Journal of Dental Implantology - http://www.implantoloji.info
Collagen Membrane Resorption in Dogs: A Comparative Study
http://www.implantoloji.info/articles/6/1/Collagen-Membrane-Resorption-in-Dogs-A-Comparative-Study/Page1.html
By JDI editor
Published on 01/15/2001
 
Kris W. Owens, DDS, MS
Postgraduate Periodontics Student, Dept. of Periodontics, Louisiana State University School of Dentistry, New Orleans, LA, USA.

Raymond A. Yukna, DMD, MS
Professor and Coordinator Postgraduate Periodontics, Dept. of Periodontics, Louisiana State University School of Dentistry, New Orleans, LA, USA.


Guided tissue barriers using materials such as collagen are used in the hope of excluding epithelium and the gingival corium from the root surface or alveolar bone to facilitate regeneration. Convention suggests that the longer a membrane remains intact, the better the regeneration results. The purpose of this study was to determine the resorption rates of various collagen membranes in the oral cavity of dogs. Twelve adult mongrel dogs had three different collagen membranes (BioGide, AlloDerm porcine-derived, and Allo-Derm human-derived) randomly inserted and secured into surgical pouches made in their palates. Full-thickness tissue punch biopsy specimens taken at 1, 2, 3, or 4 months after surgery were evaluated histologically for membrane intactness and other associated changes. At 1 month, all membranes had slight to moderate deg radation. At 2 months, all membranes had moderate to severe degradation with the exception of one AlloDerm human-derived membrane that was intact. At 3 months, all membranes had severe degradation to not identifiable. At 4 months, all membranes had severe degradation to completely absent. Blood vessel penetration varied from none to moderate. Inflammation was found in only two samples. In the dog, all three tested collagen membranes showed slight to moderate degradation at 1 month and were severely degraded to completely absent at 4 months. Within the limits of transferring animal data to humans, clinicians need to be aware of these resorption rates when selecting membranes for guided tissue and bone regeneration.

Introduction.
Kris W. Owens, DDS, MS
Postgraduate Periodontics Student, Dept. of Periodontics, Louisiana State University School of Dentistry, New Orleans, LA, USA.

Raymond A. Yukna, DMD, MS
Professor and Coordinator Postgraduate Periodontics, Dept. of Periodontics, Louisiana State University School of Dentistry, New Orleans, LA, USA.


