Microcomputed Tomographic and Histomorphometric Analyses of Novel Titanium Mesh Membranes for Guided Bone Regeneration: A Study in Rat Calvarial Defects Yunia Dwi Rakhmatia, DDS, PhD1/Yasunori Ayukawa, DDS, PhD2/ Akihiro Furuhashi, DDS, PhD3/Kiyoshi Koyano, DDS, PhD4 Purpose: The objective of this study was to evaluate the optimal thickness and porosity of novel titanium mesh membranes to enhance bone augmentation, prevent soft tissue ingrowth, and prevent membrane exposure. Materials and Methods: Six types of novel titanium meshes with different thicknesses and pore sizes, along with three commercially available membranes, were used to cover surgically created calvarial defects in 6-week-old Sprague-Dawley rats. The animals were killed after 4 or 8 weeks. Microcomputed tomographic analyses were performed to analyze the three-dimensional bone volume and bone mineral density. Soft tissue ingrowth was also evaluated histologically and histomorphometrically. Results: The novel titanium membranes used in this study were as effective at augmenting bone in the rat calvarial defect model as the commercially available membranes. The greatest bone volume was observed on 100-μm-thick membranes with larger pores, although these membranes promoted growth of bone with lower mineral density. Soft tissue ingrowth when 100-µm membranes were used was increased at 4 weeks but decreased again by 8 weeks to a level not statistically significantly different from other membranes. Conclusion: Membrane thickness affects the total amount of new bone formation, and membrane porosity is an essential factor for guided bone regeneration, especially during the initial healing period, although the final bone volume obtained is essentially the same. Newly developed titanium mesh membranes of 100 µm in thickness and with large pores appear to be optimal for guided bone regeneration. Int J Oral Maxillofac Implants 2014;29:826–835. doi: 10.11607/jomi.3219 Key words: calvarial defect, guided bone regeneration, microcomputed tomography, titanium mesh
T
itanium has been used extensively for numerous surgical applications and dental procedures because of its high strength and rigidity, its low density and corresponding low weight, its ability to withstand high temperatures, and its resistance to corrosion.1,2
1Researcher,
Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. 2 Assistant Professor, Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. 3Research Assistant, Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. 4Professor, Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. Correspondence to: Dr Yunia Dwi Rakhmatia, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Fax: +81-92-642-6380. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
Titanium mesh (Ti-mesh) has also given excellent results when used to enhance bone formation for guided bone regeneration (GBR) treatment.2–5 The basic principle of GBR involves the placement of mechanical barriers to protect the blood clot and to seal off the bone defect from the surrounding soft tissue, thus providing exclusive access for osteogenic cells into the space intended for bone regeneration.6 Accordingly, Ti-mesh has been used as a barrier membrane to facilitate the augmentation of alveolar ridge defects, induce complete bone regeneration, improve the outcome of bone grafting, and treat failing implants.7 Its rigidity provides better space maintenance and contour maintenance than other materials, its elasticity prevents mucosal compression, and its stability prevents graft displacement. Furthermore, its plasticity permits bending, contouring, and adaptation of the mesh to any unique bony defect.8,9 Various studies have reported that Ti-mesh maintains space more predictably, even in cases with a large bone cavity, and is more resistant to collapse than expanded polytetrafluoroethylene (e-PTFE) and resorbable membranes.10,11 In addition, smooth-surfaced Ti-mesh
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is thought to be less susceptible to bacterial contamination than resorbable materials.12 However, the use of Ti-mesh has some drawbacks, notably exposure as a result of soft tissue dehiscence, which leads to local infection and soft tissue ingrowth and can result in incomplete bone regeneration within the space provided by the membrane. The likelihood of exposure of the Ti-mesh increases as the membrane becomes more rigid but can also be ascribed to mechanical irritation of mucosal flaps caused by the sharp edges created by cutting, trimming, or bending the membrane.3,4,13,14 However, such exposure generally does not lead to infection,8 which makes the Ti-mesh superior to e-PTFE barriers, which often become infected after exposure.15,16 Several characteristics of barrier membranes are known to be fundamental to successful GBR outcomes, such as their ability to occlude the treatment area from cellular invasion, their barrier stability, the size of their perforations, the peripheral seal that they form around the bone, and the ease with which they allow access of osteogenic and angiogenic cells to the regeneration site.17–19 Several different designs of Ti-mesh membrane have been studied in efforts to optimize the promotion of new bone formation and stabilization of bone grafts below the membrane and to minimize the risk of exposure, collapse, and/or soft tissue ingrowth.20,21 One favorable characteristic of the Ti-mesh is its rigidity and ability to maintain space and resist collapse, and this property is influenced by its thickness. However, the maximization of its rigidity should not adversely affect its clinical properties22; therefore, the thickness must be optimized when developing the ideal membrane for GBR. Certain commercially available Ti-mesh membranes are macroporous, with pore diameters in the millimeter range. This porosity is thought to be critical in maintaining blood supply to the regenerating bone and is believed to enhance regeneration by improving wound stability through tissue integration and allowing diffusion of extracellular nutrients across the membrane.23,24 Another advantage of this macroporosity is that the attachment of the membrane to tissue may stabilize and restrict the migration of epithelial cells. However, this very property of tissue integration, despite helping to stabilize the wound, makes these membranes hard to remove. Macroporous and multiporous membrane characteristics are also related to the creation of sharp edges after cutting or bending the membrane and may facilitate microbial contamination of the healing site, especially in the oral cavity. The development of a Ti-mesh membrane with fewer and smaller pores with maximal healing properties and fewer drawbacks (such as bacterial contamination
and tedious surgical removal) would be an important advance in the field of GBR.25 Thus, the aim of this study was to investigate the effects of differences in the physical variables (namely, thickness and porosity) of newly developed Ti-mesh membranes to determine the optimal structure of these membranes for GBR treatment.
MATERIALS AND METHODS The experiment was carried out on 100 male 6-weekold Sprague-Dawley rats (body weight 160 to 185 g), which were divided into 20 study groups (nine test materials and one passive control for each of two time courses) of five animals each. Animals were kept in a standard cage in an experimental animal room, fed with standard laboratory food pellets, and given water ad libitum. This study was approved by the Committee for Animal Research of Kyushu University (Approval Number: A24-182-0).
Membranes and Group Distribution
One group of animals was assigned to each of six different designs of novel Ti-mesh, three commercially available membranes, and one control (uncovered defect). The six novel membranes (Kyocera Medical Corporation) had a diameter of 12 mm; a thickness of 20, 50, or 100 µm; and either 12 pores or multiple pores with none at the coronal border (groups designated by T). Every mesh has a thickness with the same value as its pore diameter. Thus, six groups with novel Ti-mesh were established: 20, T20, 50, T50, 100, and T100. The three commercially available membranes used in this study were Titanium Micro Mesh (TM group), BioMend (BM group), and Cytoplast (CP group). Titanium Micro Mesh (ACE Surgical Supply Co) has a 100-µm thickness and 1,700-µm pore diameter. BioMend (Zimmer Dental Inc) is a type I resorbable collagen membrane derived from bovine tendon and has a 170-µm thickness and 0.004-µm pore diameter. Cytoplast GBR200 (Osteogenics Biomedical Inc) is a dense PTFE–type membrane with 200-µm thickness and pore diameter < 0.2 µm. The control (C) group consisted of animals with uncovered defect sites. All membranes had a diameter of 12 mm and were adapted to the surrounding bone and tissue at the experimental sites.
