Differential biodegradation kinetics of collagen membranes for bone regeneration

polymers Article Differential Biodegradation Kinetics of Collagen Membranes for Bone Regeneration Manuel Toledano 1, Samara Asady 1, Manuel Toledano-Osorio 1,*, Franklin García-Godoy 2, María-Angeles Serrera-Figallo 3, JoséA. Benítez-García1and Raquel Osorio 1 1Faculty of Dentistry, Dental Materials Section, University of Granada, Colegio Máximo de Cartuja s/n, 18071 Granada, Spain; [email protected] (M.T.); [email protected] (S.A.); [email protected] (J.A.B.-G.); [email protected] (R.O.) 2College of Dentistry, Health Science Center, University of Tennessee, 875 Union Avenue, Memphis, TN 381632110, USA; [email protected] 3Faculty of Dentistry, Oral Surgery Section, University of Sevilla, Avicena s/n, 41009 Sevilla, Spain; [email protected] *Correspondence: [email protected].es; Tel.: +34-958-243-789 Received: 24 April 2020; Accepted: 2 June 2020; Published: 4 June 2020   Abstract: Native collagen-based membranes are used to guide bone regeneration; but due to their rapid biodegradation, this treatment is often unpredictable. The purpose of this study was to investigate the biodegradability of natural collagen membranes. Three non-cross-linked resorbable collagen barrier membranes were tested: Derma Fina (porcine dermis), Evolution Standard (equine pericardium) and Duo-Teck (equine lyophilized collagen felt). 10 × 10 mm 2 pieces of membranes were submitted to three different degradation procedures: (1) hydrolytic degradation in phosphate buffer solution, (2) enzyme resistance, using a 0.25% porcine trypsin solution, and (3) bacterial (Clostridium histolyticum) collagenase resistance test. Weight measurements were performed with an analytic microbalance. Thickness was measured with a digital caliper. Membranes were analyzed at different time-points, up to 21 d of immersion. A stereomicroscope was used to obtain membranes’ images. ANOVA and Student Newman Keuls were used for mean comparisons (p<0.05), except when analyzing differences between time-points within the same membrane and solution where pair-wise comparisons were applied (p<0.001). Derma Fina attained the highest resistance to all degradation challenges. Duo-Teck was the most susceptible membrane to degradation, complete degradation occurred as soon as 8 h. The bacterial collagenase solution performed as the most aggressive test as all membranes presented 100% degradation before 21 d. Keywords: collagen; membrane; biodegradation; bone regeneration 1. Introduction The aging population is the principal reason to advance the understanding of dental biomaterials aimed to achieve periodontal regeneration [ 1 ]. Chronic periodontitis is one of the most aggressive pathologies and it refers to the inflammation and progressive destruction of the supporting tissues of the periodontium resulting in loss of the teeth and the alveolar bone. Restoration of the lost periodontium is a paramount goal of periodontal therapy [ 2 ], where the guide bone regeneration (GBR) concept plays a capital roll. GBR is a surgical procedure that uses membranes as barriers to prevent the ingrowth of fibroblasts and to maintain a space for osteogenesis. The use of a barrier membrane to promote the selective repopulation of a periodontal/bone defect by cells with regenerative potential has been successfully applied for more than 40 years when the first application of a cellulose acetate laboratory filter by Millipore for GBR occurred [ 3 ]. Currently, GBR is a method successfully employed in dental practices aimed to increase the volume of the host bone at sites with insufficient bone quantity [4]. Polymers 2020,12, 1290; doi:10.3390/polym12061290 www.mdpi.com/journal/polymers Polymers 2020,12, 1290 2 of 17 Two main types of barrier membranes are available on the