The use of tissue compartment separating barriers/membranes in periodontal, ridge augmentation, and dental implant therapy is based on the biologic principles of guided tissue regeneration (GTR). This concept is based on the assumption that only the periodontal ligament cells and/or bone cells have the potential for regeneration of the attachment apparatus of the tooth. Barrier membranes are used to separate tissue compartments during healing so that the desired tissues/cells (periodontal ligament in periodontal therapy; bone in ridge augmentation and implant therapy) occupy the space provided first. Excluding the epithelium and gingival connective tissue from the area during the postsurgical healing phase favors repopulation of the area by the periodontal ligament and/or bone cells. This has been histologically shown to occur to some degree in animals and in humans.
Clinical results with GTR barriers have been quite favorable.12–19 The type of barrier used may not be as important as simply having a barrier in place. Guided tissue and bone regeneration membranes are classified into two major groups, nonresorbable and resorbable. Nonresorbable membranes include Millipore filters20 and expanded and nonexpanded polytetrafluoroethylene (ePTFE) types.21 Resorbable membranes include polylactic acid22 and collagen-based materials.23–26 Resorbable membranes have advantages such as being biodegradable and not requiring a second surgical procedure to remove them.
Allogeneic collagen membranes have been used in several studies. Freeze-dried dura mater is a human allograft material that is mainly composed of collagen, is devoid of immunogenecity, and has shown favorable regenerative results.24–26 Piattelli et al26 also found that freeze-dried dura mater resorbs slowly, and even at 12 months, it still played a role in cell occlusion.
Several investigators have examined nonhuman type I collagen as a possible membrane barrier for use in GTR procedures.22,27–35 Collagen has several advantages because it is absorbable, does not require a second surgical procedure for removal, and has some unique biologic properties. It is the major extracellular macromolecule of the periodontal connective tissue and bone and is physiologically metabolized by these tissues; it is chemotactic for fibroblasts; it has been reported to act as a barrier for migrating epithelial cells in vitro; and it has been used experimentally in animals and humans.27 Collagen is pliable when moist and conforms well to the surgical area, provides a thrombogenic surface that is sealed coronally to the root surface by a fibrin clot, and does not elicit any allergic responses.28 There have been several dental uses for collagen membranes, such as for GTR, GBR, soft-tissue augmentation, and recession treatment.
One issue concerning collagen membranes is whether they act as an intact barrier long enough to have predictable outcomes in periodontal and other oral surgery procedures. Clinical and histologic evaluations demonstrate that premature dissolution of membranes can be detrimental to the formation of cementum and/or bone. When Wilderman36 reviewed the effects of bone exposure in periodontal surgery, he found that the healing sequence included the presence of a fibrin clot beneath the flap and inflammation in the marrow spaces and Haversion canals. Granulation tissue invades the clot at 4 days and bone resorption was observed during days 4 to 8. Apposition of new bone occurred from days 10 to 21. Hiatt et al37 studied repair after mucoperiosteal flap surgery and observed cementum formation as early as 3 weeks, which continued through the sixth month.37 Most research has shown that a GTR membrane must be in place intact for at least 30 days for bone and periodontal ligament cells to fill the space. If the membrane dissolves or fragments before 4 weeks, then it does not accomplish its goal.38,39 Bragger et al40 considered that all alveolar bone adjacent to sites exposed to GTR demonstrated a slow consolidation of the tissues represented by delayed mineralization compared with the changes in probing attachment levels. Sigurdsson et al41 using ePTFE membranes have shown that dogs produce no regeneration at 3 weeks, whereas at 8 weeks, 75% of the bone has repaired together with 40% of the cementum. If collagen barriers are completely resorbed before day 30, new cementum may be found in the healing area, but no new bone.
Miller et al. found that certain cross-linked collagen membranes were resorbed within 2 weeks in rabbits. Sevor et al. evaluated the usefulness of resorbable collagen membranes for regeneration of dehisced alveolar bone adjacent to endosseous dental implants in dog mandibles. They found that the collagen membrane was present 4 weeks after placement; and that after 8 weeks, the membrane was undergoing remodeling and was not clearly delineated, indicating that resorption had occurred.
Manufacturers of several collagen membranes were asked to support this study; only two provided it. The three membranes used in this experiment are processed in different ways. BioGide (BG) (Osteohealth, Shirley, NY) is composed of porcine-derived dermal collagen. It is a bilayer membrane of collagen types I and III. The compact layer protects against soft tissue invasion, whereas the porous layer enables integration of newly formed bone. AlloDerm human-derived (A-H) (LifeCell, Branchburg, NJ) and AlloDerm porcinederived (A-P) (LifeCell) are both allogeneic acellular dermal grafts. A-H is aseptically processed from donated human skin and is a protein framework without any human cells, creating an acellular, biocompatible, human connective tissue matrix. The basement membrane complex is retained to facilitate epithelial migration and attachment.45 A-P is processed in a similar manner but is porcine-derived. Silverstein and Callan46 described the use of A-H for soft-tissue augmentation without having to use the patient’s own palate to procure the donor site. They feel it has tremendous potential clinical advantages, such as improved color and contour match, elimination of multiple surgeries because of unlimited availability, decreased patient morbidity, decreased chairside time, and less postoperative pain than is experienced with palatal autografts.
The manufacturer of BioGide claims that resorption is first observed at the light microscopic level at 4 months, whereas the producer of AlloDerm makes no claims about the resorption rate of its products. Collagen membranes have many potential applications in periodontal regenerative surgery and other oral surgery. They have several advantages such as ease of placement and resorbability. However, there is little comparative information on the resorption rates of collagen membranes in the oral cavity.
The purpose of this study was to determine the rate at which three specific collagen membranes (BioGide, AlloDerm porcine-derived, and AlloDerm human-derived) resorb in the oral cavity of dogs and to examine whether they are present long enough to allow desirable healing events to occur.

Materials and methods.
Twelve young adult mongrel dogs, 35 to 50 kg each, were used in this study. The dogs were acclimated for 10 days before any procedures were performed. Dogs were provided with regular food and water and were exercised during the course of this study, but were placed on a modified soft diet for 2 weeks after surgical procedures were performed.