Anesthesia and Surgical Procedures
All surgery was performed in aseptic conditions under systemic and local anesthesia. The forehead of the rat was shaved and disinfected with tincture of iodine. During surgery, an incision about 2 cm long was made with a scalpel along the sagittal suture. Then, a dermoperiosteal flap was reflected to reveal the parietal bone. The International Journal of Oral & Maxillofacial Implants 827
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Fig 1a A 7-mm-diameter bone defect was created on the calvarium.
Fig 1b Titanium membrane was laid over defect area and margins were glued with Histoacryl to prevent dislodgement. In the control group, the defect site was left empty.
7 mm
Fig 1c Micro-CT view of titanium membrane covering the defect area.
Fig 1d The region of interest incorporating the calvarial defect is shown as delineated for CT scan analysis. 7 mm
A circular, full-thickness, 7-mm-wide bone defect was created with a surgical trephine bur under saline irrigation to prevent overheating of the bone margins. Care was taken during the surgical procedure to prevent damage to the dura. The defect site was covered with the test membrane, and the membrane borders were bonded to the bone with Histoacryl glue (B. Braun) to prevent membrane movement (Figs 1a and 1b). Finally, the skin flaps were carefully sutured in place with a nonresorbable suture material. All animals received a postsurgical antibiotic dose of oxytetracycline (0.5 mL/kg body weight). After 4 or 8 weeks, the animals were euthanized and perfused with a fixative solution consisting of 0.1 mol/L phosphate-buffered 4% paraformaldehyde (pH 7.4). Block biopsy specimens that included the membranes, the surrounding soft tissue, and the underlying calvarial bone were obtained.
Microcomputed Tomography Analysis
Unprocessed bone biopsy specimens were imaged and analyzed using in vivo microcomputed tomography (micro-CT) (SkyScan 1076, SkyScan) at 60 kV/167 µA and a Ti-0.5 filter. The specimens were fitted into a cylindric sample holder in a horizontal position and scanned parallel to the coronal aspect of the calvarial bone. High-resolution scanning with an in-lane pixel
size and slice thickness of 18 µm was performed. The micro-CT scanner software was used to make a threedimensional (3D) reconstruction from each set of scans. From the entire 3D data set, a cylindric region of interest with a diameter of 7 mm and a height comprising the total depth scanned was selected for analysis (Figs 1c and 1d). Region of interest analysis was performed to assess primary parameters, namely, bone volume (BV) and total tissue volume (TV), both measured in mm3. BV was calculated as the volume of the region characterized as bone (defined as the number of voxels, with gray values in the range 30 to 130) and normalized ratiometrically against the total volume of the region of interest (BV/TV) to derive the percentage bone volume (%BV). Bone with different degrees of mineralization displays different densities and linear attenuation coefficients, resulting in gray value variations in the CT scans, the distribution of which is an indication of the degree of mineralization, ie, bone mineral density (BMD) (g/cm3).
Histologic and Histomorphometric Evaluation
Upon completion of micro-CT scanning, the specimens were dehydrated in a graded series of ethanols and embedded in methacrylate resin. Undecalcified sagittal sections (thickness ~60 µm) were cut, polished,
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20–4w
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Fig 2 Micro-CT 3D reconstructions of typical rat calvarial defects 4 weeks after membrane placement. Newly formed bone is shown between the red lines.
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Fig 3 Micro-CT 3D reconstructions of typical rat calvarial defects 8 weeks after membrane placement. Newly formed bone is shown between the red lines.
and stained with Masson’s trichrome. To evaluate the bone and cellular tissue responses histologically, the samples were examined under a light microscope. The center of the test membrane from one histologic section of each specimen was selected to represent that group for evaluation. Histomorphometric evaluation was used to trace and measure the area of connective tissue beneath the membrane.
Statistical Analysis
The mean values and standard deviations for each parameter were calculated for each group/healing time. A one-way analysis of variance with post hoc Tukey test was performed to analyze 3D BV and BMD values in the defect region of interest from the micro-CT analysis. Statistical analysis was also performed to evaluate the area of soft tissue beneath the membrane. Values of P < .05 were considered to indicate statistical significance.
RESULTS All rats recovered well and gained weight throughout the postoperative healing period. There were no clinical signs of infection or rejection of the membranes or other ailments during the healing period. Among the different membrane materials tested, the TM, 100, and T100 were the stiffest and were rather difficult to adapt to the bone surface contour compared with the 50, T50, 20, and T20 membranes. The margins of the BM and CP membranes, after bending, were felt to have inadequate stiffness. Three-dimensional micro-CT reconstructions of a typical rat calvarial defect were created for all groups. Bone regeneration progressing toward the center of the defect was commonly observed at 4 weeks and increased by 8 weeks in the defect areas with membranes (Figs 2 and 3). In contrast, no or minimal bone regeneration was observed in the C groups after either 4 or 8 The International Journal of Oral & Maxillofacial Implants 829
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100 90 80 70 60 50 40 30 20 10 0
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Figs 4a and 4b Percentage of new bone volume elucidated from 3D micro-CT reconstruction of a typical rat calvarial defect at (a) 4 weeks and (b) 8 weeks after membrane placement (n = 5 per group). C = control (no membrane). *,**: compared to C; †,††: compared to 100; ‡,‡‡: compared to TM; §,§§: compared to 50; || || : compared to CP; ¶, ¶ ¶: compared to T100. *, †, ‡, §, ¶ : P < .05; **, ††, ‡‡, §, || ||, ¶ ¶: P < .01.
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Figs 5a and 5b BMD from 3D micro-CT reconstructions of a rat calvarial defect at (a) 4 weeks and (b) 8 weeks after membrane placement (n = 5 per group). C = control (no membrane). *,**: compared to CP; †,††: compared to 100; ‡,‡‡: compared to TM; §,§§: compared to BM. *, †, ‡, §: P < .05; **, ††, ‡‡, §§: P < .01.
weeks of healing (Figs 2 and 3). The quantitative results derived from micro-CT analysis are shown in Figs 4 and 5. The novel Ti-mesh with 12 pores (20, 50, 100) showed a higher %BV compared with the mesh with multiple pores (T20, T50, T100) at both 4 and 8 weeks. Values for %BV at 8 weeks tended to increase as the thickness of the mesh increased. BMD values were derived by comparing x-ray attenuation in the scanned bone samples with that in hydroxyapatite standards. CP had the highest mineralization level; for the other membranes, the mineralization level tended to decrease as the thickness of the mesh increased. BMD was higher in the novel membranes with multiple pores than in those with only 12 pores. Significant differences (P < .01) were found with CP versus 100, TM, and BM. Histologic analysis of all groups (Figs 6 and 7) complemented the micro-CT findings. In some instances, the gap containing the Histoacryl tissue adhesive used to anchor the membrane and stop it from moving was evident between the border of the membrane and the bone margin. Some of the membranes deemed
to have inadequate stiffness (20, T20, BM, and CP) had visibly collapsed into the defect site. At 4 weeks, bone formation with more intense red staining was observed in sites with thicker membranes and in sites with membranes with larger pore diameters. Moreover, more bone formation was observed beneath the novel membranes with 12 pores compared with those with multiple pores. In the control group, the defect sites were filled mainly with fibrous connective tissue, and negligible new bone had formed. After 8 weeks of healing, all groups exhibited a greater and more vascularized body of new bone with a higher degree of mineralization than in the respective group after 4 weeks of healing, and the defect margins were indistinguishable from the newly formed bone. In all of the groups with 12 pores, the area of new bone formed was more expansive than in the membranes with multiple pores. Membranes with the greatest thickness (100, T100, and TM) showed the most extensive new bone formation, with some specimens exhibiting complete resolution of the defect.
