Journal of Dental Sciences

INTRODUCTION

Based on the application of a barrier membrane to cover an osseous defect, Guided Bone Regeneration (GBR) has become a widely used bone augmentation technique in oral and maxillofacial surgery [1, 2]. Naturally collagen-derived membranes, due to collagen being a principal component of connective tissue with excellent biocompatibility, have garnered significant interest [1, 3]. However, certain studies have reported that the collagen membranes have unfavorable mechanical properties, such as rapid degradation by collagenases, which compromises their barrier function [4, 5]. Therefore, it is of great importance to promote their degradation behavior while preserving barrier functionality and promoting tissue integration [6].

Consolidation techniques, such as chemical cross-linking, have been used to enhance the stability of collagen membranes. However, these techniques have cytotoxicity and are associated with poorer tissue integration and delayed vascular invasion6. Previous studies have demonstrated that the choice of donor organisms (e.g., allogeneic or xenogeneic sources) and, more importantly, the tissue sources (e.g., dermis, pericardium, or tendon tissue) of collagen-based biomaterials significantly impact their physicochemical properties, integration behavior, and vascularization in a specific clinical context [7, 9].

It is known that porcine dermis-derived collagen membranes are most often prematurely resorbed in 4-8 weeks, but it has been shown that they are “optimally” degraded via more or less physiological processes providing a good biocompatibility [10]. However, some scholars suggest that ideal GBR membranes should maintain its barrier function for 16-24 weeks to meet the requirement of different bone augmentation methods [11]. Researches have shown that collagen membranes derived from pericardium exhibited longer degradation time and enhanced barrier properties compared to those derived from dermis [8, 12]. Natural porcine-based pericardium membranes typically resorbed within 8 to 12 weeks in a dog model [12]. On the other hand, pericardium membranes from bovine had a resorption profile of 4 to 6 months [13]. However, bovine- based pericardium membranes exhibited delayed vascular penetration, which may impede bone regeneration [12, 14]. Nevertheless, some studies have reported positive effects of bovine pericardium bioavailability in enhancing bone reinforcement [15, 16]. Moreover, bovine-derived membranes have lower immunogenicity compared to porcine-derived collagen membrane, with only 3% of the population being allergic [17]. Thus, understanding the differences in tissue reaction to these materials can help in customizing composite materials for various applications in hard and soft tissue regeneration, based on clinical requirements such as fast vascularization and rapid degradation versus slow vascularization and tissue integration within the implantation bed [10].

However, for collagen-based membranes, only a few studies have analyzed the differences in various xenogeneic sources, and scarce knowledge exists about the consequences of the usage of different animal sources [18, 19]. Most of the membranes used in GBR procedures are based on porcine donor tissue. Fewer barrier membranes are used that originated from other animals or tissue origin [20]. To fill this knowledge gap, we applied three xenogenic collagen materials made from Bovine Pericardium (BP), from Bovine Dermis (BD) and from Porcine Dermis (PD) in this study. These membranes were implanted over a period of up to 16 weeks by means of an established cranial bone defect model in SD rats. The collagen-induced tissue reactions as well as their tissue integration patterns that involve cellular infiltration, vascularization, degradation and bone regeneration were investigated used by histological, immunohistochemical and bone histomorphometrical analysis in vivo.

MATERIALS AND METHODS

Material

This study utilized three representative commercial collagen membranes: 1) Bovine-sourced Pericardium membrane (BP, Megreen®, Shaanxi Reshine Biotech Co., Ltd, China); 2) Bovine-sourced Dermis membrane (BD, Heal-All®, Yantai Zhenghai Biotechnology Co., Shandong, China); and 3) Porcine-sourced Dermis membrane (PD, Bio-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland). All three collagen membranes underwent similar preparation processes and were used as received, without any further modifications.

Materials characterization

The microstructure of the three membranes was observed under a Field-Emission Scanning Electron Microscope (FE-SEM, SU8010, Hitachi, Japan). Optical images were visualized by a stereo microscope (S9I, Leica, Germany) to show the surface morphology of the three membranes. The surface roughness, denoted as the arithmetic mean deviation of the profile (Ra), was calculated using a white-light interference microscope (NT9100, Veeco, America) (n = 30). Infrared spectra were collected using a Fourier Transform Infrared Spectroscopy (FTIR, Nicolet iS50, Thermo Fisher Scientific, USA) from 400 to 4000 cm^-1.

