Evaluation of 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Cross-Linked Collagen Membranes for Guided Bone Regeneration in Beagle Dogs

The purpose of this study was to evaluate the bone regeneration efficacy of an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-cross-linked collagen membrane for guided bone regeneration (GBR). A non-cross-linked collagen membrane (Control group), and an EDC-cross-linked collagen membrane (Test group) were used in this study. In vitro, mechanical, and degradation testing and cell studies were performed. In the animal study, 36 artificial bone defects were formed in the mandibles of six beagles. Implants were inserted at the time of bone grafting, and membranes were assigned randomly. Eight weeks later, animals were sacrificed, micro-computed tomography was performed, and hematoxylin-eosin stained specimens were prepared. Physical properties (tensile strength and enzymatic degradation rate) were better in the Test group than in the Control group. No inflammation or membrane collapse was observed in either group, and bone volumes (%) in defects around implants were similar in the two groups (p > 0.05). The results of new bone areas (%) analysis also showed similar values in the two groups (p > 0.05). Therefore, it can be concluded that cross-linking the collagen membranes with EDC is the method of enhancing the physical properties (tensile strength and enzymatic degradation) of the collagen membranes without risk of toxicity.


Introduction
Implant placement with guided bone regeneration (GBR) is performed to provide sufficient bone volume and quality at implant recipient sites [1]. GBR requires the use of barrier membranes to prevent the penetration of epithelial cells into bone defects and to maintain defect spaces until they are filled with mature newly formed tissues [2][3][4][5]. An ideal barrier membrane should meet physical and biochemical requirements, be biocompatible, have adequate tissue integration ability, cell occlusion capacity, dimensional stability, and be easily manipulated [6][7][8].
The nonresorbable membranes first used to guide bone regeneration were made of polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), or titanium [1,9]. These materials are

Energy-Dispersive X-ray Spectroscopy (EDS) Analysis
An energy-dispersive X-ray spectrometer (EDS) equipped with a silicon drift detector (X-MaxN 80, Oxford, Cambridge, UK) and an energy microanalysis program (Aztec, Oxford, Cambridge, UK), connected to an FE-SEM, was used to determine membrane elemental distributions.

Tensile Strength Test
A universal testing machine (TO-102, Testone, Korea) was used to determine membrane tensile strengths. Samples (10 mm × 40 mm) were immersed in distilled water for 24 h prior to testing. Ten millimeters of each end of the membranes were pegged, and testing was conducted at an elongation rate of 20 mm/min [42].

Degradation Test
To evaluate membrane degradation, membranes (n = 3) were cut to 10 × 10 mm and weighed. The test solution (5 mL), which contained 18 units/mg collagenase, was incubated at 37 • C for 1 h. After 1 h, the membrane samples were put into each solution [39]. After incubating samples for 1, 2, 4, 6, 9, 12, 24, or 48 h at 37 • C, the solution was carefully removed, and samples were freeze-dried for 24 h at −40 • C. The weights of dried samples were measured, and membrane degradations were expressed as percentage (%) weight losses.

Cell Cultures
Human gingival fibroblasts (hGnFs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in Fibroblast Medium (FM; ScienCell Research Laboratories, Carlsbad, CA, USA) in a humidified 5% CO 2 atmosphere at 37 • C. Culture medium was renewed every 48 h.