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20–4w
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Fig 6 Histologic sections from each specimen at 4 weeks (bar = 300 µm). Double-headed arrows = new bone formation; single arrows = membrane collapse into the defect site.
Fig 7 Histologic sections from each specimen at 8 weeks (bar = 300 µm). Double-headed arrows = new bone formation; single arrows = membrane collapse into the defect site.
The connective tissue area beneath the membranes was measured (Fig 8). Fibrous connective tissue was found to have infiltrated into the gaps between the bone and the membrane structure, and also through the pores, and tended to be most prolific in membranes that were thick and/or had large pores. After 4 weeks, the 100 and T100 membranes displayed the largest areas of soft tissue ingrowth compared with the other groups. However, at 8 weeks, there was no significant difference between the specimens. Fibers and cells were observable in the defect area, having penetrated the pores of some titanium membranes, especially those in the T20, T50, T100, and TM groups. No such ingrowth was evident in the defect area beneath the
novel membranes with 12 pores (20, 50, 100), CP, and BM, which all therefore remained free of cell ingrowth, although these did display some soft tissue ingrowth through the gaps at the margins of the membrane.
DISCUSSION Rat calvaria were used in this study to test biomaterials intended for use in the regeneration of critical-size bone defects. Calvarial defects are a highly suitable experimental model because their poor blood supply and the membranous structure of the bone (low mechanical stimulation) preclude any spontaneous The International Journal of Oral & Maxillofacial Implants 831
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Connective tissue area (× 106 μm2)
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Fig 8 Connective tissue area beneath the membrane estimated by histomorphometric measurement of the middle sagittal section of the rat calvarial defect after 4 weeks and 8 weeks of healing. C = control (no membrane). *P < .05 compared to 100.
healing.26 A critical defect size of 5 mm in rat calvaria has been defined as the smallest intraosseous wound that does not heal spontaneously during the lifetime of the animal,27 although another study estimated this to be larger, at 8 mm in diameter.28 Given the skull size of the animals used (and thus the available calvarial area), the defects created in this study were as large as practicable to still fulfill the requirements for a criticalsize defect for testing the biomaterials used here. The results of this study proved that a 7-mm defect healed with fibrous tissue when left uncovered but healed with bone in the presence of a membrane. Despite the use of Histoacryl to prevent membrane dislodgement, some movement still occurred. Therefore, sufficient fixation of the membrane is vital for proper wound healing and blood clot stabilization. This fixation also prevents the membrane micromovement that has been shown to be detrimental to bone formation and can result in the development of fibrous tissue instead of bone, particularly if movement occurs during the healing process.29 The present study confirms earlier reports that a membrane must be sufficiently rigid to maintain the space of the regenerating bone area beneath it. A tendency to collapse into the bone defect limits the space into which the bone can regenerate.3,4,30 Nonabsorbable dense PTFE membranes (such as the CP used here) tended to collapse in this way, resulting in less bone formation. Despite the good BVs that were seen beneath the resorbable membrane (the collagen-based BM) after 4 weeks, some specimens at 8 weeks appeared to have lost their stiffness, resulting in lower BVs at this time point compared with titanium membranes. As indicated in previous reports, biodegradation of BM is caused by the enzymatic activity of macrophages and polymorphonuclear leukocytes, resulting in poor membrane resistance to collapse and
therefore limiting bone formation.31 The degradation of cross-linked BM membranes was also associated with decreased tissue integration and vascularization.32 Moreover, the CP and BM membranes have a fairly soft consistency, which may have limited resistance to the soft tissue pressure exerted on them from above, resulting in their collapse. These findings suggest that autogenous bone or bone substitutes should be used beneath CP and BM barriers to provide additional support. The rigidity of Ti-mesh allows it to overcome these problems of membrane collapse and thus to maintain space without the requirement for additional materials.13 The thickness of any material is a primary factor in its stiffness. A range of thickness between 100 and 200 µm was reported to be ideal for the reconstruction of large bone tissue defects and for advanced deformative and moderate atrophic alterations.33 The current findings concur that a thickness of 100 µm in titanium membranes (100, T100, and TM) results in more extensive bone formation than thinner membranes. However, the T100 membranes broke easily during surgical manipulation, which resulted in membrane collapse and subsequent decrease of the bone volume at 4 weeks compared with other titanium membranes that were 100 µm in thickness. The 20 and T20 groups had the lowest stiffness; consequently, some collapsed into the defect area, resulting in the lower BVs compared with the thicker titanium membranes. Others have reported that a 50-µm membrane made of titanium foil has the least tendency to collapse compared with other types of membrane.34 A balance must be found between the thickness of mesh that is required for stability and the need for it to be malleable enough to be adapted to the contours of the adjacent bone.3 Despite membrane permeability not being a prerequisite for GBR,35 previous investigations have concluded that the pore size must be large enough to allow angiogenesis into the new bone (ie, at least 40 µm); limited pore size can hinder angiogenesis.36 This must be weighed against the need to restrict the invasion of fibrous connective tissue into the defect area. GBR may require selectively occlusive barriers for successful bone regeneration,37 although good results have been observed with perforated titanium membranes that are nonocclusive,38,39 so this requirement is clearly not absolute. It has been suggested that occlusive membranes may hamper the penetration of nutrients and growth-regulatory factors into the protected site, increasing the frequency of soft tissue dehiscence and thereby inhibiting bone formation.24,34 Here, an increased BV beneath those membranes with larger pores (TM, 100 groups) was consistent with other studies reporting greater bone development
