How to cite this article: Asoda S, Arita T, Takakuda K. 2013. Mechanical attachment of soft tissue to dental and maxillofacial implants with mesh structures: An experiment in percutaneous model. J Biomed Mater Res Part B 2013:101B:553–559.
Mechanical attachment of soft tissue to dental and maxillofacial implants with mesh structures: An experiment in percutaneous model†
Article first published online: 20 DEC 2012
Copyright © 2012 Wiley Periodicals, Inc.
Journal of Biomedical Materials Research Part B: Applied Biomaterials
Volume 101B, Issue 4, pages 553–559, May 2013
How to Cite
Asoda, S., Arita, T. and Takakuda, K. (2013), Mechanical attachment of soft tissue to dental and maxillofacial implants with mesh structures: An experiment in percutaneous model. J. Biomed. Mater. Res., 101B: 553–559. doi: 10.1002/jbm.b.32855
- Issue published online: 9 APR 2013
- Article first published online: 20 DEC 2012
- Manuscript Accepted: 10 OCT 2012
- Manuscript Revised: 23 SEP 2012
- Manuscript Received: 9 MAY 2012
- Grant-in-Aid for Scientific Research on Priority Areas (Ministry of Education, Culture, Sports, Science and Technology, Japan). Grant Number: 15086206
- Grant-in-Aid for Scientific Research (B) (Ministry of Education, Culture, Sports, Science and Technology, Japan). Grant Number: 17300145
- dental implant;
- maxillofacial implant;
- soft tissue;
- mechanical attachment;
- mesh structure;
- implant interface
Soft tissue attachment is a major concern for the improved design of dental and maxillofacial implants. This study evaluated the efficacy of mesh structures for soft tissue attachment in a rat percutaneous model. Four kinds of implant specimens were prepared — TI implants made of titanium cylinders, HA implants of hydroxyapatite-coated titanium, TI-Mesh implants with a titanium mesh covering a groove machined around a titanium cylinder, and similar HA-Mesh implants with a hydroxyapatite-coated mesh. These specimens were implanted percutaneously into the skin tissue of rats. The detachments of the implants were examined during the experimental period of 4 weeks. Survived implants were subjected to mechanical tests for the attachment strength and histological examinations. TI and HA implants demonstrated 0% of survival rates, while TI-Mesh and HA-Mesh showed significantly higher rates of 93.3% and 100% respectively. The attachment strengths were 159 ± 47 kPa in the TI-Mesh and 135 ± 16 kPa in the HA-Mesh. Histological observations revealed that collagen fibers originating from surrounding subcutaneous tissues were anchored to the mesh structures of the TI- and HA-Mesh implants. The results demonstrated the efficacy of the mesh structures for the attachment of soft connective tissues to implants. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.
Dental and maxillofacial implants currently utilized in clinical applications are based on well-proven technologies enabling firm bonding of artificial materials to the bone tissues, namely osseointegration.1, 2 On the other hand, the bonding of materials to soft tissues have not achieved yet and considered to be of significant importance for the improved design of implants.
In fact, soft tissue attachment is established between a healthy tooth and gingiva. The dentogingival fibers that extend from subepithelial connective tissue into the cementum of the root's cervix firmly attach the tooth to gingiva. In the case of the gingiva around the implants, however, the collagen fibers in the peri-implant gingival tissues cannot anchor to the implants and run parallel to their surface.3–6 Hence, soft tissue attachment cannot be realized between the implant and the gingiva. This lack of tissue attachment is considered to cause several adverse effects such as increased risks of micro-organism's invasion that induces inflammation reaching to bone tissues7 and bone resorption.8–11
The technology realizing the bonding of soft tissues to artificial materials will enable us to create a novel implant to which surrounding soft tissues will spontaneously attach. Although only few investigations have been reported on this subject, we recently have performed experiments12 to elucidate effective structure for the anchoring of collagen fibers. The animal experiments demonstrated that placement of a mesh with a spacing of approximately 200 μm on the implant's surface was favorable for soft tissue attachment. The anchoring of collagen fibers to the mesh was demonstrated and a significant increase in the attachment strength was attained.