The water contact angle was measured at the moment a water droplet made contact with the sample using a contact angle goniometer (JC2000C, POWEREACH, China), and the average value was recorded. And the duration for a sample to absorb one droplet of water was recorded (n = 3).

The water absorption was determined by weighing the three materials before (Wdry) and after (Wwet) immersion in Milli-Q water for 5 seconds (n = 4). Water absorption rate = (Wwet _Wdry)/Wdry. Wwet is the weight of the wet sample, while Wdry is the weight of the dry sample pre-weighed prior to immersion in Milli-Q water [21].

A hydroxyproline assay kit (A030-2-1, Nanjing Jiancheng Bioengineering Institute, China) was used to quantify the collagen content in the residual materials (n =3) after being incubated for 0, 5, 10, 15, 20 or 25 min. Degradation ratio was calculated based on the OD values using the equation: Degradation Rate= (ASample- ABlank)/(AStandard-ABlank) *Cstrandard * VHydrolysis/WSample.

Where:

A Sample is the OD value of the sample's hydrolyzed supernatant. ABlank is the OD value of the blank's hydrolyzed supernatant.

AStandard is the OD value of the standard's hydrolyzed supernatant. Cstrandard is the concentration of the standard (5 µg/ml).

VHydrolysis is the total volume of the added hydrolysis solution.

WSample is the initial weight of the three membranes, which generally ranged between 13 and 15 mg.

Rat Cranial Defect Model

All experiments were conducted following the guidelines of the Institutional Animal Care and Use Committee (IACUC), ZJCLA, Hangzhou, China (Approval No. ZJCLA-IACUC-20030064). Male Sprague-Dawley (SD) rats (approximately 180-250 g) received intraperitoneal injections of sodium pentobarbital (50 mg/kg) and local lidocaine injections before surgery. After shaving and disinfection of the surgery area, the top of the rat skull was exposed and received a circular incision just before the sagittal suture on the left side. A cranial bone defect reaching the dura mater was then established using a circular drill with a diameter of 6 mm under continuous irrigation with physiologic saline solution. Subsequently, defects were stuffed with Gegreen calcined bovine bone (Shaanxi Reshine Biotech Co., Ltd, China) pre-immersed in physiologic saline solution and covered with the membranes of BP, BD, and PD, respectively [Fig. 2]. The PD membrane was as the control group. Postoperatively, penicillin (100,000 U/d) was administered intramuscularly to prevent infection. Housing and husbandry conditions were approved by IACUC.

A total of 93 SD rats participated in the experiment and then randomly divided into three groups, with 5 rats per group at each time point (except for the 8-week time point, 6 rats per group). SD Rats were included in the study if they were healthy. At 16 weeks post-implantation, a total of 3 rats died naturally, comprising 2 from the PD group and 1 from the BP group. Rats were euthanized at designated time points for tissue analysis. Tissue healing on the smooth surface of the membranes was photographed using a digital camera. Cranial bones with substitute materials were then removed, dissected, fixed in 10% buffered formaldehyde and submitted to a 10% Ethylene Diamine Tetraacetic Acid (EDTA, Servicebio, China) for 3 weeks. The decalcified specimens were dehydrated in graded series of ethanol, then embedded in paraffin. Sections of 3-5 µm thick were cut for subsequent staining analysis.

Histological analysis

On week 1, 2, 4, 8, 12 and 16 after implantation, the specimens of each material were sectioned to prepare decalcified slices. Sections were used for histochemical staining, i.e., hematoxylin and eosin (H&E). All slices were analyzed by using a light microscope (BX53, Olympus, Japan).

The histological analysis was carried out according to the previous study [22]. The parameters included the infiltrating inflammatory cells (i.e., monocyte/macrophages, granulocytes, lymphocytes, Multinucleated Giant Cells (MNGCs), fibroblasts, osteoblasts and connective tissue invasion on the different surfaces of membrane, so as to assess its histocompatibility and barrier function.

Moreover, the membrane's thickness was measured with ImageJ 1.53a software at the different time points. The membrane thickness was measured 3 times at different spots for each slide, using straight lines that were perpendicular to the membrane. And the number of samples in each group is 5-6.

