Bone regeneration by the osteopromotion technique using bioabsorbable membranes: An experimental study in rats

J Oral Maxillofac Surg

51:1106-1114,1993

Bone Regeneration by the Osteopromotion Technique Using Bioabsorbable Membranes: An Experimental Study in Rats EVA SANDBERG, DDS,* CHRISTER DAHLIN, DDS,t AND ANDERS LINDE, MSc DDS, OOONT DR:f: An osteopromotive technique has been developed that allows improved bone regeneration as well as bone neogenesis using porous, inert, nondegradable membranes made of expanded polytetrafluoroethylene (e-PTFE). For certain applications, however, it would be advantageous to use bioabsorbable membranes (BAMs), thus avoiding a surgical reentry for membrane removal. In this randomized comparison study the osteopromotive potential of BAMs was investigated in standardized "critical size defects" (5 mm in diameter) in the rat mandible. Three membrane types were tested and comparisons were made with e-PTFE membrane. The BAMs consisted of polylacticjpolyglycolic acid copolymers designed to give the membranes different absorption times when implanted in the tissue. Histologic analysis after healing periods of 1 to 12 weeks demonstrated the BAMs to be well tolerated by the tissue, causing just a mild inflammatory reaction along the membrane surfaces as long as the material remained in the tissue. The BAMs were found to be as efficient as e-PTFE membranes in that the bone repair was not significantly different with any of the four membrane types. However, healing in conjunction with one type of BAM seemed to occur somewhat more rapidly. Some cartilage was present at the early healing stages in the defects treated with BAMs, but disappeared at later stages. The results of this study show that BAMs are a valid alternative to e-PTFE membranes to improve bone regeneration, but indicate that further technical development of the membrane material is necessary.

In previous studies, we have described a new membrane technique for improved healing of bone defects and generation of new bone. I In brief, this technique involves placing a mechanical barrier in such a way that a secluded space is achieved into which only cells with an osteogenic potential can migrate. Thus, ingrowth ofconnective tissue into the bone defect is preReceived from the Department of Oral Biochemistry, Faculty of Odontology, University of Goteborg, Sweden. * Research Associate. t Assistant professor. Professor and Chairman. This study was supported by the Faculty of Odontology, University of Goteborg, and the Swedish Medical Research Council (grant no 2789). Address correspondence and reprint requests to Dr Linde: Department of Oral Biochemistry, Medicinarcgatan 7B, S-413 90 Gothenburg, Sweden.

*

© 1993 American Association of Oral and Maxillofacial Surgeons 0278-2391/93/5110-0009$3.00/0

vented. The amount of bone regeneration with the membrane technique was investigated in ditTerent animal experiments.v" The potential of this membrane technique to generate bone around exposed threads of titanium implants was originally tested in the rabbit.' and has later been repeated in other animal studies with similar results.v" Recently, this technique has been introduced clinically as an augmentation and regeneration technique in conjunction with the use of oral

implants.t '? Throughout these studies, porous expanded polytetrafluoroethylene (e-PTFE) membranes (Gore-Tex, W.L. Gore, Flagstaff, AZ) were used, requiring removal of the membrane after bone healing was sufficient. When used in combination with oral implants, this is not considered a problem, since the majority of implant systems require a two-stage surgical procedure, providing a logical opportunity for removal at the secondstage operation. However, several indications exist 1106

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SANDBERG, DAHLIN, AND LINDE

where the membrane technique could be beneficial, but where a reentry should be avoided. Examples of this are defects caused by cysts or tumors, as well as augmentation procedures for cranial and maxillofacial contour. The use ofbioabsorbable membrane materials for the regeneration and creation of new bone tissue is thus tempting, but little is found in the literature in this respect. The potential use of such material in the treatment of advanced periodontal lesions has, however, been suggested recently.P:" Polyglycolic acid (PGA) and polylactic acid (PLA) are resorbable a-polyester homopolymers, that have been used as suture material,'? in the form of plates and screws for the treatment of jaw fractures," and as sheets to repair blowout fractures of the orbital floor. 19 The degradation of such materials in tissues is accomplished by a slow hydrolysis under physiologic conditions.i" Minimal inflammation is seen and bone tissue seems to accept the material well." Thus, the chemical composition and the biologic response make it potentially possible for such materials to be used as a mechanical barrier for the treatment of osseous lesions. In the present study the efficacy of bioabsorbable membranes (BAMs) in the treatment of well-defined, standardized osseous lesions was evaluated, and the healing and predictability were compared with the use .of e-PTFE membranes. Three types of BAMs were tested, all being copolymers of PGA/PLA in different proportions, thus providing membrane types with different longevity in the tissue. The objective was also to study tissue response with regard to inflammation and stabilization of the blood clot, as well as the stability of the membrane material at various healing periods.