market: resorbable and non-resorbable. Non-resorbable membranes (as polytetrafluoroethylene -PTFE-) maintain their structural integrity for as long as they are left in the tissue, providing the operator with complete control over the time of application [ 5 ]. They also guarantee space maintenance for the duration of healing, but require a second surgery for their removal. Complications have also been reported such as frequent soft tissue dehiscences or infections during membrane exposure, which can negatively influence clinical outcomes or regenerative procedures, jeopardizing osteogenesis [ 6 – 8 ]. The resorbable membranes are those composed by synthetic polymers like polylactic or polyglycolic acid or by biopolymers like collagen [ 3 ]. Collagen membranes are the most frequently employed, and the ones with the highest number of reported clinical studies available [ 3 ]. Collagen membranes exhibit several advantages, compared to synthetic polymeric membranes, such as easy manipulation, weak immunogenicity, a direct effect on bone formation and chemotaxis of gingival and periodontal ligament fibroblasts. Some other benefits include promoting wound healing and stability through isolation, clot stabilization, hemostasis, semi-permeability, support of nutrient transfer and augmenting flap thickness by providing a collagenous scaffold [ 9 – 11 ]. The source of the collagen varies, but it is typically obtained from bovine tendon, bovine dermis, calf skin or porcine dermis [ 12 ]. Porcine skin-derived collagen membranes are widely used in GBR procedures because porcine dermis primarily consists of type I collagen and its 3D architecture is similar to that of native extracellular matrix [ 13 ]. Purified bovine and porcine collagen derived from tendons, dermis (skin), pericardium and other regions has also been shown to be suitable as donor material [ 1 ]. However, the major drawback of native collagen is the rapid biodegradation by the enzymatic activity of macrophages and polymorphonuclear leucocytes. Then, the potential of losing space maintenance ability in physiological conditions is high [ 3 ]. If the membrane dissolves quickly, clinical treatment goals will not be achieved and GBR will be unpredictable [ 13 ]. An ideal barrier for GBR should resorb gradually over time [ 9 ]. Although the biodegradable nature of biological membranes eliminates the need for surgical membrane retrieval, these collagen membranes present limitations in terms of controlling degradation [ 2 ]. It has been suggested that these membranes must stay physically and mechanically intact for at least 4–6 weeks for regenerative therapy to be successful [ 2 ]. Therefore, unsuitable degradation rates may significantly hamper the regenerative potential of currently available GBR membranes [14]. Only a limited number of studies have investigated the resorption patterns of collagen membranes, showing that their degradation might start within 4 days to 6 weeks after surgical placement. However, in most of the published studies, resorption of collagen membrane was evaluated in subcutaneous tissue membranes implantation in rats. Moreover, the available data mainly consist of qualitative histologic observations and/or the measurement of membrane thickness, with little characterization of the enzymatic or hydrolytic degradation process [ 9 ]. In vitro findings suggest that the composition and/or structure of the membrane may play an important role in the clinical outcomes of degradation. The activity of bacteria and their enzymes in sites of regeneration may contribute to the rapid elimination of membrane material and shorten the desired period of regeneration considerably [15]. Resorption time of collagen membranes may be extended by cross-linking of the fibers through physical or chemical procedures. These