Proliferation Assay
hGcF proliferative activity was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, cells were seeded at a density of 2000 cells per well in 48-well plates. Control group and Test group membranes were preincubated with 500 µL FM before being seeded with cells. All conditions were evaluated in triplicate. After 24, 48, and 72 h, medium was discarded and replaced with 200 µL FM containing 10% CCK-8 solution, according to the manufacturer's instructions, and were incubated at 37 • C for 2 h in a humidified 5% CO 2 atmosphere. Absorbances at 450 nm were measured using a Benchmark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Immunocytochemistry
The adhesion of hGnFs to membranes 2 h after seeding was observed by immunofluorescence imaging. First, hGnFs were seeded at a density of 2000 cells per membrane in 48-well plates. After 2 h, the cells were washed twice with phosphate buffered saline (PBS) and fixed in 3.7% formaldehyde solution in PBS for 20 min at room temperature. Cells were then washed three times with PBS, incubated in PBS containing 0.1% TritonX-100 (Sigma, St. Louis, MO, USA) for 5 min, washed three times with PBS, mounted on slide glasses on membranes, and incubated with 100 nM rhodamine phalloidin (Invitrogen, Corp., Carlsbad, CA, USA) for 30 min. After washing with PBS, cells were incubated with 10 µg/mL 4 ,6-diamidino-2-phenylindole (DAPI; Invitrogen, Corp., Carlsbad, CA, USA) for 10 min. Images were acquired using a Nikon Eclipse TE2000-E confocal microscope (Nikon, Tokyo, Japan).
Implant surgery was performed 2 months after extraction. A crestal incision and a vertical releasing incision were made at edentulous sites. Three rectangular bone defects (8 mm wide, 5 mm high, 5 mm deep) were prepared under saline irrigation. A total of 36 implants (diameter: 4 mm, length: 8 mm, Cowell Medi Co, Ltd., Busan, Korea) were inserted with 3 threads exposed ( Figure 1a). All peri-implant defective sites were grafted with porcine-derived xenografts (Bone-XP, MedPark, Busan, Korea) (Figure 1b), and then randomly covered with either Control group or Test group membranes ( Figure 1c). Surgical sites were sutured with 4-0 Vicryl (Mersilk, Ethicon Co., Livingston, UK). Before surgery, each dog was injected with 20 mg/kg of antibiotic cefazolin sodium (Chong Kun Dang, Seoul, Korea), and after surgery with 10 mg/kg of methylprednisolone succinate sodium (Reyon Pharm, Seoul, Korea). Post-operative treatments were orally administered for 2 weeks, that is, amoxicillin-clavulate (Amocla, Kuhnil Pharm, Chungnam, Korea) as an antibiotic, firocoxib (Previcox, Merial, France) 5 mg/kg as an anti-inflammatory, and 0.5 mg/kg famotidine (Nelson famotidine, Nelsonkorea, Seoul, Korea) for gastrointestinal protection. All dogs were monitored daily, and sutures were removed one week after surgery. Dogs were sacrificed at 8 weeks after surgery, and mandibles were carefully harvested and fixed in formalin (Sigma Aldrich Co., St. Louis, MO, USA) for 2 weeks.

Micro-Computed Tomography (µCT) Analysis
Harvested mandibles were prepared for micro-CT analysis to determine bone volumes in the regions of interest (ROIs), 1 mm around the implant excluding the base of the implant. All specimens were scanned at 130 kV and 60 µA using a micro-CT scanner (Skyscan-1173, ver. 1.6, Bruker-CT Co., Kontich, Belgium) and a pixel size of 24.15 µm. All images were reconstructed using Nrecon reconstruction software ver. 1.7.0.4 (Bruker-CT Co., Kontich, Belgium).
To make polymerized specimen blocks, specimens were fixed on a frame and then embedded using the UV embedding system (Exakt 520, Kulzer), according to the manufacturer's instructions. Polymerized blocks were sectioned at implant centers using a diamond cutter (KULZER EXAKT 300 CP Band System, Exakt Apparatebau, Norderstedt, Germany). Sectioned specimens were ground to 30 µm using a grinding machine (KULZER EXAKT 400CS, Exakt Apparatebau, Norderstedt, Germany), mounted on slides, and stained with hematoxylin and eosin (H&E). Images of stained specimens were captured using a computer connected to a light microscope (Olympus BX, Olympus, Tokyo, Japan). Percentage New Bone Areas (NBA%), Inter-Thread Bone Densities (ITBD%), and percentage Bone-Implant Contacts (BIC%) were determined using i-solution (IMT, Daejeon, Korea) by a single investigator ( Figure 2).

