Materials

In the study, three different CS were examined. Two of these were of porcine origin (p-CM and p-ADM), and one was of human origin (a-ADM). P-CM was a cross-linked collagen matrix (Fibro Gide®, Geistlich Pharma AG, Wollhusen, Switzerland), while p-ADM and a-ADM represented acellular dermal matrices (Mucoderm®, Botiss, Zossen, Germany, and Puros® Dermis Tissue Matrix, Tutogen Medical GmbH, Neunkirchen, Germany). Table 1 provides an overview of the materials used and their properties.

Table 1 Overview of the test-materials

Native connective tissue from the porcine palate (p-CTG) served as the control and was stored at − 18 °C. Notably, animals of this study were raised and slaughtered for food production according to the Swiss standards for animal welfare. The study protocol did not in any way influence premortal fate of the animals or the slaughtering process. Therefore, this investigation was not classified as an animal study and the institutional ethics committee did not have any objections to the protocol.

Solutions

Four reagents were used to test the resorption behavior. These solutions, designed to mimic the oral environment, included simulated body fluid (SBF) and artificial saliva (Klimek), each with and without collagenase (0.5 unit/ml), respectively. SBF and Klimek were prepared exactly according to the instructions provided by Kokubo et al. [27] and Klimek et al. [28], respectively. The ingredients of the used reagents are listed in Tables 3 and 4 in Appendix.

The swelling and cyclic compression tests were conducted in an external laboratory (Geistlich Pharma AG, Wollhusen, Switzerland). This company uses an isotonic phosphate buffered salt solution (PBS) as a reagent for these tests. Therefore, PBS was also employed for these two tests. PBS was prepared according to the company’s recipe.

Collagenase

When used clinically, the tested materials undergo natural degradation. To simulate this biodegradation in vitro, collagenase was employed as an enzyme. The collagenase used in this experiment was of bacterial origin, obtained from Clostridium histolyticum. Available as a lyophilized powder (C9891, Sigma Aldrich, St. Louis, USA), it was mixed with either SBF or Klimek (5 unit/ml) before being diluted to 0.5 unit/ml.

Experimental procedureResorption behavior

Standardized 5 × 5 mm test specimens of the included materials were prepared. The thickness of the samples was measured using an ethanol-purified digital external micrometer (Mitutoyo, Urdorf, Switzerland) to avoid compressing the materials. Five samples (n = 5) of each of the four tested materials (three artificial matrices and one p-CTG as control) were exposed to the four solutions at three timepoints (4, 8 and 24 h). Samples were placed in a 24-well plate for 15 min, each in 1 ml of 0.9% sodium chloride solution. After 15 min, the samples were dried and weighed (see “Procedure for standardized drying and weight measurement” section). The sodium chloride solution was removed and replaced by the respective test solutions. At each timepoint, the solutions were exchanged. The 24-well plate containing the samples and replaced solutions was sealed with tape and incubated at 37 °C. At each solution change, CS and control samples were dried and weighed with a Mettler AT261 DeltaRange® scale (Mettler, Greifensee, Switzerland). The resorption rate of the materials was investigated based on the weight measurements.

Surface analysis

The surface morphology of the test materials was examined using a scanning electron microscope (SEM; Zeiss, Oberkochen, Germany) at a magnification of 2000×. SEM images were taken in the untreated condition (out of the package) and after 24-h exposure to both SBF with collagenase (0.5 unit/ml) and Klimek with collagenase (0.5 unit/ml), respectively. Before imaging, CS were soaked in deionized water for 3 min and then dried (see “Procedure for standardized drying and weight measurement” section). Subsequently, samples were affixed to an SEM carrier using a self-adhesive carbon pad and sputter-coated with 10 nm of gold.

Swelling behavior

In this experiment, three CS (p-CM, p-ADM, a-ADM) were cut to a size of 15 × 20 mm, with three samples (n = 3) of each matrix prepared. Dimensions (length, width and height) were measured at the highest point of the samples using a digital external micrometer (Mitutoyo, Urdorf, Switzerland) at the first visual contact. A pencil line was drawn on each matrix 2 mm from the bottom edge. The prepared samples were vertically mounted on a sample holder attached to a XS204 DeltaRange® scale (Mettler, Greifensee, Switzerland) (Fig. 1). A vessel containing PBS was placed on the balance plate and the bottom 2 mm of each matrix was immersed in PBS. The time and mass change from the immersion of the matrix in the solution until the solution reached the top of the matrix were assessed. Once the matrix appeared fully swollen, the balance plate was lowered, so that the matrix was no longer in contact with the solution. After this, weight change was recorded for an additional 2 min to measure any evaporation. The liquid absorption capacity per volume was calculated by subtracting the mass of the dry matrix from the mass of the swollen matrix and then dividing by the volume of the matrix.

Fig. 1

Scheme of the experimental setup

Cyclic compression test

The test materials, along with the control material, were cut to a size of 10 × 20 mm. Three specimens (n = 3) of each material were prepared. These materials were transferred with forceps into tubes filled with 50 ml of PBS. These tubes were then incubated in a water bath at 37 °C for 2 h. After this 2-h incubation, specimens were placed using bent tweezers under a serrated press plate on the metal plate of the material testing machine (Zwick Roell, Ulm, Germany), fully immersed in PBS at 37 °C. The tool gap was visually adjusted to secure the specimen in place. Cyclic testing (force: 12.1 kPa) of the specimens was performed in PBS at 37 °C for a total of 49 cycles. In the 50th cycle, each specimen was completely pushed through.

Procedure for standardized drying and weight measurement

The standardized drying procedure was carried out as follows: at the time of measurement, each sample was removed from the perforated plate using diamond tweezers (Intensiv SA, Collina d’Oro, Switzerland), cleaned with ethanol and placed on a Tela napkin. The napkin was then folded over and weighted down for 5 s with a multi-kilogram lead cylinder. This procedure was repeated on a dry area of the napkin.

Prior to each weight measurement, the scale was calibrated with a plastic pan. Using the diamond tweezers, the specimen was then placed on this pan in the balance for weighing.

Statistical analysis

The analysis was conducted using the statistical software R. Median, interquartile range (IQR), and the smallest and largest values were calculated from the relative values of the weight changes of the different specimens and compared. Pairwise Wilcoxon Rank Sum Tests were conducted to determine the differences between the materials in the various solutions at the 24-h timepoint. Statistical significance was indicated with an “*” and was always assumed at p < 0.05. For the test materials, area, volume change and fluid absorption capacity per volume for each material were calculated. Median, IQR and the smallest and largest values were determined and compared in each case. Pairwise Wilcoxon Rank Sum Tests were used to calculate pairwise comparisons between material levels for swelling behavior. Due to the number of specimens (n = 3) no p value was obtained.