Materials and Methods ANIMALS AND ANESTHESIA

Thirty-six 5-month-old male albino Sprague-Dawley rats (body weight 425 to 500 g) were used. Before surgery, the animals were sedated with an intraperitoneal injection (3.3 mL/kg body weight) consisting of 25% Hypnorm vet (Janssen Pharmaceuticals, Beerse, Belgium), 25% Dormicum (Hoffman-La Roche, Basel, Switzerland), and 50% sterile water. During surgery, supplemental sedation was administered when needed. Local anesthesia, I mL of a 2% lidocaine-adrenalin solution (Astra, Sodertalje, Sweden), was given regularly at the site ofsurgery. Postoperatively, the animals were given Temgesic (Reckitt & Colman, Hull, Great Britain) daily for I week for pain relief.

from both the buccal and lingual aspects at the angles of the mandible. A trephine bur was used to create similar standardized, round, through-and-through osseous defects (5 mm in diameter) on both sides of the jaw. The size of the defect is consistent with a so-called "critical size defect" in rats, and was chosen to minimize the possibility for spontaneous repair. 2,22 All defects were covered, both buccally and lingually, with a membrane extending 2 to 3 mm outside the margins of the defect. Fixation was obtained by means ofa transosseous e-PTFE suture (Gore-Tex). The flap, including the periosteum, was carefully repositioned on the outer side of the membrane and sutured to provide full coverage. The periosteum thus was located on the peripheral surface of the membrane without any direct contact with the bone defect. The animals were divided into five groups, and healing was allowed for 1,2,3,6, and 12 weeks, respectively. MEMBRANE QUALITIES

Four membrane types were used, three BAMs and one e-PTFE membrane. The three types of BAM consisted ofcopolymers ofPLA and PGA in different proportions. Due to their respective resorption times when implanted in the tissue (8-12 weeks, 20 weeks, and 40 weeks), they were denoted BAM-8, BAM-20 and BAM40, respectively. After these intervals, the membranes had essentially disappeared from the tissue and were not detectable by histological technique. As a control, and serving as a comparison with our earlier results.' nonbiodegradable e-PTFE (Gore-Tex) membranes also were installed. For each membrane type, three sites were evaluated after each healing interval (I, 2, 3, 6, and 12 weeks, respectively), except for the 6-week period, when six sites with each membrane type were evaluated. TISSUE PROCESSING

The animals were killed with an overdose of carbon dioxide. Block biopsies, comprising the bone with surrounding tissue, were fixed by immersion in 4% buffered formalin, decalcified in ethylenediamine tetraacetic acid, and embedded in paraffin. Serial 5-Jlm horizontal sections, encompassing the entire defect, were made, stained with hematoxylin-eosin, alcian blue, or toluidine blue, and subjected to microscopic examination. Each defect was located by means of serial histologic sectioning, and the three most central sections were selected for histologic evaluation.

SURGICAL PROCEDURES HISTOLOGIC EVALUATION AND CLASSIFICATION

The region of the mandibular angle was shaved bilaterally and washed with 70% ethanol. After the skin incisions were made, mucoperiosteal flaps were raised

The appearance of the blood clot, the inflammatory response, the formation of the compact bone layer and

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OSTEOPROMOTION WITH BIOABSORBABLE MEMBRANES

bone marrow, the degree of bone union, and the presence ofcartilage in the wound area were recorded after different healing intervals. To evaluate the histologic findings and provide a basis for statistical comparison, a modified numerical scale 23 was used (Table 1). Two different methods, analysis of variance for incomplete balanced block design and the Newman-Keuls' test,24,25 were used to evaluate these data. The experimental protocol for this study was approved by the Animal Research Ethics Committee at the University of Goteborg.

Results All animals recovered well after the surgical procedure. No postoperative infections were evident, and all the animals gained weight during their respective healing periods. The e-PTFE sutures, used for fixation of the membranes, were fully accepted by the tissue (Fig 1), and at some sites were even embedded in newly formed bone. In all but one of the specimens, the membranes had remained in a proper position. In general, both with the BAMs and the e-PTFE membranes, there was a pronounced inflammatory response 1 week after surgery. After the second week, a considerably less severe infiltration of inflammatory cells was present, and then primarily in relation to the membranes (Figs 2-6). With the BAMs, a minor inflammatory cell infiltration was seen along the membrane surfaces as long as the membranes remained in the tissue. Thereafter, no signs of inflammation were detectable in the wound area. The e-PTFE material

Table 1.