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underneath membranes with larger pores.40,41 Bartee and Carr described complete healing of a rat mandibular defect treated with a dense PTFE membrane for 10 weeks after surgery,23 but the present study suggests that membranes with small pores (such as the dense PTFE–based CP material used here) seem to block the integral vascularization process and therefore inhibit bone formation in the defect area. Other studies investigating bone ingrowth into systems with different pore sizes showed that a pore size of 100 µm allows bone ingrowth, but that a pore size greater than 150 µm is required for osteon formation.42,43 In contrast, the present study found no significant pore size–related difference in bone development at 8 weeks, consistent with other studies showing that bone ingrowth is possible through any pore larger than 50 µm.43 Another previous study went further, concluding that pore size has no influence on the healing response or on the clinical outcome.40 Increasing barrier porosity may promote early osteogenesis but not the final amount of new bone that is formed,34 concordant with the results showing that membrane porosity is an essential factor for GBR in the initial healing period but that the final BV obtained is essentially the same. The fact that different rates of bone formation were found indicates that pore size is not the only relevant factor of membranes in osteogenesis. Indeed, bone formation underneath a membrane is governed by a variety of factors, including the physical and surface characteristics of the material, its thickness and porosity, its chemical composition, and the depth at which it is placed within the tissue.44 Other local environmental factors may also influence membrane effects. Hydrophilicity of the membrane is part of the physical and surface characteristics of the material. This characteristic may more take into account biocompatibility of a membrane, which allows the cells to grow and subsequently to create integration between membranes and surrounding tissue compared with membrane’s other characteristics. Moreover, hydrophilicity affects cell adherence to a membrane’s surface. However, CP, because it is a dense PTFE membrane, has lower hydrophilicity and therefore may decrease cell adhesion, as shown by a previous study that investigated cell attachment to various membranes, including PTFE.45 The minimal tissue integration of the CP membrane may be advantageous for membrane retrieval. However, it may also create potential problems for initial clot formation, wound stabilization, and membrane stability and thus may interfere with wound healing. BM was reported to be more hydrophilic and is known to modulate various cell behaviors, such as adhesion, proliferation, and migration, because of its chemotactic capability.46 Hydrophilic titanium membrane
surfaces were also shown to influence cell differentiation and growth factor production.47 Interestingly, despite the correlation between large pore sizes and greater new BV, membranes with large pore sizes also appeared to be linked with lower bone density, especially compared to the CP membranes at 4 weeks (P < .01). Membranes with multiple pores had higher BMD values than membranes with only 12 pores. If bone is growing from the edges of the pores into the center, it is intuitive that a material with many small pores will have a greater number of “growth centers,” thus producing better-quality (ie, higher-density) bone. It is possible that mineral apposition might be incomplete when materials with a large pore size are used because the new bone takes time to grow into these “unsupported” areas.40,41 Previous studies have shown that soft tissue ingrowth is greater when using Ti-mesh with a large pore size (> 2 mm) than with a small pore size (< 2 mm).9 Histologic results showed that membranes with small pore sizes tended to permit less ingrowth of connective tissue. The soft tissue layer beneath the CP, 20, and T20 membranes, which have few pores of minimal size, was thinner and had parallel-structured fibers, compared with the dense and multilayered fibrous tissue found under the 100, T100, and TM membranes. A previous study confirmed this correlation between pore size and connective tissue ingrowth; it found that such growth was absent beneath a completely occlusive titanium barrier (ie, where pore size = 0).18 It is suggested that pores in excess of 100 µm are required for the rapid penetration of highly vascular connective tissue, and small pores tend to become filled with more avascular tissue.48 The dense connective tissue that was found beneath some membranes could be the result of insufficient adaptation of the membrane to the surrounding bony tissue. Connective tissue growth under the membrane is thought to be caused by various factors, including insufficient peripheral healing (ie, sealing) between the material and the bone, ingrowth of connective tissue through the pores of the barrier, and insufficient stability of the wound area.49 However, after 8 weeks, the connective tissue content had decreased and bone was increased in the defect area, in agreement with a previous study observing that, with sufficient initial stability, the early tissue infiltrate will differentiate into bone, either by direct bone formation or via appositional growth from adjacent bone.42 There are no well-defined rules for determining the sample size needed for a reliable study. The relatively small sample size of five rats in each group decreases the statistical power of this study. Therefore, within the lack of quantitative variables, the results must be recognized to have limitations. The International Journal of Oral & Maxillofacial Implants 833
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CONCLUSIONS The novel titanium meshes used here augmented new bone in this critical-sized rat calvarial defect model as effectively as commercially available membranes after 4 and 8 weeks of healing. Of the thicknesses tested here, 100-µm-thick titanium mesh is recommended for the reconstruction of bone tissue defects because of its ability to support new bone growth into the defect area. Moreover, membrane porosity is another essential factor for guided bone regeneration, especially during the initial healing period. Further studies of larger defects in larger animals will be required to further advance the knowledge of the optimal properties of titanium meshes for the resolution of bone defects.
ACKNOWLEDGMENTS This study was conducted with financial assistance from Kyocera Medical, Osaka, Japan. The authors reported no conflicts of interest related to this study.
REFERENCES 1. Wang RR, Fenton A. Titanium for prosthodontic applications: A review of the literature. Quintessence Int 1996;27:401–408. 2. Degidi M, Scarano A, Piattelli A. Regeneration of the alveolar crest using titanium micromesh with autologous bone and a resorbable membrane. J Oral Implantol 2003;29:86–90. 3. Von Arx T, Kurt B. Implant placement and simultaneous peri-implant bone grafting using a microtitanium mesh for graft stabilization. Int J Periodontics Restorative Dent 1998;18:117–127. 4. Maiorana C, Santoro F, Rabagliati M, Salina S. Evaluation of the use of iliac cancellous bone and anorganic bovine bone in the reconstruction of the atrophic maxilla with titanium mesh: A clinical and histologic investigation. Int J Oral Maxillofac Implants 2001;16:427–432. 5. Corinaldesi G, Pieri F, Sapigni L, Marchetti C. Evaluation of survival and success rates of dental implants placed at the time of or after alveolar ridge augmentation with an autogenous mandibular bone graft and titanium mesh: A 3- to 8-year retrospective study. Int J Oral Maxillofac Implants 2009;24:1119–1128. 6. Dahlin C. Scientific background of guided bone regeneration. In: Buser D, Dahlin C, Schenk RK (eds). Guided Bone Regeneration in Implant Dentistry. Chicago: Quintessence, 1994:32–34. 7. Sottosanti JS. Calcium sulfate: A valuable addition to the implant/ bone regeneration complex. Dent Implantol Update 1997;8:25–29. 8. Von Arx T, Hardt N, Wallkamm B. The TIME technique: A new method for localized alveolar ridge augmentation prior to placement of dental implants. Int J Oral Maxillofac Implants 1996;11:387–394. 9. Her S, Kang T, Fien MJ. Titanium mesh as an alternative to a membrane for ridge augmentation. J Oral Maxillofac Surg 2012;70:803–810. 10. Becker W, Becker B, Mellonig J. A prospective multi-center study evaluating periodontal regeneration for class II furcation invasions and intrabony defects after treatment with a bioabsorbable barrier membrane: 1-year results. J Periodontol 1996;67:641–649. 11. Zitzmann NU, Naef R, Scharer P. Resorbable versus nonresorbable membranes in combination with Bio-Oss for guided bone regeneration. Int J Oral Maxillofac Implants 1997;12:844–852.