Immunohistochemical analysis

In weeks 1, 2, and 4 after implantation, sections of each material were subjected to immunohistochemical staining for the CD31 marker, which is widely recognized as a specific indicator of endothelial cells. All slices were examined using a light microscope (BX53, Olympus, Japan). The vessel counting method was adapted from Said Alkildani [23], modifying the field of view from per mm² to 100x magnification. The intra-membrane vessel density (vessels per 100x) was quantified by counting the number of vessels in two randomly selected fields from each sample at 100x magnification, with five samples from each group analyzed using ImageJ software. Single endothelial cells or clusters of endothelial cells positive for CD31 was considered as individual vessels. The cells poorly resolved or stained were not counted.

Micro-computed tomography (Micro-CT) analysis

At the 8 and 12 weeks after surgery, the cranial bones were harvested and fixed by 4% paraformaldehyde (n = 6). The specimens were scanned using Micro-CT (U-CT- XUHR, Milabs, Netherlands) at high resolution. Images were reconstructed into a three-dimensional structure. New bone volume fraction (Bone Volume/Votal Volume, BV/TV) was calculated in the total defect area (diameter of 6 mm, height of 2 mm) and the area adjacent to the membrane (diameter of 6 mm, height of 0.5 mm), respectively. Further, the proportion of bone formation adjacent to the membrane to the total bone formation in the defect area was evaluated.

Data analysis

Quantitative results were presented as mean ± standard deviation. In SPSS 25.0 software, the data were checked for normality (Shapiro-Wilk test) and homogeneity (Levene test). If both matched, statistical significance was determined by one-way analysis of variance (ANOVA) followed by LSD post-hoc multiple tests. If the data do not conform to the normality or do not conform to the homogeneity of variance, the nonparametric test (Kruskal-Wallis test) was used, and the multiple tests were corrected by Bonferroni to determine statistical significance. Values of p < 0.05 were considered statistically significant. The sample size per group at each time point in this study was similar to those generally employed in the field [6, 24] and was not pre-determined by a sample size calculation. Potential confounders (e.g., animal/cage location) were not controlled.

Results

Physico-chemical characteristics

The cross-sectional structure of the membranes was observed using FE-SEM [Fig. 1]. The results showed all membranes had a bilayer structure with smooth surface on the top and relative rough surface on the bottom. The cross-sectional analysis of the PD membrane revealed a more pronounced bilayer structure compared to the BP and BD membranes [Fig. 1]. The rough surfaces of all three groups displayed gross undulations and a fibrous texture [Fig. 1], while the smooth surfaces exhibited a less exaggerated topography [Fig. 1]. It is noteworthy that the smooth surfaces of the BP and PD membranes manifested a higher density in comparison to that of the BD membrane, indicating a potentially superior barrier function for the BP and PD membranes [Fig. 1]. Optical images and roughness measurements supported the FE-SEM results, showing a lower Ra value for the smooth surfaces of the BP and PD membranes in comparison to the BD membrane [Fig. 1]. On the other hand, the PD membrane had the highest Ra value on the rough surface, suggesting a more spatially loose filamentous structure.

In Figure 1C, the BD membrane demonstrated a more rapid degradation rate in vitro. After 5 minutes of enzymatic digestion, the BD membrane became invisible, and was completely degraded after 15 minutes, a timeline comparable to that of the PD membrane. In comparison, the BP membrane underwent a slower degradation process, showing ongoing degradation even beyond the final time point [Fig. 1].

FTIR was used to assess the integrity of the collagen triple helical structure in all samples. The peaks corresponding to amide A (3300 cm^-1), I (1630 cm^-1), II (1545 cm^-1), and III (1235 cm^-1) remain unchanged, indicating that the processing methods for all three membranes did not compromise the collagen structure [Fig. 1].

Additionally, the typical amide B band of collagen of the BP and BD membranes was observed at 2919 and 2939 cm^-1, respectively. Notably, the FTIR spectra obtained from the BD membrane showed a distinct peak at a wavelength of 2116 cm^-1. Contact angle measurements in Figure 1E-F showed that the BP membrane exhibited the highest contact angle and required the longest duration to absorb a water droplet compared to the PD membrane, indicating diminished hydrophilicity.

Despite this, the PD and BP membranes had significantly higher water absorption rates than the BD membrane, implying enhanced water retention capabilities for the PD and BP membranes.