were initially used as wound cover in burns and other large wounds [4]; however, they are now also finding numerous other clinical applications as a scaffold allowing for new tissue ingrowth: it is the case of wound healing (diabetic ulcers or burns), tendon and ligament repair [5], soft-tissue supplementation [6], and joint resurfacing [7]. Methods of decellularization can have different processing times and they can employ different means, eventually producing alterations of the matrix structure; the most common processes include a combination of physical, chemical, and enzymatic agents. The use of NaOH medium for the decellularization of human skin has been reported in the literature as an effective low-cost method, which is able to remove donor cells and hairs [8]. The dermal tissue requires a more extensive biochemical exposure and longer rinse times, like other laminated thicker tissues (e.g. cardiac muscle tissue) [3]. However, bases run the risk of decreasing the extracellular matrix (ECM) mechanical properties due to the cleavage of collagen fibrils and disruption of collagen crosslinks [1]. The authors herein have tried to elucidate to what extent this process of decellularization affects the mechanical behavior of the dermal tissue, in relation to its duration. Apposite tensile tests have been planned both along Langer lines orientation and perpendicularly, and decellularized tissue properties have been compared with the native tissue ones. As a result, indications about the optimal decellularization treatment length have been given. In literature, the tensile properties of ex vivo native skin samples have been studied by Nì Annaidh et al. [8] who calculated engineering stresses and strains, and the elastic modulus for different back areas and different orientations. They demonstrated the variability of tissue properties in relation to specimen orientation (referred to Langer lines), body area, and among different donors; therefore, the influence of the decellularization treatment length has been tested on groups of specimens coming from the same donor, with the same orientation, and belonging to the same body area. Yoder and Elliott performed tensile tests on various graft materials used in soft-tissue applications, having outlined the relevance of mechanical properties of these tissues for a successful surgical reconstruction [9]. All these authors reported ultimate engineering stresses and this might be a limit since the specimen section undergoes a significant striction during tensile tests up to rupture; therefore, true stresses have also been calculated in this work, measuring specimen section in real time.

2. MATERIALS AND METHODS

3. RESULTS AND DISCUSSION

Results concerning the ultimate tensile stress have been reported in Fig. (1) and Fig. (2), respectively, for specimens cut parallel and perpendicular to Langer lines, while results concerning the elastic modulus have been reported in Fig. (3) and Fig. (4), respectively, for 0° and 90° specimens. The comparison of mechanical properties measured on specimens parallel or perpendicular to the Langer lines is in accordance with the well-known dermal tissue anisotropy, and is related to the prevalent orientation of collagen fibers [9, 11]: with reference to data preceding decellularization treatment (“T0”), specimens cut along Langer lines have exhibited a higher ultimate stress (33.95 MPa true stresses, and 10.28 MPa engineering stress) and a higher elastic modulus (21.8 MPa) when compared with specimens cut perpendicularly to Langer lines (24.11 MPa ultimate true stresses, 7.01 MPa ultimate engineering stress, and 12.31 MPa elastic modulus)

(Figs. 1 and 2) testify that referring to engineering stress would lead to a considerable underestimation of the ultimate stress for tests like those performed here (white columns are always higher than grey columns), due to the significant specimen striction taking place before specimen rupture, and to the small section of the specimens tested here (the thickness of dermal tissue is lower than 3 mm). Nevertheless, the patterns of ultimate stress versus decellularization treatment length have been found to be consistent between engineering and true stress formulations for the tensile tests (5-6 weeks values are similar and are smaller than ‘T0’ values both in Figs. 1 and 2).

All pictures testify that the decellularization treatment set up with sodium hydroxide has weakened the mechanical properties of the tissue (‘T0’ values remain the highest for whatever treatment length both in Figs. (1) and (2)), and its effect is more deleterious for specimens cut along Langer line orientation where collagen fiber bundles are stressed more closely to their axes (the difference between ‘T0’ values and all treated specimens is higher in Fig. (2) compared with Fig. (1)). Effectively, it is known that alkaline bases are able to degrade the structural components of the matrix including collagen to some extent: Sheridan et al. [12] used sonication and 0.5 M NaOH to controllably degrade small collagen fibrils of decellularized pig arterial tissue; in more detail, bases reduce the mechanical properties due to the cleavage of collagen fibrils and disruption of collagen nonreducible crosslinks [13].

