Histologic studies on osseointegration: Soft tissues response to implant surfaces and components. A review

Abstract

It is important to clarify the potential response of different types of cells to different implant materials and topographies. Thus, in vitro studies are performed using cell cultures, in order to evaluate, among other characteristics, the morphology, orientation, proliferation and adhesion of the cells. Histology evaluation are performed in animals or humans to describe the physiological response to different surfaces.

Introduction

Titanium surfaces

Soft tissues around teeth and implant present anatomic similarities represented by the presence of an oral epithelium, continuous with a junctional epithelium. Differences are observed concerning the positioning of the most apical portion of the junction epithelium, which in tooth sections is approximated at the level of cementum–enamel junction; and in implant sections, it is at a variable distance from gingival margin. Another difference concerns the peri-implantar absence of cementum layer and Sharpey’s fibers. Consequently, collagen fiber bundles in teeth (dentogingival fibers, dento-periodontal fibers and circular fibers) are inserted perpendicularly to the surface, while in implant sites, a dense network of collagen fibers is observed, extending from the alveolar bone crest to the gingival margin, arranged parallelly in relation to the implant surface .

Connective tissue in contact to implant can be divided into 2 parts. The upper part, located under the JE, presents collagen fibers associated to Type III collagen, and is relatively rich in fibroblasts (with a great number of secretory elements, which may reflect an important turnover in this area). The lower part, closely bound to the implant, is poor in cells, and the extracellular matrix is represented by large and dense bundles of thick Type I collagen fibers, which may contribute to mechanical resistance and stability of the tissues . The fibers of connective tissue are closely adapted to the titanium, and are mainly oriented parallel to the implant surface, and there is no evidence of fiber insertion into the surface .

Histological analysis in dogs revealed that peri-implant tissues presents an inflammatory cell infiltrate at the level of implant/abutment junction, even at sites which had been exposed to plaque control. It is suggested that this infiltrate represents an effort by the host to limit the bacteria invasion, and this may contribute to the crestal bone loss observed after implant placement .

In histological analysis of human tissues, absence of inflammatory infiltrate was observed in the oral epithelium and in its underlying connective tissue, nevertheless lymphocytes and macrophages could be found in adjacent connective tissue. Moreover, the presence of epithelium was observed to be restricted to the abutment surface, not reaching the implant shoulder .

The dynamics of epithelium formation was demonstrated in a study developed in rats. After implant placement, an epithelial downgrowth occurs along the implants from the 3rd to 10th day post-implantation, and complete regeneration process completely finished by the 15th day post-implantation . The epithelium formed after implant placement, consists of an internal basal lamina, composed by a lamina densa and lamina lucida, similarly to that observed on the interface teeth-implant. The contact to implant surface is reinforced by the presence of hemidesmosomes, and the secretion of laminins and fibronectin. In implant sites, hemidesmosomes are observed only in the lower region and rarely in the middle region; while in teeth sites, they are found throughout the interface . Laminins are a component of the basement membrane that contribute to epithelial cell migration and adhesion, and which may be detected at periodontal and peri-implant sites; and fibronectins are extracellular matrix proteins present in serum, which mediate cell attachment to subtract .

The observation of the cellular behavior is important to clarify the potential response of different types of cells to different implant materials and topographies. Thus, laboratorial studies are performed using cell culture, in order to evaluate, among other characteristics, the morphology, orientation, proliferation and adhesion of them; while histologic evaluation are performed in animals or humans to describe the physiological response to different surfaces.

Considering specifically the epithelial cells, their phenotype, and attachment and spreading characteristics varies according to the surface . The initial attachment of cells on titanium ( R a = 0.05 μm), for example, is inferior to polystyrene ( R a = 0.03 μm) and glass surfaces ( R a = 0.03 μm) used as control , and higher than ceramic surfaces as alumina and dental porcelain .

Oral epithelial cells growth was evaluated in titanium sandblasted ( R a = 2.14 μm) and turned surface ( R a = 0.8 μm). In sandblasted surfaces cells presented varied morphology with numerous, long and branched or dendritic filopodia closely adapted to the surface; while in turned surfaces they were displayed in a flat morphology . A comparison among epithelial behavior in sandblasted (Al 2 O 3 particles), plasma-sprayed and polished titanium surfaces was performed and concluded that those cells attached, spread and proliferated with the greatest extension on the polished than on plasma-sprayed surfaces .

The attachment and proliferation of fibroblast on titanium surfaces blasted with TiO 2 particles (mean 45 μm, 45–63 μm, or 63–90 μm) were compared turned surface, used as control. Human oral fibroblast culture was used, and the highest percentage of cell attachment was observed on the turned and on surface blasted by 8–88 μm (mean 45 μm) TiO 2 particles. No significant difference in the percentage of fibroblast cell attachment was observed between these two groups. In the present study, an increase in diameter of the blasting particles inhibited cellular attachment. At 7 days analysis, no significant difference was found in the percentage of cell attachment for any of the surface preparations .

The influence of titanium surface characteristic on gingival fibroblast morphology was demonstrated by using sand-blasted and acid-etched ( R a = 4.14 μm) and turned titanium surfaces ( R a = 0.54 μm). Sand-blasted acid-etched surface showed cells orienting themselves along surface irregularities, and smooth surface exhibited a flat monolayer of cells .

Currently, specific modifications have been proposed in the surfaces in order to create an ideal surface that could “modulate” the cellular behavior, for example by using laser ; however, further studies are necessary.