Insertion of Membranes.
The protocol design and procedures for this study were reviewed and approved by the Louisiana State University Medical Center Institutional Animal Care and Use Committee. All procedures were performed under sterile conditions in an operating room using intramuscular pentobarbital (30 mg/kg) for general anesthesia supplemented with local anesthesia in the form of 2% xylocaine with 1:100,000 epinephrine before any of the surgical procedures. Individual split-thickness flap pouched tunnels 3 mm deep were made over the lateral part of the palate at the first and third rugae posterior to the canine within the connective tissue to provide a good blood supply. Three different membranes (BG, A-P, and A-H) were used, and each dog received a pair of one of the membrane types on opposite sides of their mouth. The tunnel opening was sutured to obtain primary closure.
All dogs received buprenorphine (0.1 mg) im q12 h for pain and 2,000,000 units of bicillin im for 7 days postoperatively. The dogs were evaluated for healing and discomfort every day postoperatively until the palate was healed enough to return to the regular diet.

Biopsies.
According to a presurgically determined treatment code, the dogs were randomly assigned a procedure number, indicating material use and healing times. The treatment code was developed to ensure a balance in materials used, side of jaw treated, and healing times. The following was performed at 1 and 3 or 2 and 4 months, depending on to which group the dog was assigned. Dogs were reanesthetized as above. A biopsy of each tissue-containing rugae was performed using a 4-mmdiameter soft tissue punch (Miltrex Instrument, Lake Success, NY) to obtain a full-thickness cylindrical tissue sample from the surface tissue to the bone. Collagen powder (Avitene, Davol, Cranston, RI) was adapted to the wound for hemostasis. The dogs were evaluated daily postoperatively for healing and placed on a soft diet until the palate was healed enough to return to the regular diet.

Histologic Preparation.
The soft tissue samples were placed in individual vials containing fresh 10% neutral buffered formalin. Sections were mounted so that apical-occlusal cross sections were cut at 6 to 8 mm. A total of 192 sections of 48 biopsies were included in the analysis. Two samples of each biopsy were stained with silver stain (for elastic fiber identification), and two samples of each biopsy were stained with hematoxylin and eosin (for general morphology). Elastic fiber identification aided in distinguishing the membrane from surrounding tissue because those fibers are found in the membranes and very few are found in the palate of the dog.

Histologic Analysis.
Slides were evaluated at 310 magnification using a light microscope (Microstar, American Optical, Buffalo, NY). Two calibrated investigators independently evaluated each slide, and the scores were later compared. If the two investigators were discrepant, they reviewed the slide together to come to a consensus on the rating. The biopsies were compared with a normal dog palate and unused membrane samples. The membrane in each specimen was given a score from 0 to 4 for membrane condition (0 5 intact, easily identifiable; 1 5 slight degradation (,35%); 2 5 moderate degradation (35%–65%); 3 5 severe degradation (.65%); and 4 5 absent, not identifiable). The membrane area in each specimen was evaluated separately for blood vessel penetration, elastic fiber presence, and inflammation. The scoring system used for each of these evaluations was 0 5 none, 1 5 slight, 2 5 moderate, and 3 5 abundant.

Results - Discussion - References.
RESULTS.
Clinical Findings.
Postoperative healing after insertion of the membrane was uneventful in all dogs except two. One dog showed necrosis of all four sites on the palate, whereas another dog showed necrosis on one side of the palate. New membranes were inserted into the second and fourth rugae posterior to the canine using the same surgical techniques described in the Material and Methods section, and subsequent healing was uneventful in those dogs. Evaluation of these later-placed samples were not different, so they were combined with the others.

Histologic Analysis.
Membrane degradation rates were variable at different time periods and among dogs within the same time period. Six samples were unreadable, but their absence did not significantly affect the results. Scores ranged from 1 to 2 for all membranes at the 1-month time period. Scores ranged from 2 to 3 for all membranes at the 2-month time period, except for one A-H membrane that seemed to be intact and was given a score of 0. All membranes were given a score of 3 to 4 at both the 3 month and 4 month time periods, indicating almost total degradation after 3 months.
Blood vessel penetration varied among the samples. Blood vessels were found to be abundant in one of the BG samples at 3 months. Only two samples showed any signs of inflammation.