cross-linked collagen membranes are not usually employed clinically, because it has been shown that cross-linking using glutaraldehyde decreased the biocompatibility whereas enzymatic cross-linking negatively reduced the tissue integration and biodegradation pattern. The degree of chemical cross-linking caused severe inflammatory reactions [ 11 , 16 ]. Even more, one of the questions is whether non cross-linked membranes really display differences in terms of degradation behavior. Therefore, non-crosslinked collagen membranes were selected for the present study. Absorbable barrier membranes offer limited control over the length of application because their inherent nature allows the disintegration process to start upon placement in the tissue. Porosity is an important feature of membranes as they allow for the infiltration of nutrients into the defect, which promotes bone growth [ 17 ]; however, the excessively large pores after biodegradation might make Polymers 2020,12, 1290 3 of 17 the membranes less effective as a barrier against soft tissue cells. Provided that the longevity of the barriers’ function is an important aspect of their clinical performance, the loss of the structural integrity of collagen membranes due to fast biodegradation by enzymatic activities becomes a major problem of this type of bioabsorbable devices [ 5 ]. The membrane thickness and weight affect the mechanical properties of the membrane and define the diffusion distance between tissue compartments and, therefore, provide a rationale for selecting membranes in view of specific applications in GBR [ 18 ]. Then, investigations on variations in these properties or in the possibility of microstructural membranes defects formation during their degradation processes is a crucial point to be considered in order to increase the success of GBR therapy. The necessity and novelty of the present study is justified. Hence, the aim of this study was to investigate the degradation kinetic, from 4 h to 21 d, of three non-crosslinked collagen membranes from different origin. A qualitative microstructural assessment and a quantitative analysis of the collagen membrane degradation were performed. The null hypotheses to be tested were that: (i) the three membranes for guided bone regeneration do not degrade in the same extent, over time; and (ii) the three membranes do not resist similarly the different degradation processes (hydrolytic, bacterial collagenase and trypsin). 2. Materials and Methods 2.1. Membranes Tested Three GBR collagen membranes were tested. Membranes are commercially available and CE-certified for oral applications and all have heterologous origin. The membranes tested were: (1) Derma Fina (OsteoBiol ® by Tecnoss, Torino, Italy); (2) Evolution Standard (OsteoBiol ® by Tecnoss, Torino, Italy); (3) Duo-Teck (OsteoBiol ® by Tecnoss, Torino, ® by Tecnoss, Torino, Italy). According to the manufacturer, Derma Fina is derived from porcine dermis after removal of the epithelial layer. The membrane is composed of a network of highly purified non-cross-linked porcine collagen fibers intermingled with porcine elastin fibers. This membrane is made of non-cross-linked porcine Type I and III collagens and has a bi-layered structure. The processing technique is performed at room temperature (cold process). Evolution Standard is a resorbable dense collagen mesh barrier derived from heterologous mesenchymal equine pericardium tissue, and Duo-Teck is a resorbable membrane derived from equine lyophilized collagen felt, one of the external surfaces is covered by micronized equine bone particles (up to 300 µm). 