These findings were, however, obtained for the case of specimens implanted subcutaneously. The percutaneously implanted specimens are considered to be in an environment closely resembling that of dental implants and are in the same environment of maxillofacial implants utilized onto the skin tissues to attach epithese for oral and facial deficiencies. Hence, in this study, we prepared implant specimens and percutaneously implanted them in the skin of rats. After 4 weeks of the experimental period, the specimens were harvested with the surrounding tissues and evaluated mechanically and histologically to elucidate the efficacy of the mesh structures in anchoring the collagen fibers. Furthermore, implants with surfaces coated by hydroxyapatite (HAp) were also prepared and the effect of the coating on soft tissue attachment was examined.
MATERIALS AND METHODS
Preparation of specimens
The materials used for the specimens were commercially pure titanium rods and meshes (Nilaco Co., Tokyo, Japan). The dimensions of the titanium mesh were inspected with a measuring microscope (VH-8000; Keyence, Osaka, Japan); the fiber diameter and mesh spacing were 120 μm and 213 μm, respectively.
Four types of implant specimens were prepared in this study, they were designated as TI, HA, TI-Mesh, and HA-Mesh. Photographs of these specimens are shown in Figure 1. The dimensions of them are presented in Figure 2, together with the schema of implantation. TI was the basic implant specimen and had a cylindrical form with a diameter of 10 mm and a height of 6 mm. It also had six small holes on its upper part for securing the specimen to surrounding tissues with sutures. Further, a female screw hole with a diameter of 5 mm was provided on the upper surface, which was used to connect a jig in mechanical testing as described later. HA was a specimen made of hydroxyapatite (HAp) coated titanium, while its geometry was the same as TI specimens. The coating was performed with a thermal decomposition method (Platon Japan, Tokyo, Japan) and the thickness of the coating layer was 3–5 μm. TI-Mesh was a titanium specimen having a titanium mesh covering a groove, 3 mm in width and 0.5 mm in depth, machined on its lateral surface. The mesh was adhered to the main body of the specimen by a dental adhesive (Super-Bond C&B; Sun medical, Shiga, Japan). Similarly, HA-Mesh was a titanium specimen having a HAp coated titanium mesh covering a lateral groove. The specimens thus prepared were rinsed with 70% ethanol, air dried, sterilized with EOG, and used for further experiments.
Implantation of specimens
Twenty-five, 8-week-old male Sprague Dawley rats (body weight: 250–300 g) were used in the experiment. Each animal was implanted with randomly selected two specimens in the dorsal back skin. Pieces of full-thickness cutaneous tissues 10-mm in diameter were resected from the left- and right-side skins. Subsequently, the specimen was placed on the exposed subcutaneous tissue and sutured to the skin surrounding it with 4-0 Nylon, as shown in Figure 2. The number of specimens was 10 for the TI and HA specimens and 15 for the TI-Mesh and HA-Mesh specimens. The implantation period was 4 weeks. The experiment was conducted in compliance with the protocol that had been approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University and was in compliance with the committee's guidelines.
Daily observation after implantation and harvest of specimens
After the surgery, the implant specimens and the surrounding tissues were inspected everyday and the specimens that got detached from the skin were registered. Thus the survival rates were obtained for four types of specimens. After 4 weeks of implantation, the survived specimens were harvested together with the sheet of the surrounding cutaneous tissues and were used for mechanical tests or histological examinations.
The attachment strengths of TI-Mesh and HA-Mesh specimens to the surrounding cutaneous tissues were measured by pull-out tests (n = 10, respectively) in which a testing machine (Model 1185; Instron, Canton, MA, USA) and a load cell (LUR-A-50NSA1; Kyowa Electronic Instruments, Tokyo, Japan) were used. The schema of mechanical test is illustrated in Figure 3. The cutaneous tissues were sandwiched by stainless steel plates with center holes of 12-mm diameter and fixed to the lower crosshead of the testing machine. The implant specimen was connected to the load cell fixed on the upper crosshead via a universal joint. The mechanical test was performed with a crosshead speed of 2 mm/min. Noticing that the nominal area of the mesh facing the tissues was calculated to be 94.2 mm2 [3 mm (the width of the effective mesh) × 3.14 × 10 mm (the diameter of the specimen)], the peak load measured was divided by this nominal mesh area and the resulting value was determined as the attachment strength.