BioGide.
The BG samples showed slight to moderate degradation at 1 month after insertion. Blood vessels ranged from none to moderate. No inflammation or giant cells were found. The BG samples at 2 months showed slight to moderate degradation. Blood vessels ranged from slight to moderate, whereas only slight inflammation was found in one sample. The BG samples at 3 months showed severe degradation. No inflammation was found in any samples. Blood vessels were found to be abundant in one of the BG samples at 3 months. BG samples at 4 months revealed severe degradation to completely absent. Blood vessels ranged from none to moderate, and no inflammation was found.

AlloDerm Human-Derived.
The A-H samples showed slight to moderate degradation at 1 month. Blood vessels ranged from slight to moderate, whereas no inflammation was found in any of the samples. The A-H samples at 2 months ranged from intact and easily identifiable to severe degradation. No inflammation was found. Blood vessels ranged from none to moderate amounts. The A-H samples at 3 and 4 months ranged from severe degradation to absent. No inflammation was found. Blood vessels ranged from none to moderate.

AlloDerm Porcine-Derived.
A-P samples showed slight to moderate degradation at 1 month. Slight inflammation was found in one sample. Blood vessels ranged from slight to moderate. A-P samples revealed moderate to severe degradation at 2 months. No inflammation was found with only a slight amount of blood vessels detected. A-P samples showed severe degradation to not identifiable at 3 and 4 months. No inflammation was seen. Blood vessels ranged from none to moderate.

Statistical Analysis.
Statistical analysis was not performed. The n was too low to perform any meaningful statistical analysis, and this study was intended to be a descriptive study.

DISCUSSION.
One of the pivotal issues concerning GTR and GBR is how long the barrier should stay in place. The amplifying divisions of periodontal ligament cells may be complete at 21 days47; how long these cells need to be protected after this has not been determined. Computer-assisted densitometric image analysis and clinical probing correlate weakly at 3 months and still only moderately at 12 months. Bragger et al. considered that all alveolar bone adjacent to sites exposed to GTR demonstrated a slow consolidation of the tissues represented by delayed mineralization compared with the changes in probing attachment levels. Sigurdsson et al41 used ePTFE membranes and showed that dogs produce no regeneration at 3 weeks, whereas at 8 weeks, 75% of the bone has repaired together with 40% of the cementum. If collagen barriers are completely resorbed before day 30, neocementum can be found in the healing area, but no new bone. However, 10 days duration seems long enough to avoid apical migration of the epithelium.
The collagen membranes used in this experiment have different characteristics, but similar purported resorption rates. Different animal models surely have an influence on the speed of resorption. Periodontal healing rates have not been classified among potential models, and the effects of their various metabolisms are still greatly unknown. This might explain why the polylactic acid membrane used by Gottlow in monkeys was still intact at 3 months and was considerably degraded in the rabbit model after the same length of time. It is quite possible that the collagen barriers studied in this project would not resorb as quickly in humans. Therefore, one needs to be careful when applying data found in this project to humans.
The length of time the membrane must stay in place is estimated very differently, depending on which materials researchers are utilizing. Because the cell-mediated breakdown of this material produces only a slight inflammatory reaction, if at all, there seems to be no contraindication to having a very slow biodegradation. However, clinical and histologic evaluations as those described above seem to demonstrate that premature removal or dissolution can be detrimental to the formation of cementum and/or bone.
Because research on bioresorbable membranes is in constant progress, it is often difficult to compare results with an identical product. Research concerning collagen membranes involves materials from many different origins. Apparently not only the degree of cross-linkage is important, but also the percent of collagen in the initial solution. Kodama et al49 studied the effects of three concentrations of atelocollagen and found that the highest concentration produced the best results. When freeze-dried cross-linked bovine collagen was tested by Hyder et al, it was found to be totally dissolved in bacterial collagenase 5 times slower than non–cross-linked collagen. Modifications of collagen membranes could include more crosslinking to slow down resorption rates.
Suggestions for future studies include evaluation of additional types of collagen membranes. A different model system that mimics the human in healing rates would provide better data. Evaluation with something that marks the membrane and truly distinguishes it from the surrounding tissues would make identification of the membrane easier.

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