2.2. Degradation Assays Membrane samples were cut to a size of 10 × 10 mm 2 . Three specimens of each membrane type were employed for each test and further measured in weight and thickness. For weight (W) measurements, an analytic scale (A&D-Instruments, Frankfurt, Germany) was used, with an accuracy of 0.0001 g; the complete device was mounted on an antivibratory table. Thickness (Th) was measured at random positions by means of a digital caliper (Mitutoyo 293-561, Tokyo, Japan). Three different degradation tests were performed: (1) Hydrolytic degradation test: In vitro hydrolytic degradation behavior of membranes was analyzed in phosphate buffer solution (PBS) at 37 ◦C [19]. (2) Enzyme resistance test: Samples were immersed in a 0.13% porcine trypsin solution (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 37 ◦C [13]. (3) Bacterial collagenase resistance test: A collagenase solution from Clostridium histolyticum bacteria Type V (Sigma Aldrich, St Louis, MO, USA) was used. It is actually a mixture of several different enzymes including collagenase, which act together to break down tissue. This preparation contains collagenase, non-specific proteases, clostripain, neutral protease, and aminopeptidase activities. Specific activity is ≥ 125 CDU/mg solid. A collagenase concentration of 2 IU/mL in 50 mM Tris HCl (pH 7.4) containing 10 mM CaCl 2 was used [ 15 , 16 ]. After each 48 h, degradation solutions were removed carefully through suction and renewed [ 2 ]. After each immersion time point (4 h, 8 h, Polymers 2020,12, 1290 4 of 17 12 h, 16 h, 24 h, 48 h, 72 h, 7 d, 14 d, and 21 d), samples were dried by placing them into a vacuum chamber at 37 ◦ C for 72 h. Next, the weight and thickness of the dried samples were measured. Three measurements were taken from each specimen. All degradation experiments were performed in triplicate. At the end of the storage, pictures of the membranes’ surfaces were obtained by means of an Olympus SZ-CTV stereomicroscope (Olympus, Tokyo, Japan) a digital signal processor DSP 5050 (Olympus, Tokyo, Japan) was used. 2.3. Statistical Analysis Multiple ANOVA models were used to assess the influence of the independent variables (degradation solution, type of membrane and immersion time) on the dependent variables (weight and thickness). Analyses of interactions were also performed. ANOVA and Student Newman Keuls post-hoc comparisons were performed to determine differences between materials and degradation solutions. To permit for these comparisons, the variables weight and thickness were converted to percentage of variation respect to the initial measurement following the equation: Percentage of loss =[(X0−Xt)/X0]×100, (1) where, X 0 is the initial weight or thickness of specimens; and X t is the specimen’s weight or thickness at each time-point (t). Pairwise comparisons were performed to ascertain for differences between immersion time-points within the same membrane and solution experimental group. Normal distribution of data was probed before the analyses and statistical significance was always considered at p<0.05 except for pairwise comparisons where a Bonferroni’s correction was applied and p<0.001 was set. Statistical analysis was performed using SPSS 25.0 (SPSS Inc., Chicago, IL, USA) software package. 2.4. Light Microscopy Analysis Before immersion and at the end of the storage period (21 d), specimens were observed under a stereomicroscope Olympus SZ-60 (Olympus, Tokyo, Japan) for microstructural analysis. Images were taken at 60×and 120×magnifications. 