12. Schopper CH, Goriwoda W, Moser D. Long-term results after guided bone regeneration with resorbable and microporous titanium membranes. J Oral Maxillofac Surg Clin North Am 2001;13:449–458. 13. Watzinger F, Luksch J, Millesi W. Guided bone regeneration with titanium membranes: A clinical study. Br J Oral Maxillofac Surg 2000;38:312–315. 14. Proussaefs P, Lozada J. Use of titanium mesh for staged localized alveolar ridge augmentation: Clinical and histologic-histomorphometric evaluation. J Oral Implantol 2006;32:237–247. 15. Buser D, Dula K, Hirt HP, Schenk RK. Lateral ridge augmentation using autografts and barrier membranes: A clinical study with 40 partially edentulous patients. J Oral Maxillofac Surg 1996;54:420–432. 16. Simion M, Jovanovic SA, Trisi P, Scarano A, Piattelli A. Vertical ridge augmentation around dental implants using a membrane technique and autogenous bone or allografts in humans. Int J Periodontics Restorative Dent 1998;18:9–23. 17. Kostopoulos L, Karring T. Augmentation of the rat mandible using guided tissue regeneration. Clin Oral Implants Res 1994;5:75–82. 18. Lundgren AK, Lundgren D, Taylor A. Influence of barrier occlusiveness on guided bone augmentation. An experimental study in the rat. Clin Oral Implants Res 1998;9:251–260. 19. Schenk RK. Bone regeneration: Biologic basis. In: Buser D, Dahlin C, Schenk RK (eds). Guided Bone Regeneration in Implant Dentistry. Chicago: Quintessence, 1994:49–100. 20. Roccuzzo M, Ramieri G, Spada MC, Bianchi SD, Berrone S. Vertical alveolar ridge augmentation by means of a titanium mesh and autogenous bone grafts. Clin Oral Implants Res 2004;15:73–81. 21. Assenza B, Piattelli M, Scarano A, Lezzi G, Petrone G, Piattelli A. Localized ridge augmentation using titanium micromesh. J Oral Implantol 2001;27:287–292. 22. Malchiodi L, Scarano A, Quaranta M, Piattelli A. Rigid fixation by means of titanium mesh in edentulous ridge expansion for horizontal ridge augmentation in the maxilla. Int J Oral Maxillofac Implants 1998;13:701–705. 23. Bartee BK, Carr JA. Evaluation of a high-density polytetrafluoroethylene (n-PTFE) membrane as a barrier material to facilitate guided bone regeneration in the rat mandible. J Oral Implantol 1995;21:88–95. 24. Linde A, Thoren C, Dahlin C, Sandberg E. Creation of new bone by an osteopromotive membrane technique: An experimental study in rats. Int J Oral Maxillofac Surg 1993;51:892–897. 25. Rakhmatia YD, Ayukawa Y, Furuhashi A, Koyano K. Current barrier membranes: Titanium mesh and other membranes for guided bone regeneration in dental applications. J Prosthodont Res 2013;57:3–14. 26. Develioglu H, Unver Saraydin S, Kartal U. The bone healing effect of a xenograft in a rat calvarial defect model. Dent Mater J 2009;28:396–400. 27. Bosch C, Melsen B, Vargervik K. Importance of the critical size bone defect in testing bone-regenerating materials. J Craniofac Surg 1998;9:310–316. 28. Hollinger JO, Kleinschmidt JC. The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1990;2:237–243. 29. Cameron H, Pilliar R, MacNab I. The effect of movement on the bonding of porous metal to bone. J Biomed Mater Res 1973;7:301–311. 30. Fritz ME, Malmquist J, Koth D. The use of guided bone regeneration to fill large mandibular defects in monkeys: A pilot study. Int J Oral Maxillofac Implants 1994;9:644–652. 31. Tatakis DN, Promsudthi A, Wikesjö UM. Devices for periodontal regeneration. Periodontology 2000 1999;19:59–73. 32. Rothamel D, Schwarz F, Sager M, Herten M, Sculean A, Becker J. Biodegradation of differently cross-linked collagen membranes: An experimental study in the rat. Clin Oral Implants Res 2005;16:369–378. 33. Vovk V, Vovk Y. Results of the guided bone regeneration in patients with jaw defects and atrophies by means of Mondeal occlusive titanium membranes. Int J Oral Maxillofac Surg 2005;34:74. 34. Zellin G, Linde A. Effects of different osteopromotive membrane porosities on experimental bone neogenesis in rats. Biomaterials 1996;17:695–702.
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35. Schmid J, Hämmerle CHF, Olah AJ, Lang NP. Membrane permeability is unnecessary for guided generation of new bone. An experimental study in the rabbit. Clin Oral Implants Res 1994;5:125–130. 36. Zhang M. Biocompatibility of materials. In: Shi D, Wang M, Zhang M, Clare A, Kasuga T, Liu Q (eds). Biomaterials and Tissue Engineering. Berlin, Heidelberg: Springer-Verlag, 2004:83–143. 37. Van Steenberghe D, Johansson C, Quirynen M, Molly L, Albrektsson T, Naert I. Bone augmentation by means of a stiff occlusive titanium barrier. Clin Oral Implants Res 2003;14:63–71. 38. Lemperle SM, Calhoun CJ, Curran RW, Holmes RE. Bony healing of large cranial and mandibular defects protected from soft-tissue interposition: A comparative study of spontaneous bone regeneration, osteoconduction, and cancellous autografting in dogs. Plast Reconstr Surg 1998;101:660–672. 39. Dupoirieux L, Pourquier D, Picot MC, Neves M. Comparative study of three different membranes for guided bone regeneration of rat cranial defects. Int J Oral Maxillofac Surg 2001;30:58–62. 40. Bobyn J, Stackpool G, Hacking S. Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial. J Bone Joint Surg [Br] 1999;81:907–914. 41. Gutta R, Baker RA, Bartolucci AA, Louis PJ. Barrier membranes used for ridge augmentation: Is there an optimal pore size? J Oral Maxillofac Surg 2009;67:1218–1225. 42. Spector M, Flemming W, Kreutner A. Bone growth into porous high-density polyethylene. J Biomed Mater Res 1976;10:595–603.
43. Welsh R, Pilliar R, Macnab I. Surgical implants. The role of surface porosity in fixation to bone and acrylic. J Bone Joint Surg [Am] 1971;53:963–967. 44. Galgut P, Pitrola R, Waite I, Doyle C, Smith R. Histological evaluation of biodegradable and non-degradable membranes placed transcutaneously in rats. J Clin Periodontol 1991;18:581–586. 45. Wang HL, Miyauchi M, Takata T. Initial attachment of osteoblasts to various guided bone regeneration membranes: An in vitro study. J Periodontal Res 2002;37:340–344. 46. Locci P, Calvitti M, Belcastro S, et al. Phenotype expression of gingival fibroblasts cultured on membranes used in guided tissue regeneration. J Periodontol 1997;68:857–863. 47. Schwarz F, Wieland M, Schwartz Z, et al. Potential of chemically modified hydrophilic surface characteristics to support tissue integration of titanium dental implants. J Biomed Mater Res B Appl Biomater 2009;88:544–557. 48. Chvapil M, Holusa R, Kliment K, Stoll M. Some chemical and biological characteristics of a new collagen-polymer compound material. J Biomed Mater Res 1969;3:315–332. 49. Simion M, Trisi P, Maglione M, Piattelli A. A preliminary report on a method for studying the permeability of expanded polytetrafluoroethylene membrane to bacteria in vitro: A scanning electron microscopic and histological study. J Periodontol 1994;65:755–761.