Statistical Analysis
The Mann-Whitney U test was used to analyze in vitro and in vivo results. Results are presented as means ± standard deviations (SD) and the analysis was conducted using SPSS ver. 25.0 (SPSS Inc, Chicago, IL, USA). Statistical significance was accepted for p values < 0.05.

Morphological Findings
FE-SEM images of cross-sections and surface morphologies of Control group and Test group membranes are shown in Figure 3. Both membrane types showed similarly shaped contacts with outer (contact with soft tissue) and inner surfaces (contact with the hard tissue), respectively, but the surface density of the Test group was higher than that of the Control group (Figure 3c,e,g,i). Both groups were interwoven individual collagen fibers that formed irregular collagen strands (Figure 3d,f,h,j).  ×200 (a,b); ×500 (c,e,g,i); ×5000 (d,f,h,j). Control is represented by non-cross-linked collagen membrane; Test is represented by EDC-cross-linked collagen membrane.

Tensile Strength Test
Means ± standard deviations (SD) of tensile strengths of the Control group and Test group membranes were 11.46 ± 1.90 and 16.70 ± 2.43, respectively (Table 2), and these values were significantly different (p < 0.05). Control is represented by non-cross-linked collagen membrane; Test is represented by EDC-cross-linked collagen membrane.

Enzymatic Degradation Test
The results of enzyme resistance testing are shown in Figure 4. Control group membranes continuously degraded during testing and disappeared at 48 h, whereas Test group membranes degraded more slowly until 24 h and then degraded more rapidly. At 48 h, Test membranes were partially degraded. These results showed that the Test group membrane was significantly more resistant to enzymes than the Control group membrane (p = 0.043).

Proliferation Assay
One day after seeding, hGnF cells appeared to have attached to the membrane surfaces. Cell proliferations on Control group and Test group membranes were similar at all times (p > 0.05) ( Figure 5).

Immunofluorescent Staining
Immunocytochemical staining of total actin and DAPI revealed that cells adhered equally to both membranes 2 h after seeding ( Figure 6).

Clinical Findings
All six experimental animals survived, and no infection or inflammation was observed at surgical sites. The 36 implants were collected without issue.

µCT Findings
Well-formed bone was observed in both groups. Bone volume results are summarized in Table 3. Mean (±SD) bone volumes (%) in the Control and Test groups were similar (62.19 (±9.97) and 60.98 (±10.02), respectively).

Histological Findings
The histological specimens are shown in Figure 7. In both groups, no adverse reaction was observed (Figure 7a,d). Both membrane types partially survived for 8 weeks in situ (Figure 7b,e), and good healing with new bone formation was observed in both groups (Figure 7c,f).