Inflammatory response

Bone marrow

Bone union

I-WEEK SPECIMENS

At the time of death, all animals showed a tendency to be swollen in the wound areas. In spite of this tissue swelling, all of the membranes were in a proper position. No signs of new bone were observed inside any of the defects. In all defects, regardless of membrane type, a blood clot was present, which was encapsulated by the membrane. The defects contained sparse, primitive blood vessels.

Numerical Scores for Histologic Classification

Blood clot

Bone cortex

was well tolerated; from the third week on, inflammatory cells were seen only occasionally. The BAMs showed a tendency to collapse or to be pushed into the defects. This was obvious at about one third of the sites, whereas the shape of the membranes was essentially unaltered in the remaining defects. This slight tendency to collapse was never seen in conjunction with the e-PTFE membranes. At almost 80% of the sites covered with e-PTFE membranes, small islands of cartilage were present within the membrane structure. This was detectable at all sites at week 3, but was also seen in some cases at earlier observation periods. In contrast to the e-PTFE membranes, cartilage was not seen inside the BAM structure itself. However, in about half of the BAMtreated defects, cartilage was found inside the healing bone defects during the first 3 weeks (Figs 3, 7), at some sites comprising a considerable portion of the defect:The numerical scores for the histologic findings at each healing period for each membrane type are given in Table 2.

No organization of clot Organization well under way (vessels, fibers) Clot completely organized (coarse bundles)

o

Minor signs of inflammation Small number of inflammatory cells along the membrane surface Moderate signs/general presence of inflammatory cells Intense local reaction-heavy invasion of inflammatory cells into the surrounding tissue-tissue damage

o

None Beginning to appear Formation well under way Complete organization

o

None Hemopoietically active, large number of blood-forming cells Decreased number of blood cells, increased number of adipose cells Adult type of fatty marrow

o

No sign of bone union, rounded margins Bone formation at the rims, but two thirds of the defect remains Bone formation at the rims, but one third of the defect remains Complete bone union

o

NOTE: To statistically evaluate the healing process at different periods, the above scoring system, modified after Heiple et al,23 was used.

2

4 1

2 4 I

2 4 I

2 4 2

5 10

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SANDBERG, DAHLIN, AND LINDE

FIGURE I. After 2 weeks of healing, using biodegradable membranes, newly formed bone (NS) can be seen at the rims of the defect and on the surface of the old bone (a). The edges of the original defect are marked with arrows. Most of the defect is still unossified and filled with connective tissue (cr), The membrane can be seen in the tissue only as a series of empty spaces (arroll' heads). At this stage, there is some inflammatory cell infiltration, primarily confined to the membrane surfaces. There is virtually no inflammatory reaction to the suture material (5). (BAM-8, 2 weeks, hematoxylin-eosin stain, original magnification X90).

In six of the nine sites covered with BAMs, some cartilage was found at the original bone edges. At two sites, these membranes showed a tendency to shrink into the preformed bone gaps. In one of the three ePTFE-covered defects, small patches of cartilage were present inside the membrane structure itself. 2-WEEK SPECIMENS

Newly formed bone was evident at the rims of the defects in four of the 12 sites (Fig I). This was seen

FIGURE 3. Varying amounts of cartilage (arroll' heads) are present inside the newly formed bone (NS), within a BAM-covered defect after 2 weeks of healing. Note the difference in quality between the existing mandibular bone (s) and the newly formed, polymorphic bone. Very little inflammation can be seen in relation to the membrane in this specimen (upper part ofillustrationi. (BAM-20, 2 weeks, hematoxylin-eosin stain, original magnification X450.)

both in the BAM-covered defects and in defects covered with e-PTFE membrane. After 2 weeks, the inflammatory reaction in general was abated, and fewer inflammatory cells were seen, mainly along the membrane surfaces (Figs 2, 3). However, in one case where the membrane had been displaced, the wound was heavily invaded with inflammatory cells (BAM-20). At five of the nine sites covered with BAMs, varying amounts of cartilage were noticed within the defect area (Fig 3). In one of the three wounds treated with e-PTFE membrane, cartilage was present inside the membrane structure itself and also inside the defect (Fig 8).