TI-Mesh and HA-Mesh specimens (n = 5, respectively) were harvested with surrounding cutaneous tissues and fixed with 10% formalin for 7 days. They were then dehydrated in a graded series of ethanol, defatted with acetone, and embedded in methylmethacrylate resin. The resin block was sectioned longitudinally with a diamond saw and grounded to a thickness of 150 μm. The nondecalcified ground sections thus obtained were stained with toluidine blue and observed with a light microscope. Further, the collagen fibers in these sections were observed with polarized light.
For the survival rates of TI, HA, TI-Mesh, and HA-Mesh specimens, Fisher's exact test extended to a multiway contingency table was performed to detect significant effects of the specimen's type on the survival rates. Fisher's exact tests in combination with Bonferroni correction were then carried out for the post hoc multiple comparisons of the survival rates among experimental groups. The attachment strength of TI- and HA-Mesh specimens was analyzed by two-tailed unpaired Welch's t test for the difference between the means of the two groups. Statistical differences were assumed for p < 0.05. All statistical analyses were performed using an open-software R.
Survival rates of implants
No infection was observed in each experiment during the entire period of implantation. The sutures fixing the specimens to the surrounding tissues broke 3–10 days after surgery. Thus these sutures did not contribute to the fixation of the specimens to the skin thereafter. The survival rates of the four types of specimens are presented in Figure 4. All of TI and HA specimens (n = 10, respectively) got detached from the skin in 3–14 days (mean: 9.3 days) and 7–16 days (mean: 11.3 days) after implantation respectively. On the other hand, only 1 of 15 TI-Mesh specimens got detached out of the skin on 6th day, and none of 15 HA-Mesh specimens dropped. Thus, the survival rates were 0% for TI and HA, 93.3% for TI-Mesh, and 100% for HA-Mesh. Fisher's exact test revealed significant effects of the specimen type on the survival rates (p < 0.01). Multiple comparisons performed by using Fisher's exact tests in combination with Bonferroni correction demonstrated that the survival rates of TI- and HA-Mesh specimens were significantly greater than those of TI and HA specimens (p < 0.01). However, no significant difference was observed between TI and HA specimens and between TI-Mesh and HA-Mesh specimens.
Attachment strengths of the specimen to surrounding soft tissues
Attempts to perform the mechanical evaluation TI and HA specimens for the attachment strength to surrounding cutaneous tissues were abandoned since all specimens were detached from the skin. Therefore, only TI- and HA-Mesh specimens were subjected to mechanical tests (n = 10, respectively). The typical load-displacement curves observed in the test are presented in Figure 5. In every test, the soft tissue attached to the mesh was elevated according to the upward displacement of the implant specimen. The peak load was recorded when the displacement reached approximately 2 mm, and the soft tissue was torn from the mesh. All the failure took place at just outside of the mesh; the meshes themselves were kept intact and the tissues were found within the groove. The mean peak load and the attachment strength were 15.0 ± 4.7 N (mean ± standard deviation) and 159 ± 47 kPa respectively in TI-Mesh specimens, and they were 12.7 ± 1.6 N and 135 ± 16 kPa in HA-Mesh specimens, as shown in Figure 6. No statistical difference was observed between the two groups (p > 0.05).