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T
itanium has been used extensively for numerous surgical applications and dental procedures because of its high strength and rigidity, its low density and corresponding low weight, its ability to withstand high temperatures, and its resistance to corrosion.1,2
1Researcher,
Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. 2 Assistant Professor, Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. 3Research Assistant, Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. 4Professor, Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. Correspondence to: Dr Yunia Dwi Rakhmatia, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Fax: +81-92-642-6380. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
Titanium mesh (Ti-mesh) has also given excellent results when used to enhance bone formation for guided bone regeneration (GBR) treatment.2–5 The basic principle of GBR involves the placement of mechanical barriers to protect the blood clot and to seal off the bone defect from the surrounding soft tissue, thus providing exclusive access for osteogenic cells into the space intended for bone regeneration.6 Accordingly, Ti-mesh has been used as a barrier membrane to facilitate the augmentation of alveolar ridge defects, induce complete bone regeneration, improve the outcome of bone grafting, and treat failing implants.7 Its rigidity provides better space maintenance and contour maintenance than other materials, its elasticity prevents mucosal compression, and its stability prevents graft displacement. Furthermore, its plasticity permits bending, contouring, and adaptation of the mesh to any unique bony defect.8,9 Various studies have reported that Ti-mesh maintains space more predictably, even in cases with a large bone cavity, and is more resistant to collapse than expanded polytetrafluoroethylene (e-PTFE) and resorbable membranes.10,11 In addition, smooth-surfaced Ti-mesh
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Rakhmatia et al
is thought to be less susceptible to bacterial contamination than resorbable materials.12 However, the use of Ti-mesh has some drawbacks, notably exposure as a result of soft tissue dehiscence, which leads to local infection and soft tissue ingrowth and can result in incomplete bone regeneration within the space provided by the membrane. The likelihood of exposure of the Ti-mesh increases as the membrane becomes more rigid but can also be ascribed to mechanical irritation of mucosal flaps caused by the sharp edges created by cutting, trimming, or bending the membrane.3,4,13,14 However, such exposure generally does not lead to infection,8 which makes the Ti-mesh superior to e-PTFE barriers, which often become infected after exposure.15,16 Several characteristics of barrier membranes are known to be fundamental to successful GBR outcomes, such as their ability to occlude the treatment area from cellular invasion, their barrier stability, the size of their perforations, the peripheral seal that they form around the bone, and the ease with which they allow access of osteogenic and angiogenic cells to the regeneration site.17–19 Several different designs of Ti-mesh membrane have been studied in efforts to optimize the promotion of new bone formation and stabilization of bone grafts below the membrane and to minimize the risk of exposure, collapse, and/or soft tissue ingrowth.20,21 One favorable characteristic of the Ti-mesh is its rigidity and ability to maintain space and resist collapse, and this property is influenced by its thickness. However, the maximization of its rigidity should not adversely affect its clinical properties22; therefore, the thickness must be optimized when developing the ideal membrane for GBR. Certain commercially available Ti-mesh membranes are macroporous, with pore diameters in the millimeter range. This porosity is thought to be critical in maintaining blood supply to the regenerating bone and is believed to enhance regeneration by improving wound stability through tissue integration and allowing diffusion of extracellular nutrients across the membrane.23,24 Another advantage of this macroporosity is that the attachment of the membrane to tissue may stabilize and restrict the migration of epithelial cells. However, this very property of tissue integration, despite helping to stabilize the wound, makes these membranes hard to remove. Macroporous and multiporous membrane characteristics are also related to the creation of sharp edges after cutting or bending the membrane and may facilitate microbial contamination of the healing site, especially in the oral cavity. The development of a Ti-mesh membrane with fewer and smaller pores with maximal healing properties and fewer drawbacks (such as bacterial contamination
and tedious surgical removal) would be an important advance in the field of GBR.25 Thus, the aim of this study was to investigate the effects of differences in the physical variables (namely, thickness and porosity) of newly developed Ti-mesh membranes to determine the optimal structure of these membranes for GBR treatment.
MATERIALS AND METHODS The experiment was carried out on 100 male 6-weekold Sprague-Dawley rats (body weight 160 to 185 g), which were divided into 20 study groups (nine test materials and one passive control for each of two time courses) of five animals each. Animals were kept in a standard cage in an experimental animal room, fed with standard laboratory food pellets, and given water ad libitum. This study was approved by the Committee for Animal Research of Kyushu University (Approval Number: A24-182-0).
Membranes and Group Distribution
One group of animals was assigned to each of six different designs of novel Ti-mesh, three commercially available membranes, and one control (uncovered defect). The six novel membranes (Kyocera Medical Corporation) had a diameter of 12 mm; a thickness of 20, 50, or 100 µm; and either 12 pores or multiple pores with none at the coronal border (groups designated by T). Every mesh has a thickness with the same value as its pore diameter. Thus, six groups with novel Ti-mesh were established: 20, T20, 50, T50, 100, and T100. The three commercially available membranes used in this study were Titanium Micro Mesh (TM group), BioMend (BM group), and Cytoplast (CP group). Titanium Micro Mesh (ACE Surgical Supply Co) has a 100-µm thickness and 1,700-µm pore diameter. BioMend (Zimmer Dental Inc) is a type I resorbable collagen membrane derived from bovine tendon and has a 170-µm thickness and 0.004-µm pore diameter. Cytoplast GBR200 (Osteogenics Biomedical Inc) is a dense PTFE–type membrane with 200-µm thickness and pore diameter < 0.2 µm. The control (C) group consisted of animals with uncovered defect sites. All membranes had a diameter of 12 mm and were adapted to the surrounding bone and tissue at the experimental sites.
Anesthesia and Surgical Procedures
All surgery was performed in aseptic conditions under systemic and local anesthesia. The forehead of the rat was shaved and disinfected with tincture of iodine. During surgery, an incision about 2 cm long was made with a scalpel along the sagittal suture. Then, a dermoperiosteal flap was reflected to reveal the parietal bone. The International Journal of Oral & Maxillofacial Implants 827
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Rakhmatia et al
Fig 1a A 7-mm-diameter bone defect was created on the calvarium.
Fig 1b Titanium membrane was laid over defect area and margins were glued with Histoacryl to prevent dislodgement. In the control group, the defect site was left empty.
7 mm
Fig 1c Micro-CT view of titanium membrane covering the defect area.
Fig 1d The region of interest incorporating the calvarial defect is shown as delineated for CT scan analysis. 7 mm
A circular, full-thickness, 7-mm-wide bone defect was created with a surgical trephine bur under saline irrigation to prevent overheating of the bone margins. Care was taken during the surgical procedure to prevent damage to the dura. The defect site was covered with the test membrane, and the membrane borders were bonded to the bone with Histoacryl glue (B. Braun) to prevent membrane movement (Figs 1a and 1b). Finally, the skin flaps were carefully sutured in place with a nonresorbable suture material. All animals received a postsurgical antibiotic dose of oxytetracycline (0.5 mL/kg body weight). After 4 or 8 weeks, the animals were euthanized and perfused with a fixative solution consisting of 0.1 mol/L phosphate-buffered 4% paraformaldehyde (pH 7.4). Block biopsy specimens that included the membranes, the surrounding soft tissue, and the underlying calvarial bone were obtained.
Microcomputed Tomography Analysis
Unprocessed bone biopsy specimens were imaged and analyzed using in vivo microcomputed tomography (micro-CT) (SkyScan 1076, SkyScan) at 60 kV/167 µA and a Ti-0.5 filter. The specimens were fitted into a cylindric sample holder in a horizontal position and scanned parallel to the coronal aspect of the calvarial bone. High-resolution scanning with an in-lane pixel
size and slice thickness of 18 µm was performed. The micro-CT scanner software was used to make a threedimensional (3D) reconstruction from each set of scans. From the entire 3D data set, a cylindric region of interest with a diameter of 7 mm and a height comprising the total depth scanned was selected for analysis (Figs 1c and 1d). Region of interest analysis was performed to assess primary parameters, namely, bone volume (BV) and total tissue volume (TV), both measured in mm3. BV was calculated as the volume of the region characterized as bone (defined as the number of voxels, with gray values in the range 30 to 130) and normalized ratiometrically against the total volume of the region of interest (BV/TV) to derive the percentage bone volume (%BV). Bone with different degrees of mineralization displays different densities and linear attenuation coefficients, resulting in gray value variations in the CT scans, the distribution of which is an indication of the degree of mineralization, ie, bone mineral density (BMD) (g/cm3).
Histologic and Histomorphometric Evaluation
Upon completion of micro-CT scanning, the specimens were dehydrated in a graded series of ethanols and embedded in methacrylate resin. Undecalcified sagittal sections (thickness ~60 µm) were cut, polished,
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20–4w
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Fig 2 Micro-CT 3D reconstructions of typical rat calvarial defects 4 weeks after membrane placement. Newly formed bone is shown between the red lines.