NB

FIGURE 2. When using a bioabsorbable membrane, there was a clear but relatively mild inflammatory reaction in the tissue at 2 weeks. This was primarily localized adjacent to the membrane (M). The membrane can be seen as a series of empty spaces in the tissue. The newly formed bone (NS) is ofa rather primitive type at this stage. (BAM-20, 2 weeks. hematoxylin-eosin stain, original magnification X450).

B

Ne

FIGURE 4. Site covered with a BAM showing a complete bone union after 3 weeks of healing. The rim of the original defect is marked with arrows. Apposition ofa considerable thickness of new bone (NS) has also occurred on the surface of the old bone (n). Note the more primitive character of the newly formed bone. Some new bone (arrOlI' heads) has also been formed outside the membrane. (BAM-8, 3 weeks, hematoxylin-eosin stain, original magnification X90).

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OSTEOPROMOTION WITH BIOABSORBABLE MEMBRANES

FIGURE 5. Complete union in a defect covered with a BAM. The new bone (NB) is still ofa relatively primitive character without any distinctly developed cortex and marrow spaces. Only minor signs of inflammation are found close to the membrane. The membrane (M) can be seen only as large voids in the section. (BAM-8, 3 weeks, hematoxylin-eosin stain, original magnification X180).

3-WEEK SPECIMENS

At all 12 sites except for one, various amounts of new bone formation were present within the defects. This bone tissue varied in degree of maturity (Figs 47). Osteogenic activity was obvious in several-specimens. Cartilage was detectable in relation to all kinds of membranes at this time (Fig 7). Generally, only minor signs of an inflammatory reaction were found (Figs 4-6). At this stage, eight of the nine defects covered with BAMs showed equal or better results than the e-PTFEcovered defects in terms of how far bone regeneration had reached. Only one site (BAM-40) displayed less bone formation than the e-PTFE sites. In two (BAM-

FIGURE 6. This figure shows the difference in quality between the bone formed after 3 weeks (NB) and the existing mandibular bone (B). The new bone is ofa woven type. Very little inflammatory reaction is seen adjacent to the membrane (M) structure. (BAM-40, 3 weeks, hematoxylin-eosin stain, original magnification X180).

FIGURE 7.

After 3 weeks of healing, varying amounts of cartilage .

(e) can be seen within the healing defect in relation to the BAM.

(BAM-20, 3 weeks, hematoxylin-eosin stain, original magnification XI80).

8) of the nine sites, a complete union was seen (Figs 4,5). In four of the nine BAM-covered defects, the wounds contained cartilage tissue (Fig 7). In several specimens, cancellous bone with an obvious osteogenic activity was present on the peripheral side ofthe BAMs (Fig 4). At the three sites covered with e-PTFE, new bone formation was beginning to occur at the rims of the defects, but two thirds of the gap still remained filled with soft tissue. All the e-PTFE membranes showed cartilage within the membrane structure itself; on the outside of the membrane there were various amounts of bone and cartilage. 6-WEEK SPECIMENS

At this time the compact bone layer had reached complete organization at almost all ofthe sites, showing osteocytes embedded in the lacunae. The marrow spaces showed varying degrees of maturity in the different specimens, with no direct relationship to the membrane types placed. Some displayed a hemopoietic character, whereas others contained mainly adipose tissue. Complete bone union was seen in to of the 18 specimens treated with BAMs. In one of these animals, the site was macroscopically swollen, presumably due to infection. During histologic examination, however, this defect also showed complete bone union. Of the eight remaining specimens, six showed a large amount of new bone formation. At three of these sites less than one third of the defect remained unossified (Fig 9), whereas in the other three defects more than one third of the defect remained open. At one of the two remaining sites, where no signs of new bone formation were found, an intense local inflammatory reaction was seen (BAM-20).

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Table 2.

Healing of Rat Mandibular Trephine Defects at Different Times after Surgery Membrane Types

Weeks Postsurgery

2

3

6

12

e-PTFE

BAM-S

BAM-20

BAM-lO

Score

Mean

Range

Mean

Range

Mean

Range

Mean

H B I H B I H B I H B I H B I

2.0 0.0 2 4.0 0.7 0.7 6.3 2.0 0 18.8 8.7 0 19.7 8.3 0

2 0 2 2 to 6 Ot02 o to I 6 to 7 2 0 II to 22 2to 10 0 17 to 22 5 to 10 0

2.0 0.0 2 6.7 1.3 I 14.3 7.3 I 16.7 7.5 I 13.0 3.0 0

2 0 2 4 to 8 o to 2 I 9 to 17 2 to 10 I 5 to 22 5to 10 I 12 to 15 2 to 5 0

2.0 0.0 2 4.0 0.7 2 10.7 4.0 1.3 13.7 6.2 1.5 18.3 8.3 I

2 0 2 o to 8 o to 2 I t04 9 to 12 2 to 5 I to 2 o to 19 o to 10 I to 4 15 to 20 5 to 10 I