However, all results have demonstrated a partial recovery of mechanical properties for longer treatment lengths (with specific reference to the decellularization treatment performed here through incubation in NaOH 0.06 N culture medium at room temperature, in continuous tilting): according to Fig. (1), the ultimate engineering tensile stress of untreated tissues cut perpendicular to Langer lines is not significantly different from those of specimens treated for 5 weeks (Tukey-Kramer test, p<0.05), or for 6 weeks (Tukey-Kramer test, p<0.05). These observations have induced one to perform more detailed analyses where real-time specimen width and thickness have been acquired to calculate true stresses. These tests have further confirmed the similarity between untreated specimens and specimens treated for 5-6 weeks through incubation in NaOH 0.06 N in continuous tilting (Fig. 1, ‘T0’ and ‘T5-6’ white columns). On the contrary, the ultimate tensile stress of tissues cut along Langer line orientation has got significantly lower, for whatever treatment duration (Fig. 2, ‘T0’ and ‘T5-6’ grey columns), and this observation has also been confirmed referring to true stresses (Fig. 2, ‘T0’ and ‘T5-6’ white columns). The observation of results concerning the elastic modulus (and consequently to physiologic stress levels) has led to similar conclusions: with reference to 90° orientation (Fig. 3), the performance of specimens treated for 5-6 weeks in NaOH 0.06 N culture medium is again similar to the performance of untreated specimens (‘T5-6’ vs. ‘T0’, Fig. 3); however, specimens treated for 4 weeks (‘T4’ in Fig. 3) are most similar to untreated specimens. With reference to Langer line orientation (Fig. 4), whatever treatment length has degraded the tissue mechanical properties (‘T0’ column is the highest in Fig. 4), 5 and 7 week treatments in NaOH 0.06 N culture medium are slightly more conservative (‘T5’ and ‘T7’ in Fig. 4). In summary, the experiments have demonstrated that there is an improvement in the mechanical properties mainly along 90° orientation for decellularization treatments in sodium hydroxide lasting 5-6 weeks (Fig. 3): this result can be explained assuming the recovery of intermolecular interactions between low-order collagen structures, resulting in fiber reassembly (the reversible dissociation of collagen has been clearly demonstrated [14]).

Other authors agree with the final findings of the present experimental work: they evaluated a cost-effective method based on low concentrations of NaOH for the decellularization of human donor skin preserved in 85% glycerol [10] and found that a 6-week incubation period was optimal. It should be stressed how decellularization through chemical agents is commonly considered to be a cost-effective method [10, 15]; nevertheless, its long duration could have inherent problems from a commercialization standpoint.

Differences of behaviors between 0° and 90° orientation samples (Fig. 1 vs. Fig. 2, and Fig. 3 vs. Fig. 4) can be explained considering that 0° samples are stressed along the main collagen fibers orientation, and are therefore more sensitive to collagen molecules denaturation, cleavage, or partial disruption, which can take place during treatments in alkaline culture media.

The obtained results refer to “ex vivo” tests on pre-implant specimens; when these grafts are implanted, remodeling processes take place, and tensile mechanical properties are likely to undergo significant variations; some clues can be gained from the functional tests performed in a rat subcutaneous implant model and in a porcine transplantation model by Richters et al. [10]. In future, it would be very interesting to perform in vivo tensile tests [16, 17] on implanted dermal substitutes.

CONCLUSION

This work has focused on the study of the consequences of an alkaline decellularization process (incubation in NaOH 0.06 N culture medium for 1-7 weeks, at room temperature in continuous tilting) on the structural function of dermal tissue, referring to the tissue strength (that is the ultimate stress measured during tensile tests at 3.2%/s strain rate), and to the tissue behavior at physiologic stress levels (elastic modulus of the linear portion of stress/strain curve).

The mechanical properties of the decellularized tissue have been compared with those of the native tissue, and considering two orthogonal orientations (parallel or perpendicular to the main orientation of collagen fibers), for different lengths of the decellularization treatment.

According to the results, the optimal duration of the decellularization treatment in NaOH is 5-6 weeks, since these treatment lengths have produced the most similar results to the native tissue.

The reliable quantitative measurement of structural properties of dermal tissue up to rupture requires an experimental setup able to capture specimen geometry variations in real time, while engineering stress and strain data have resulted in providing significant indication for comparative analyses among different decellularization treatment lengths.

REFERENCES