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Fig 3 Micro-CT 3D reconstructions of typical rat calvarial defects 8 weeks after membrane placement. Newly formed bone is shown between the red lines.
and stained with Masson’s trichrome. To evaluate the bone and cellular tissue responses histologically, the samples were examined under a light microscope. The center of the test membrane from one histologic section of each specimen was selected to represent that group for evaluation. Histomorphometric evaluation was used to trace and measure the area of connective tissue beneath the membrane.
Statistical Analysis
The mean values and standard deviations for each parameter were calculated for each group/healing time. A one-way analysis of variance with post hoc Tukey test was performed to analyze 3D BV and BMD values in the defect region of interest from the micro-CT analysis. Statistical analysis was also performed to evaluate the area of soft tissue beneath the membrane. Values of P < .05 were considered to indicate statistical significance.
RESULTS All rats recovered well and gained weight throughout the postoperative healing period. There were no clinical signs of infection or rejection of the membranes or other ailments during the healing period. Among the different membrane materials tested, the TM, 100, and T100 were the stiffest and were rather difficult to adapt to the bone surface contour compared with the 50, T50, 20, and T20 membranes. The margins of the BM and CP membranes, after bending, were felt to have inadequate stiffness. Three-dimensional micro-CT reconstructions of a typical rat calvarial defect were created for all groups. Bone regeneration progressing toward the center of the defect was commonly observed at 4 weeks and increased by 8 weeks in the defect areas with membranes (Figs 2 and 3). In contrast, no or minimal bone regeneration was observed in the C groups after either 4 or 8 The International Journal of Oral & Maxillofacial Implants 829
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Figs 4a and 4b Percentage of new bone volume elucidated from 3D micro-CT reconstruction of a typical rat calvarial defect at (a) 4 weeks and (b) 8 weeks after membrane placement (n = 5 per group). C = control (no membrane). *,**: compared to C; †,††: compared to 100; ‡,‡‡: compared to TM; §,§§: compared to 50; || || : compared to CP; ¶, ¶ ¶: compared to T100. *, †, ‡, §, ¶ : P < .05; **, ††, ‡‡, §, || ||, ¶ ¶: P < .01.
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Figs 5a and 5b BMD from 3D micro-CT reconstructions of a rat calvarial defect at (a) 4 weeks and (b) 8 weeks after membrane placement (n = 5 per group). C = control (no membrane). *,**: compared to CP; †,††: compared to 100; ‡,‡‡: compared to TM; §,§§: compared to BM. *, †, ‡, §: P < .05; **, ††, ‡‡, §§: P < .01.
weeks of healing (Figs 2 and 3). The quantitative results derived from micro-CT analysis are shown in Figs 4 and 5. The novel Ti-mesh with 12 pores (20, 50, 100) showed a higher %BV compared with the mesh with multiple pores (T20, T50, T100) at both 4 and 8 weeks. Values for %BV at 8 weeks tended to increase as the thickness of the mesh increased. BMD values were derived by comparing x-ray attenuation in the scanned bone samples with that in hydroxyapatite standards. CP had the highest mineralization level; for the other membranes, the mineralization level tended to decrease as the thickness of the mesh increased. BMD was higher in the novel membranes with multiple pores than in those with only 12 pores. Significant differences (P < .01) were found with CP versus 100, TM, and BM. Histologic analysis of all groups (Figs 6 and 7) complemented the micro-CT findings. In some instances, the gap containing the Histoacryl tissue adhesive used to anchor the membrane and stop it from moving was evident between the border of the membrane and the bone margin. Some of the membranes deemed
to have inadequate stiffness (20, T20, BM, and CP) had visibly collapsed into the defect site. At 4 weeks, bone formation with more intense red staining was observed in sites with thicker membranes and in sites with membranes with larger pore diameters. Moreover, more bone formation was observed beneath the novel membranes with 12 pores compared with those with multiple pores. In the control group, the defect sites were filled mainly with fibrous connective tissue, and negligible new bone had formed. After 8 weeks of healing, all groups exhibited a greater and more vascularized body of new bone with a higher degree of mineralization than in the respective group after 4 weeks of healing, and the defect margins were indistinguishable from the newly formed bone. In all of the groups with 12 pores, the area of new bone formed was more expansive than in the membranes with multiple pores. Membranes with the greatest thickness (100, T100, and TM) showed the most extensive new bone formation, with some specimens exhibiting complete resolution of the defect.
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20–4w
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Fig 6 Histologic sections from each specimen at 4 weeks (bar = 300 µm). Double-headed arrows = new bone formation; single arrows = membrane collapse into the defect site.
Fig 7 Histologic sections from each specimen at 8 weeks (bar = 300 µm). Double-headed arrows = new bone formation; single arrows = membrane collapse into the defect site.
The connective tissue area beneath the membranes was measured (Fig 8). Fibrous connective tissue was found to have infiltrated into the gaps between the bone and the membrane structure, and also through the pores, and tended to be most prolific in membranes that were thick and/or had large pores. After 4 weeks, the 100 and T100 membranes displayed the largest areas of soft tissue ingrowth compared with the other groups. However, at 8 weeks, there was no significant difference between the specimens. Fibers and cells were observable in the defect area, having penetrated the pores of some titanium membranes, especially those in the T20, T50, T100, and TM groups. No such ingrowth was evident in the defect area beneath the
novel membranes with 12 pores (20, 50, 100), CP, and BM, which all therefore remained free of cell ingrowth, although these did display some soft tissue ingrowth through the gaps at the margins of the membrane.
DISCUSSION Rat calvaria were used in this study to test biomaterials intended for use in the regeneration of critical-size bone defects. Calvarial defects are a highly suitable experimental model because their poor blood supply and the membranous structure of the bone (low mechanical stimulation) preclude any spontaneous The International Journal of Oral & Maxillofacial Implants 831
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Connective tissue area (× 106 μm2)
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Membrane types
Fig 8 Connective tissue area beneath the membrane estimated by histomorphometric measurement of the middle sagittal section of the rat calvarial defect after 4 weeks and 8 weeks of healing. C = control (no membrane). *P < .05 compared to 100.