2.0 0.0 2 4.7 0.0 I 8.3 2.3 I 16.8 6.5 I 18.0 6.7 I

Range

2 0 2 4 to 0 I 5 to o to I 11 to 2to I 15 to 5 to I

6

12 5 22 10 22 10

NOTE: Through-and-through mandibular defects, 5 mm in diamter, were covered with four different types of membrane, and healing was evaluated after I to 12 weeks by two types of histologic scoring. For each membrane type, three sites were evaluated after each healing interval, except for the 6-week period, where six sites with each membrane were evaluated. Abbreviations: H, summarized scores for blood clot formation, formation of bone compacta, bone marrow, and bone union; B, score for bone union only; I, score for inflammatory response.

In general, considerable amounts of cancellous bone were present adjacent to the outer membrane surfaces. In one specimen (BAM-8), the membrane was not detectable in the tissue by microscopy; the other BAMs were still visible at this time. In five of the six specimens treated with e-PTFE membranes, the bone union was complete. A large amount of immature cancellous bone was present on the outer surfaces of these membranes. Both bone and cartilage tissue could be seen inside the e-PTFE membrane (Fig 10).

At 12 weeks, regardless of membrane type, the defect areas contained compact bone and the marrow spaces showed adipose cells of an adult type. At three of the nine BAM sites, bone union was complete. In four specimens, one third of the bone defect persisted and at the remaining two sites only one third of the defect was filled with bone. At this time, all the BAM-8 membranes had seemingly disappeared from the tissue and accordingly were not

FIGURE 8. In one of three defects covered with e-PTFE membrane (Gore-Tcx), a small aggregation of cartilage cells is seen on the surface ofthe old bone (B) at the rim of the defect. (e-PTFE, 2 weeks, toluidine blue stain, original magnification X 1800.)

FIGURE 9. After 6 weeks of healing a BAM-covered defect shows complete bone union. Due to a lack of stiffness of the membrane, the bone has taken on an hourglass shape, and slight soft connective tissue cores (arrol\'s) remain in the central part of the defect. (BAM8, 6 weeks, hematoxylin-eosin stain, original magnification X90.)

12-WEEK SPECIMENS

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OSTEOPROMOTION WITH BIOABSORBABLE MEMBRANES

FIGURE 10. Small numbers of cartilage cells (arrows) within the e-PTFE membrane (M). (e-PTFE, 6 weeks, toluidine blue stain, original magnification X450).

causing any inflammation. Along the surfaces of the BAM-20 and BAM-40 membranes, only minor signs of inflammatory reaction were seen. In two of the nine specimens, it was obvious that the membranes had collapsed into the defect. The bone healing in the center was thus incomplete, leaving one third of the gap filled with soft tissue. At two of the three sites treated with e-PTFE membranes, bone union was complete. In the third specimen, one third ofthe central part of the defect persisted. Along the outer surface of the e-PTFE membranes, a large amount of immature cancellous bone was seen. As previously mentioned, cartilage was present inside the membrane structure at all the sites. STATISTICAL ANALYSES

The H score (Table 2) comprised the sum of the scores for the appearance of blood clot, the formation ofa compact bone layer, the maturity ofthe bone marrow, as well as the degree of bone union. The B score represented the degree ofbone union only, whereas the I score described the inflammatory response. To elucidate the progress of healing during the 12week period, all the scores were summarized at each separate week regardless of the membrane type used, resulting in five groups, one for each healing interval. In the SNK grouping of the H scores, significant (P < .00 I) differences were found between the second and the third week and also between the third and the sixth week, demonstrating that the major steps in healing occurred mainly during these intervals. The test could not statistically separate weeks 1 and 2 or weeks 6 and 12 (a = .05). Regarding bone healing (the B score), a significant difference was found only between weeks 2 and 3. The B scores for the first 2 weeks could not be distinguished from each other, and weeks 3, 6, and 12 were not significantly different from one another (a =