Discussion
Ideal membranes can prevent foreign body response and epithelial cell invasion [15], and ideal membranes can remain in situ until enough periodontal tissue and bone have regenerated [43]. Moses et al. [44] reported that cross-linked collagen membranes better assist bone healing by withstanding enzymatic degradation during the bone regeneration period. EDC is one of the most widely used agents to cross-link collagen membranes. Park et al. [39] showed that the EDC-cross-linked membranes have lower cytotoxicity and greater enzymatic degradation resistance than their non-cross-linked counterparts in vitro. Others have demonstrated the biocompatibility of cross-linked collagen membranes [38,45,46].
Appropriate mechanical properties enable membranes to survive in vivo and to supply the stress needed to encourage early tissues to differentiate into pre-osteoblasts [15,31]. In the present study, EDC-cross-linked membranes had greater tensile strengths than non-cross-linked controls ( Table 2) (p < 0.05), which is consistent with the results of previous studies [39,40,47,48].
Enzymatic degradation resistance is another important contributor to surgical success [49]. During the immediate postoperative period, membrane collapse and degradation can adversely affect bone formation [50]. Therefore, to achieve satisfactory GBR results, membrane degradation rates must be regulated [50]. We found that cross-linked membranes better resisted enzymatic degradation ( Figure 4). Non-cross-linked membranes degraded steadily after implantation, whereas cross-linked membranes remained relatively intact at 20 days postoperatively. This result is in line with those of previous studies [40,47,49], and shows that EDC-cross-linking strengthened interactions between collagen molecules and increased structural integrity [50,51]. As our animal experiments were conducted using a short follow-up of 8 weeks, it was difficult to confirm the difference between the in vivo biodegradabilities of the two membranes histologically. We suggest this be further investigated by longer-term study.
The cytotoxicities of chemically cross-linked membranes are determined by the presence of residual chemical species [39], and thus, post-treatments such as washing, and evaporation are used to reduce the risk of cytotoxicity [40]. In our in vitro studies, we used human gingival fibroblasts (hGnFs), which are the main interstitial cells and important for maintaining the original shape and function of gums, and have also been used to evaluate cell proliferation on different membrane types [52]. In the present study, cell viability results confirmed that cross-linked and non-cross-linked membranes performed similarly in terms of cell adhesion and proliferation ( Figure 5). In addition, immunocytochemistry showed that hGnF cells adhered equally well to both membranes ( Figure 6). Furthermore, our cytotoxicity investigation showed cross-linked and non-cross-linked membranes were both noncytotoxic, and these results concurred with previous studies [53,54]. In addition, through the EDS analysis, it can be confirmed that both membranes (cross-linked and non-cross-linked membranes) have similar chemical compositions ( Table 2). This result appears to be that the chemical components of EDC have been removed by washing because EDC does not remain as part of the bond and turns into a water-soluble urea derivative with low cytotoxicity [39,55]. Therefore, as previously reported [39,47], we believe that EDC-cross-linked collagen membranes do not raise cytotoxicity concerns.
The histological results of bone defects around implants showed that both membranes integrated well with surrounding tissues and achieved good osseointegration (Figure 7). In addition, no membrane exposure was observed at any animal, and all defects healed without complications. Histomorphometric and micro-CT analyses showed that cross-linked and non-cross-linked membranes had similar bone regeneration abilities (Tables 3 and 4). These results are contrary to a previous study in which the cross-linked collagen membrane showed a relatively high bone regeneration rate compared to the non-cross-linked collagen membrane [30]. However, the previous study was conducted with a larger-size defect (6 × 6 × 6 mm and 9 × 9 × 9 mm) than that applied in this study (8 × 5 × 5 mm) [30]. In addition, the results of the previous study have shown that the difference in bone regeneration rates between the cross-linked collagen membrane and the non-cross-linked membrane at the relatively wide defect size (9 × 9 × 9 mm) tended be relatively higher than that of the narrow defect size (6 × 6 × 6 mm) [30]. Therefore, it is considered that the defect size applied in this study was not sufficient for the beneficial effect on bone regeneration of the cross-linked collagen membrane. In addition, the previous study had a healing period of 16 weeks [30]. Thus, it is expected that additional studies with various defect-size models and a long healing period are needed. Notably, the origins of these two collagen membranes differed, that is, Colla-D ® (cross-linked) was composed of bovine collagen, and Bio-Gide ® (non-cross-linked) was composed of porcine collagen. Bio-Gide ® was selected as controls because previous studies have reported good results in terms of biocompatibility and bone regeneration [30,38,56,57], and because it is the most widely used resorbable membrane [11].

Conclusions
Given the limitations of the present study, we conclude that: (i) EDC-cross-linked collagen membranes are not cytotoxic; (ii) EDC-cross-linked collagen membranes have better mechanical properties and enzymatic degradation resistance than non-cross-linked collagen membranes; and (iii) EDC-cross-linked and non-cross-linked collagen membranes similarly aid bone regeneration. Therefore, the EDC-cross-linking of collagen membranes can be considered a means of improving membrane physical characteristics for guided bone generation without risk of toxicity.