3. Results The thickness (Th) values in mm of the three membranes (Derma Fina, Evolution Standard and Duo-Teck) submitted to the three different degradation tests (PBS, trypsin and C. histolyticum collagenase) as a function of the different time-points are reflected in Table 1. The loss of percentage thickness (Th) values of the membranes, as a function of the different degradation tests and time-points are represented in Figure 1. Polymers 2020,12, 1290 5 of 17 Table 1. ( a ) Thickness (mm) analysis of the three experimental membranes (Derma Fina, Evolution Standard and Duo-Teck) after immersions periods up to 21 days in PBS (Hydrolytic degradation test), collagenase from Clostridium histolyticum (Bacterial collagenase resistance test) and trypsin (Enzyme resistance test). Values shown are mean and standard deviation. ( b ) Attained pvalues after pairwise comparisons between membranes’ thicknesses after the different immersion times and the initial thickness (thickness at t0: Th0). Significance was considered at p≤0.001. (a) Derma Evolution Duo-Teck Trypsin C. hystolyticum PBS Trypsin C. hystolyticum PBS Trypsin C. hystolyticum PBS t01.02 (0.13) 1.09 (0.17) 1.22 (0.04) 0.43 (0.14) 0.39 (0.05) 0.47 (0.01) 0.15 (0.00) 0.15 (0.01) 0.17 (0.00) 4 h 0.75 (0.05) 0.59 (0.05) 0.85 (0.10) 0.21 (0.02) 0.14 (0.02) 0.20 (0.03) 0.13 (0.04) 0.14 (0.01) 0.18 (0.06) 8 h 0.64 (0.07) 0.54 (0.05) 0.75 (0.08) 0.20 (0.06) 0.08 (0.01) 0.23 (0.03) 0.11 (0.03) 0 (0) 0.14 (0.05) 12 h 0.61 (0.06) 0.48 (0.07) 0.66 (0.03) 0.16 (0.04) 0.06 (0.01) 0.21 (0.04) 0.08 (0.01) 0 (0) 0.11 (0.05) 16 h 0.60 (0.05) 0.41 (0.04) 0.66 (0.05) 0.16 (0.04) 0.06 (0.01) 0.20 (0.03) 0.07 (0.02) 0 (0) 0.08 (0.02) 24 h 0.57 (0.02) 0.36 (0.03) 0.63 (0.04) 0.08 (0.02) 0.05 (0.01) 0.16 (0.06) 0.04 (0.04) 0 (0) 0.09 (0.01) 48 h 0.51 (0.04) 0.34 (0.02) 0.63 (0.03) 0.06 (0.03) 0.05 (0.01) 0.13 (0.01) 0.03 (0.03) 0 (0) 0 (0) 72 h 0.49 (0.05) 0.28 (0.03) 0.60 (0.05) 0.03 (0.02) 0.03 (0.01) 0.10 (0.03) 0.01 (0.02) 0 (0) 0 (0) 7 d 0.49 (0.02) 0.20 (0.05) 0.59 (0.03) 0.03 (0.03) 0 (0) 0.05 (0.03) 0.01 (0.01) 0 (0) 0 (0) 14 d 0.53 (0.07) 0.08 (0.04) 0.56 (0.04) 0.04 (0.03) 0 (0) 0.04 (0.02) 0 (0) 0 (0) 0 (0) 21 d 0.57 (0.07) 0 (0) 0.52 (0.08) 0.04 (0.03) 0 (0) 0.05 (0.01) 0 (0) 0 (0) 0 (0) (b) Th0-Th10.002 <0.001 <0.001 0.008 <0.001 <0.001 0.152 0.602 0.323 Th0-Th4h 0.001 <0.001 <0.001 0.002 <0.001 <0.001 0.009 <0.001 0.46 Th0Th8h <0.001 <0.001 <0.001 0.002 <0.001 <0.001 0.02 <0.001 0.052 Th0-Th12h <0.001 <0.001 <0.001 0.001 <0.001 <0.001 0.035 <0.001 <0.001 Th0-Th16h <0.001 <0.001 <0.001 0.001 <0.001 <0.001 0.001 <0.001 <0.001 Th0-Th24h <0.001 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Th0-Th48h <0.001 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Th0Th72h <0.001 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Th0-Th7d <0.001 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Th0-Th14d <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Th0-Th21d <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Polymers 2020,12, 1290 6 of 17 Polymers 2020, 12, x FOR PEER REVIEW 7 of 19 Figure 1. Degradation profile by % thickness loss analysis of the three experimental membranes (Derma Fina, Evolution Standard and Duo-Teck) over immersions periods up to 21 days in: (a) PBS, (b) trypsin and (c) collagenase from Clostridium histolyticum. Values shown are mean and standard deviation (n = 3). Significant differences between membranes within the same immersion solution are noted by low-case letters. Differences between different immersion solutions within the same membrane are pointed out with capital letters. Multiple comparisons were performed by Student-Newman-Keuls (p < 0.05). Figure 1. Degradation profile by % thickness loss analysis of the three experimental membranes (Derma Fina, Evolution Standard and Duo-Teck) over immersions periods up to 21 days in: ( a ) PBS, ( b ) trypsin and ( c ) collagenase from Clostridium histolyticum. Values shown are mean and standard deviation (n=3). Significant differences between membranes within the same immersion solution are noted by low-case letters. Differences between different immersion solutions within the same membrane are pointed out with capital letters. Multiple comparisons were performed by Student-Newman-Keuls (p<0.05). Polymers 2020,12, 1290 7 of 17 