.05). Consequently, the major increase in bone healing occurred between weeks 2 and 3. When comparing the four different membrane types, none showed any significant differences for the H score or the B score in comparison with the other membrane types (a = .05). Also, no significant differences were found in either the H or the B scores when the three different BAMs were combined, treated as one group, and compared statistically with the e-PTFE membrane group over time. Thus, the osteopromotive effect of the BAMs, as a group or individually, was not inferior to that of the e-PTFE membrane. When comparing the four membrane types with one another after each healing period, the analysis of variance showed a significant difference only at the 3-week period. According to the Newman-Keuls test (a == .05), this possible difference would appear only for the summarized healing score H, which was higher for BAM8 in comparison with BAM-40 and e-PTFE, but not compared with BAM-20. Neither the summarized H score or the B score for bone union itself were significantly different for any of the membranes at any healing period, including the B score at the third week. Discussion We have previously demonstrated, in a series of experiments, the potential of using membranes made of biologically inert e-PTFE to accomplish an improved healing in bone defects. I By using such membranes, it is also possible to create new bone at sites where bone normally is not present." The present investigation attempted to explore further the osteopromotive membrane technique using BAMs. The results of the study clearly demonstrate that it is possible to achieve bone regeneration in standardized mandibular trephine defects in adult rats using BAMs. The BAMs were found to be as effective as e-PTFE membranes in this respect. In agreement with our earlier findings.i? the e-PTFE membranes were well tolerated and the inflammatory response in the tissue was limited. In relation to the BAMs, inflammatory cells were present in a moderate amount in the early phases of healing. From the second week of healing, however, such cells were, in general, only detectable along the membrane surfaces. After degradation of the material, the morphologic signs of inflammation essentially disappeared. Indications were found that bone regeneration beneath the BAMs took place earlier than with the ePTFE membrane. Thus, the regenerative activity (Table 2) after 3 weeks of healing beneath BAM-8 was found to be somewhat more advanced than in the corresponding defects covered with e-PTFE membrane or BAM-40. If this is so, the membrane itself might be

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SANDBERG, DAHLIN, AND LINDE

stimulatory to osteogenesis. Another explanation may be that growth factors, stimulatory to osteogenesis, are produced in the area by the cells of the mild inflammatory reaction noted in conjunction with the BAMs, since inflammatory cells are known to produce factors that could influence osteogenesis.Fr" As found in our earlier studies, bone formation also occurred on the peripheral side ofthe membranes. The membrane structure itself, be it e-PTFE or BAM material, may be osteoconductive. Another possibility is that factors, stimulatory to osteogenesis, are produced by the overlying periosteum or are diffusing out from the healing defect. A conspicuous finding was the presence of areas of cartilage, either in the defect during healing (Figs 3, 7, 8) or within the membrane material (Fig 10). Cartilage formation during bone regeneration has been considered to be due to low oxygen tension in the tissue." In contrast to the e-PTFE membranes, which are porous and thus allow a free interchange of tissue fluid and macromolecules while keeping cells out, the BAM material is essentially solid during the early healing periods. The presence of cartilage inside the defects, when using BAMs, might thus be due to low oxygen tension caused by sealing off the periosteal vascular supply. The same conditions might prevail with the ePTFE membrane, while still providing conditions adequate for osteogenesis to -take place inside the bone wound. The bone healing accomplished with the use of BAMs showed the inherent capacity of such materials to prevent cells of the surrounding fibrous tissue from populating the defect area and to create a secluded space long enough to allow bone regeneration to occur. The thickness of the newly formed bone is obviously determined by the width of the space. Thus, stability in the shape of the membranes is crucial. In contrast to the e-PTFE membranes, some of the BAMs showed a lack in stiffness, resulting in a collapse of the membrane into the defect area and causing the newly formed bone to take on an hourglass shape. To prevent this, it might be necessary to use BAMs in combination with some space-holding material, for example autogeneic bone chips. Another possibility is that BAMs might be manufactured in such a way as to reinforce the membrane structure to maintain its stiffness throughout the healing period. The osteopromotion principle using e-PTFE membrane materials has proven to be of great value for different clinical applications. For those applications where there is a need to avoid a surgical reentry, the use ofBAMs might be advantageous. This investigation showed that BAMs may be produced with properties that allow ample time for bone formation. Obviously, the mild inflammatory reaction caused by these mem-

branes does not interfere with osteogenesis. It 'may thus be concluded that such membranes could be of great value in reconstructive maxillofacial and craniofacial surgery.

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OSTEOPROMOTION WITH BIOABSORBABLE MEMBRANES

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