Four of 5 TI-Mesh specimens and all of five HA-Mesh specimens survived and were subjected to histological observations. Typical microscopic observations of the sections stained with toluidine blue stain are shown in Figures 7(A,B). The higher magnification observation of collagen fibers with polarized light is shown in Figures 7(C,D). The histological findings on the TI- and HA-Mesh specimens were similar. At the bottom of the specimens, collagen fibers were aligned parallel to the specimen surface. In contrast, on the surface of the mesh, they were aligned almost perpendicular to the mesh and penetrating into the groove under the mesh, as shown in Figures 7(C,D). The epidermis, dermis, and subcutaneous tissues were also penetrating into the groove. The groove was mainly filled with the subcutaneous tissues and the gaps observed between the groove wall and the soft tissue in Figure 7 were considered as an artifact produced during the preparation of histological sections. The epidermis-dermis border was located in the upper portion of the groove and the dermis-subcutaneous border was located in the middle to upper portion of the groove. Further, no significant downgrowth of the dermal tissue was observed.
The structure of soft tissues around the dental and maxillofacial implant differed from that around the natural tooth. In the latter case, the collagen fibers from the surrounding soft tissues were anchored to the cement layer and they thus established soft tissue attachment to the tooth.13 On the other hand, the implant does not have a cement layer, hence the collagen fibers from the surrounding soft tissues are not anchored to the implant and are alligned parallel to the surface of the implant.3 Thus, the implant and the adjacent soft tissues lack integrity that leads to inferior marginal sealing and invokes potential risks including infections.7
The issue described above might be resolved if we can realize the anchoring of collagen fibers from soft tissues to artificial materials. In this context, porous materials were investigated to construct a structure for soft tissue attachment.14–19 Most of these reports, however, assumed the application to percutaneous devices. The attachment structure using porous materials was too thick for the dental and maxillofacial implant. Hence, we are investigating a mesh structure placed on the surface of the implant for soft tissue attachment. Such a use of the mesh may possibly aids the realization of an interface thinner than that obtained by using porous materials. In a previous report,12 we demonstrated that the collagen fibers from subcutaneous tissues were favorably anchored to a mesh with a 200-μm spacing. The specimens were, however, completely submerged in the subcutaneous tissue and were in a different condition to that around of dental and maxillofacial implants. Hence, in this investigation, we implanted the specimens into the dermal tissue in a percutaneous manner and examined the attachment of the dermal tissue to the specimens.
The material of the specimens was titanium, which is used in clinically available dental and maxillofacial implants. Half of the specimens were coated with HAp, with the expectation that the HAp layer and collagen fibers could possibly become attached. The coating procedure was the same as that used for clinically utilized products. The specimens were placed on the exposed subcutaneous tissues and fixed to the skin percutaneously to replicate the condition around the dental and maxillofacial implant. The implantation period was set to be 4 weeks, and then the specimens were harvested together with the surrounding tissues for mechanical and histological evaluation. Paquay et al.17 investigated their experimental percutaneous device fabricated with a sintered titanium fiber mesh and reported that acute inflammation due to surgery continued for 2 weeks and the necessary healing period was approximately 3 weeks. Hence, the 4-week implantation in this study, although short, was sufficiently long for examining the state of the attachment immediately after initial healing. In the measurement of the attachment strength, the pull-out test similar to that utilized for the evaluation of osseointegration was employed. For the evaluation of soft tissue integration, it is necessary to measure the tearing force required for the detachment of the soft tissue from the implant. The tensile test adopted in the previous report12 enabled us to evaluate the tensile strength that is corresponding to the resistance stress when the gingiva is torn from the implant. Still the test involved difficulties in the test-piece preparation and data processing. The direction of the force is perpendicular to the implant in the case of tensile tests and is tangential in the case of pull-out tests; however, both tests enable us to measure the force necessary to tear the soft tissues from implants. Therefore, in this study, the simple pull-out test was utilized.