3.1. Thickness Evaluation after PBS Degradation Assay At 4 h time-point, Duo-Teck attained lower loss of Th values tan both Derma Fina and Evolution Standard, which performed similarly. The trend was as follows Derma Fina =Evolution Standard >Duo-Teck . After 12 h of storage, the three membranes performed similar with the trend Derma Fina =Evolution Standard =Duo-Teck. After 16 h of storage, Derma Fina attained the lowest and Evolution Standard the highest loss of Th values, respectively, and Duo-Teck reached an intermediate performance between both. The trend was as follows Evolution Standard ≥Duo-Teck ≥Derma Fina (Figure 1a). After 48 h and 72 h of storage, the highest loss of Th was attained by Duo-Teck which totally degraded. Derma Fina showed the lowest loss of Th values. The trend was as follows: Duo-Teck >Evolution Standard >Derma Fina. At 7, 14 and 21 d all samples followed the same trend: i.e., Duo-Teck >Evolution Standard >Derma Fina (Figure 1a). In general terms, Duo-Teck totally degraded after 48 h of storage. Both Derma Fina and Evolution Standard membranes had an ascending and parallel loss of thickness over time, but differentiated from 24 h until 21 d. The loss of Th in the three membranes was significantly increasing according to the different time-points of the study when compared with the initial thickness, except Duo-Teck that attained significant differences after 12 h of storage (Table 1). 3.2. Thickness Evaluation after Trypsin Degradation Assay At 4 h time-point, both Derma Fina and Duo-Teck membranes reduced their percentage thickness (Th) similarly, whose values were lower than those of Evolution Standard. Similar performance was followed after 16 h and 7 d of storage, but with different percentages values (Figure 1b). At 14 d time point, the membranes performed as follows respect to the loss of Th: Duo-Teck >Evolution Standard >Derma Fina. This outcome indicated that Duo-Teck completely disappeared after immersion in trypsin degradation solution. Similar performance was followed at 21 d of storage, with a similar percentage Th loss (Figure 1b). In general terms, both Evolution Standard and Duo-Teck membranes suffered and ascending loss of Th over time, more accentuated in Duo-Teck, which totally disappeared at 14 d time-point. As early as 16 h of storage, Evolution Standard attained significant differences over time, when all time-points were compared with the initial time. Duo-Teck started to loose thickness after 24 h time-point until the end of the study (Table 1). Degradation of Derma Fina reproduced a mild parabolic track in degradation. From 8 h until 21 d, the loss Th values were significant when compared with the initial time (Table 1). 3.3. Thickness Evaluation after C. histolyticum Collagenase Degradation Assay At 4 h of storage, the membranes performed as it follows respect to the loss of Th: Evolution Standard >Duo-Teck >Derma Fina (Figure 1c). After 7 d of storage, both Evolution Standard and Duo-Teck completely degraded as the loss of Th was complete at both. Derma Fina showed the lowest loose of Th values and the trend was as follows: Derma Fina <Evolution Standard =Duo-Teck. Similar performance was followed at 14 d time-point but with different percentage. At 21 d time-point, all membranes completely degraded as the loss of Th was complete for the three membranes (Figure 1c). In general, Duo-Teck totally degraded after 8 h of storage. Both Derma Fina and Evolution Standard membranes had a parallel degradation over time, but Evolution Standard disappeared after 7 d and Derma Fina at 21 d time-points (Table 1). The loss of Th in the three membranes was significantly increasing at the different time-points of the study, when compared with the initial thickness, except Duo-Teck thickness loss that attained significance at 4 h of