healing.26 A critical defect size of 5 mm in rat calvaria has been defined as the smallest intraosseous wound that does not heal spontaneously during the lifetime of the animal,27 although another study estimated this to be larger, at 8 mm in diameter.28 Given the skull size of the animals used (and thus the available calvarial area), the defects created in this study were as large as practicable to still fulfill the requirements for a criticalsize defect for testing the biomaterials used here. The results of this study proved that a 7-mm defect healed with fibrous tissue when left uncovered but healed with bone in the presence of a membrane. Despite the use of Histoacryl to prevent membrane dislodgement, some movement still occurred. Therefore, sufficient fixation of the membrane is vital for proper wound healing and blood clot stabilization. This fixation also prevents the membrane micromovement that has been shown to be detrimental to bone formation and can result in the development of fibrous tissue instead of bone, particularly if movement occurs during the healing process.29 The present study confirms earlier reports that a membrane must be sufficiently rigid to maintain the space of the regenerating bone area beneath it. A tendency to collapse into the bone defect limits the space into which the bone can regenerate.3,4,30 Nonabsorbable dense PTFE membranes (such as the CP used here) tended to collapse in this way, resulting in less bone formation. Despite the good BVs that were seen beneath the resorbable membrane (the collagen-based BM) after 4 weeks, some specimens at 8 weeks appeared to have lost their stiffness, resulting in lower BVs at this time point compared with titanium membranes. As indicated in previous reports, biodegradation of BM is caused by the enzymatic activity of macrophages and polymorphonuclear leukocytes, resulting in poor membrane resistance to collapse and
therefore limiting bone formation.31 The degradation of cross-linked BM membranes was also associated with decreased tissue integration and vascularization.32 Moreover, the CP and BM membranes have a fairly soft consistency, which may have limited resistance to the soft tissue pressure exerted on them from above, resulting in their collapse. These findings suggest that autogenous bone or bone substitutes should be used beneath CP and BM barriers to provide additional support. The rigidity of Ti-mesh allows it to overcome these problems of membrane collapse and thus to maintain space without the requirement for additional materials.13 The thickness of any material is a primary factor in its stiffness. A range of thickness between 100 and 200 µm was reported to be ideal for the reconstruction of large bone tissue defects and for advanced deformative and moderate atrophic alterations.33 The current findings concur that a thickness of 100 µm in titanium membranes (100, T100, and TM) results in more extensive bone formation than thinner membranes. However, the T100 membranes broke easily during surgical manipulation, which resulted in membrane collapse and subsequent decrease of the bone volume at 4 weeks compared with other titanium membranes that were 100 µm in thickness. The 20 and T20 groups had the lowest stiffness; consequently, some collapsed into the defect area, resulting in the lower BVs compared with the thicker titanium membranes. Others have reported that a 50-µm membrane made of titanium foil has the least tendency to collapse compared with other types of membrane.34 A balance must be found between the thickness of mesh that is required for stability and the need for it to be malleable enough to be adapted to the contours of the adjacent bone.3 Despite membrane permeability not being a prerequisite for GBR,35 previous investigations have concluded that the pore size must be large enough to allow angiogenesis into the new bone (ie, at least 40 µm); limited pore size can hinder angiogenesis.36 This must be weighed against the need to restrict the invasion of fibrous connective tissue into the defect area. GBR may require selectively occlusive barriers for successful bone regeneration,37 although good results have been observed with perforated titanium membranes that are nonocclusive,38,39 so this requirement is clearly not absolute. It has been suggested that occlusive membranes may hamper the penetration of nutrients and growth-regulatory factors into the protected site, increasing the frequency of soft tissue dehiscence and thereby inhibiting bone formation.24,34 Here, an increased BV beneath those membranes with larger pores (TM, 100 groups) was consistent with other studies reporting greater bone development
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underneath membranes with larger pores.40,41 Bartee and Carr described complete healing of a rat mandibular defect treated with a dense PTFE membrane for 10 weeks after surgery,23 but the present study suggests that membranes with small pores (such as the dense PTFE–based CP material used here) seem to block the integral vascularization process and therefore inhibit bone formation in the defect area. Other studies investigating bone ingrowth into systems with different pore sizes showed that a pore size of 100 µm allows bone ingrowth, but that a pore size greater than 150 µm is required for osteon formation.42,43 In contrast, the present study found no significant pore size–related difference in bone development at 8 weeks, consistent with other studies showing that bone ingrowth is possible through any pore larger than 50 µm.43 Another previous study went further, concluding that pore size has no influence on the healing response or on the clinical outcome.40 Increasing barrier porosity may promote early osteogenesis but not the final amount of new bone that is formed,34 concordant with the results showing that membrane porosity is an essential factor for GBR in the initial healing period but that the final BV obtained is essentially the same. The fact that different rates of bone formation were found indicates that pore size is not the only relevant factor of membranes in osteogenesis. Indeed, bone formation underneath a membrane is governed by a variety of factors, including the physical and surface characteristics of the material, its thickness and porosity, its chemical composition, and the depth at which it is placed within the tissue.44 Other local environmental factors may also influence membrane effects. Hydrophilicity of the membrane is part of the physical and surface characteristics of the material. This characteristic may more take into account biocompatibility of a membrane, which allows the cells to grow and subsequently to create integration between membranes and surrounding tissue compared with membrane’s other characteristics. Moreover, hydrophilicity affects cell adherence to a membrane’s surface. However, CP, because it is a dense PTFE membrane, has lower hydrophilicity and therefore may decrease cell adhesion, as shown by a previous study that investigated cell attachment to various membranes, including PTFE.45 The minimal tissue integration of the CP membrane may be advantageous for membrane retrieval. However, it may also create potential problems for initial clot formation, wound stabilization, and membrane stability and thus may interfere with wound healing. BM was reported to be more hydrophilic and is known to modulate various cell behaviors, such as adhesion, proliferation, and migration, because of its chemotactic capability.46 Hydrophilic titanium membrane
surfaces were also shown to influence cell differentiation and growth factor production.47 Interestingly, despite the correlation between large pore sizes and greater new BV, membranes with large pore sizes also appeared to be linked with lower bone density, especially compared to the CP membranes at 4 weeks (P < .01). Membranes with multiple pores had higher BMD values than membranes with only 12 pores. If bone is growing from the edges of the pores into the center, it is intuitive that a material with many small pores will have a greater number of “growth centers,” thus producing better-quality (ie, higher-density) bone. It is possible that mineral apposition might be incomplete when materials with a large pore size are used because the new bone takes time to grow into these “unsupported” areas.40,41 Previous studies have shown that soft tissue ingrowth is greater when using Ti-mesh with a large pore size (> 2 mm) than with a small pore size (< 2 mm).9 Histologic results showed that membranes with small pore sizes tended to permit less ingrowth of connective tissue. The soft tissue layer beneath the CP, 20, and T20 membranes, which have few pores of minimal size, was thinner and had parallel-structured fibers, compared with the dense and multilayered fibrous tissue found under the 100, T100, and TM membranes. A previous study confirmed this correlation between pore size and connective tissue ingrowth; it found that such growth was absent beneath a completely occlusive titanium barrier (ie, where pore size = 0).18 It is suggested that pores in excess of 100 µm are required for the rapid penetration of highly vascular connective tissue, and small pores tend to become filled with more avascular tissue.48 The dense connective tissue that was found beneath some membranes could be the result of insufficient adaptation of the membrane to the surrounding bony tissue. Connective tissue growth under the membrane is thought to be caused by various factors, including insufficient peripheral healing (ie, sealing) between the material and the bone, ingrowth of connective tissue through the pores of the barrier, and insufficient stability of the wound area.49 However, after 8 weeks, the connective tissue content had decreased and bone was increased in the defect area, in agreement with a previous study observing that, with sufficient initial stability, the early tissue infiltrate will differentiate into bone, either by direct bone formation or via appositional growth from adjacent bone.42 There are no well-defined rules for determining the sample size needed for a reliable study. The relatively small sample size of five rats in each group decreases the statistical power of this study. Therefore, within the lack of quantitative variables, the results must be recognized to have limitations. The International Journal of Oral & Maxillofacial Implants 833
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CONCLUSIONS The novel titanium meshes used here augmented new bone in this critical-sized rat calvarial defect model as effectively as commercially available membranes after 4 and 8 weeks of healing. Of the thicknesses tested here, 100-µm-thick titanium mesh is recommended for the reconstruction of bone tissue defects because of its ability to support new bone growth into the defect area. Moreover, membrane porosity is another essential factor for guided bone regeneration, especially during the initial healing period. Further studies of larger defects in larger animals will be required to further advance the knowledge of the optimal properties of titanium meshes for the resolution of bone defects.
ACKNOWLEDGMENTS This study was conducted with financial assistance from Kyocera Medical, Osaka, Japan. The authors reported no conflicts of interest related to this study.
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