The experimental result on the survival rates of specimens demonstrated the statistical difference between TI and HA groups and TI- and HA-Mesh groups as Figure 4 shows. No significant difference was found between TI and HA groups and between TI-Mesh and HA-Mesh groups. Thus, the mesh structure was found to significantly increase the survival rates, whereas the HAp coating did not. We considered that this difference was attributed to the fact that TI- and HA-Mesh groups had larger attachment strengths as compared to TI and HA groups. Furthermore, as demonstrated in Figure 5, the attachment between the soft tissue and the mesh did not fail until the load and displacement reached values as large as approximately 10 N and 2 mm, respectively. Noticing the fact that the thickness of the periodontal ligament was reported to be 0.1–0.3 mm,20 the possible displacements of the tooth and the dental implant were <0.3 mm. Hence the measured maximum displacement of 2 mm was considered to be sufficient to resist the normal displacement at least in an intraoral environment.
Histological observations revealed penetration of the epidermis, dermis, and subcutaneous tissues through the mesh and filling of the space in the groove. Further, observation in the polarized light demonstrated the collagen fibers from the surrounding subcutaneous tissues were penetrating the mesh and entering into the tissues in the groove, as Figure 7 shows. Although relatively thick histological sections in this investigation hinder us to perform detailed observations, thin paraffin sections utilized in our previous study12 revealed the anchoring of collagen fibers to the mesh. Although the nylon mesh was replaced by more biocompatible Ti mesh or HAp coated Ti mesh, the mesh spacing and the size were same as the previous. The collagen fibers penetrating through the mesh structure were confirmed again, and these collagen fibers were considered to effectively anchor the surrounding tissues to the implant and increase the survival rates and attachment strength. On the other hand, the geometry of the specimen used in this study resulted in the facing of the epidermis tissues to the mesh surface of the specimen, and the invasion of epithelial tissues into the groove. The measurements of the extents of invasion were abandoned due to the difficulty in pointing out the precise location in the thick histological sections. Nevertheless, the invasion did not cause any problem such as infection even though it is considered desirable that the epidermis tissues face the upper part of the specimen and the dermis tissues face the mesh structure.
The mucosal barrier of the peri-implant tissues comprised of an epithelium layer of approximately 2-mm thickness and connective tissues of 1–1.5 mm thickness.21, 22 Considering these geometries, the most desirable width of the mesh structure in the dental implants would be 1–1.5 mm. If a mesh structure of this width was placed at a level just above the alveolar crest, the connective tissues would face the mesh and a favorable soft tissue attachment would be realized. Such requirement for the precise placement of implants will yield certain difficulty in the clinical application. On the other hand, the mesh for soft tissue anchoring can be effectively applied to the implants for maxillofacial reconstruction. In this case, the dermis tissue around the implant is thicker than the lamina propria of the mucous membrane. Hence, a favorable mesh width might be 2–3 mm. Soft tissue attachment is considered to be useful to decrease possible risks, including the downgrowth of the epidermis tissues, in such implants.
Several investigations demonstrated the improved soft tissue attachment to the modified surface of implants. For example, the efficacy of rough surfaces created by titanium plasma splay or coarse blasting was proved by Kim et al.23 and that of surfaces coated with sol–gel-derived TiO2 by Paldan et al.24, 25 These reports, however, examined the attachment only with the morphological observations of histological sections and no functional evidences based on the strength measurement were provided. The increase of attachment strength was, to the authors' knowledge, not reported elsewhere except our previous article.12 It was highly probable that the anchoring of collagen fibers shown in our studies was crucial for the increase of attachment strength. Furthermore, such a realization of soft tissue anchoring was considered to be important as the first step toward the periodontal ligaments regeneration around the dental implants.
In conclusion, the mesh structure was presented as a candidate for the mechanical attachment of soft connective tissues to dental and maxillofacial implants, and its potential efficacy was demonstrated. The favorable surface of the implant for the attachment of bone tissues or osseointegration was well established.1, 26, 27 Similarly, a favorable surface for epithelial tissues is possibly the polished surface and that for the soft connective tissues will be the mesh structure proposed in this article. We considered that improved implants could be designed by combining these suitable surfaces for the respective tissues. However, further investigations that examine long-term stability of the attachment are necessary for clinical applications.
The authors express their gratitude to Mr. Takahiro Sakata and Mr. Leo Takanohashi for their assistance in the experiments.
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