storage (Table 1). The weight (W) values in g of the three membranes (Derma Fina, Evolution Standard and Duo Teck) submitted to the three different degradation tests (PBS, trypsin and C. histolyticum collagenase) as a function of immersion time are reflected in Table 2and Figure 2. Polymers 2020,12, 1290 8 of 17 Table 2. ( a ) Weight ( µ g) analysis of the three experimental membranes (Derma Fina, Evolution Standard and Duo-Teck) after immersion periods up to 21 days in PBS (Hydrolytic degradation test), collagenase from Clostridium histolyticum (Bacterial collagenase resistance test) and trypsin (Enzyme resistance test). Values shown are mean and standard deviation. ( b ) Attained pvalues after pairwise comparisons between membranes’ weights after the different immersion times and the initial weight (weight at t0: W0). Significance was considered at p≤0.001. (a) Derma Evolution Duo-Teck Trypsin C. hystolyticum PBS Trypsin C. hystolyticum PBS Trypsin C. hystolyticum PBS t055.47 (4.04) 57.49 (1.54) 59.69 (1.13) 15.52 (3.19) 14.78 (0.70) 16.04 (0.13) 8.83 (0.40) 9.35 (0.52) 8.9 (0.28) 4 h 53.25 (3.71) 55.58 (1.61) 58.62 (1.17) 14.42 (2.96) 13.67 (0.44) 15.89 (0.10) 7.83 (0.35) 6.28 (0.31) 8.70 (0.17) 8 h 53.19 (3.68) 53.02 (0.68) 58.34 (1.33) 14.67 (2.78) 12.30 (0.46) 15.8 (0.04) 7.60 (0.54) 0 (0) 8.34 (0.09) 12 h 51.95 (3.60) 49.32 (0.39) 56.45 (1.11) 14.89 (2.86) 10.94 (0.47) 15.27 (0.12) 7.20 (0.14) 0 (0) 6.53 (1.20) 16 h 52.42 (3.64) 47.90 (0.15) 57.64 (1.24) 14.08 (2.98) 9.04 (0.07) 15.41 (0.08) 3.88 (0.75) 0 (0) 6.92 (0.79) 24 h 52.21 (3.62) 44.87 (0.04) 57.11 (1.47) 14.04 (3.06) 7.79 (0.35) 15.67 (0.07) 1.16 (1.27) 0 (0) 6.47 (0.45) 48 h 53.27 (3.79) 41.93 (0.06) 58.25 (1.78) 14.02 (3.27) 6.33 (0.79) 15.6 (0.17) 1.02 (1.12) 0 (0) 0 (0) 72 h 52.73 (3.60) 34.89 (0.49) 56.66 (1.02) 12.95 (3.45) 3.63 (1.27) 13.58 (0.55) 0.97 (1.06) 0 (0) 0 (0) 7 d 52.96 (3.64) 24.34 (1.00) 56.24 (0.68) 11.87 (3.81) 0 (0) 12.13 (0.13) 0.98 (1.07) 0 (0) 0 (0) 14 d 52.88 (3.69) 5.67 (0.80) 54.33 (1.20) 11.26 (3.23) 0 (0) 11.93 (0.07) 0 (0) 0 (0) 0 (0) 21 d 52.89 (3.70) 0 (0) 51.51 (1.55) 10.99 (2.88) 0 (0) 11.66 (0.06) 0 (0) 0 (0) 0 (0) (b) W0-W4h <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.01 W0-W8h <0.001 <0.001 <0.001 <0.001 <0.001 0.003 <0.001 <0.001 0.044 W0-W12h <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.002 W0-W16h <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 W0-W24h <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 W0-W48h <0.001 <0.001 <0.001 <0.001 <0.001 0.015 <0.001 <0.001 <0.001 W0-W72h <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 W0-W7d <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 W0-W14d <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 W0-W21d <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Polymers 2020,12, 1290 9 of 17 Polymers 2020, 12, x FOR PEER REVIEW 11 of 19 Figure 2. Degradation profile by % weight loss analysis of the three experimental membranes (Derma Fina, Evolution Standard and Duo-Teck) over immersion periods up to 21 days in: (a) PBS, (b) trypsin and (c) collagenase from Clostridium histolyticum. Values shown are mean and standard deviation (n = 3). Significant differences between membranes within the same immersion solution are noted by low-case letters. Differences between different immersion solutions within the same membrane are pointed out with capital letters. Multiple comparisons were performed by Student-Newman-Keuls p < 0.05). Figure 2. Degradation profile by % weight loss analysis of the three experimental membranes (Derma Fina, Evolution Standard and Duo-Teck) over immersion periods up to 21 days in: ( a ) PBS, ( b ) trypsin and ( c ) collagenase from Clostridium histolyticum. Values shown are mean and standard deviation (n =3). 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