The influence of the titanium surface topography has also been evaluated and brief descriptions of the studies are presented in Table 1 . In a dog model, soft tissue reactions to titanium implants were evaluated using sandblasted, a fine sandblasted, or a turned surface. After 3 months, differences concerning the healing pattern of the soft tissues were not observed among the surfaces, and the length of direct connective tissue contact was similar .

Table 1
Findings of comparative studies developed in vivo to compare differences among titanium surfaces.
Authors Surface treatment Findings Details of the study
Buser et al. (1992) (1) Sandblasted
(2) “Fine” sandblasted
(3) Turned
Differences were not observed among the surfaces, concerning the healing pattern of the soft tissues and the length of direct connective tissue contact Histological analysis in dogs
Abrahamsson et al. (1996) (1) Astra ® system
(2) Branemark ® system
(3) Bonefit ® system
Mucosal barrier had similar composition among groups. Histological analysis in dogs
Abrahamsson et al. (2001) (1) Acid-etched
(2) Turned
No significant differences were observed between soft tissues structure between the surfaces. Histological analysis in dogs
Abrahamsson et al. (2002) (1) Acid-etched
(2) Turned
Soft tissue attachment was not influenced by the roughness of the titanium surface. Histological analysis in dogs
Comut et al. (2001) (1) HA-coated by plasma-spraying
(2) HA-coated by IBAD deposition
(3) Turned
No statistically significant differences were detected among groups concerning the percentage of the extension of tissue attachment. Histological analysis in dogs
Roccuzzo et al. (2001) (1) Sandblasted and acid-etched
(2)Plasma-sprayed
No significant differences were observed concerning plaque index, bleeding on probing, mean probing depth average or marginal bone loss between the two treatment modalities. Clinical analysis in humans
Glauser et al. (2005) (1) Oxidized
(2) Acid-etched
(3) Turned
Oxidized and acid-etched implants presented less epithelial downgrowth and longer connective tissue seal than machined implants. Histological analysis in humans

HA = hydroxyapatite; IBAD = ion beam assisted deposition.

The mucosal barrier, formed around titanium implants following different clinical procedures (1-stage and 2-stage implant installations), was evaluated by using three different implant systems (Astra ® , Branemark ® , and Bonefit ® ). It was suggested that a “correctly performed implant installation may ensure proper conditions for both soft tissue healing, and that the geometry of the titanium implant seems to be of limited importance” .

Soft tissue reaction to different implant surface topography was studied in dogs. In such a study, dual surface implants in which the marginal 3 mm was turned, and the remaining part was acid-etched ( S a = 0.94 μm) were compared to entirely turned ( S a = 0.53 μm) implants. The authors did not observe significant differences regarding the dimension of the soft tissues comparing the groups .

The composition of soft tissue barrier formed around smooth or rough abutments was then evaluated. Abutments with a dual acid-etched surface were compared to others with turned surface. The authors concluded that the soft tissue attachment that formed was not influenced by the roughness of the titanium surface .

The responses to a surface modified by sandblasted and acid-etched (‘SLA’) implants in comparison to plasma-sprayed under loaded conditions. In this split-mouth design, the patients were examined 12-month after implant placement, and no significant differences were observed concerning plaque index, bleeding on probing, mean probing depth average or marginal bone loss between the two treatment modalities .

In a dog model, analyzed soft peri-implant tissues were performed around turned titanium surfaces ( R a = 0.2 μm) in comparison to hydroxyapatite coatings applied by plasma-spraying ( R a = 1.8 μm), or hydroxyapatite coatings applied by ion beam assisted deposition (‘IBAD’) ( R a = 0.2 μm). Four months after implants placement, no statistically significant differences were detected among groups concerning the percentage of the extension of tissue attachment .

The histological evaluation of the peri-implant soft tissue barrier was performed around mini-implants in humans. The topography surfaces examined were oxidized and microporous TiO 2 layer, acid-etched, and machined. Mini-implants and surrounding tissues were retrieved 8 weeks after placement. Epithelial tissue was attached to the implant surface; and an area adjacent to the metal (100–150 μm-wide) presented connective tissue mainly free from blood vessels and dominated by collagen fibers oriented parallel to the longitudinal axis to the implant. Furthermore, adjacent to this area, the connective tissue was densely packed with collagen fibers oriented circumferentially around the implants. The authors observed a shorter length of epithelial attachment, and longer connective tissue seal at the oxidized and acid-etched surfaces in comparison to machined implants .

Non-titanium surfaces

The ceramic materials alumina (Al 2 O 3 ) and zirconia (ZrO 2 ) have been used in implantology as an alternative to titanium. These materials are stable and biocompatible, with a color similar to the teeth. Alumina has been employed for sandblasting implant surface, and to produce abutments, resulting in satisfactory function and esthetics, however, clinical studies are required to confirm the long-term performance of this type of restoration . The development of an entire implant was proposed by using the so-called “single crystal sapphire” (α-Al 2 O 3 ), which revealed high success rates in long-term evaluation . A comparison, between the soft tissues formed surrounding single crystal sapphire and titanium implants, revealed no qualitative structural differences between these surfaces . Epithelial cells and fibroblasts develop more avidly on this material and on alumina in comparison with plastic dishes used as control in cell culture experiments .

On the other hand, the use of zirconia has been studied in sandblasting procedure, and in the production of entire abutments and implants . The main difference between alumina and zirconia concerns on the mechanical properties, which are better for zirconia . This material is reported to present a contact with soft tissue similar to that observed in titanium implants . Ceramic copings, constituted by a combination of zirconia (30%) and alumina (70%) were tested, and the results revealed clinical success with esthetical, functional, and harmonious replacement of missing teeth, even after a long follow-up period .

Comparison between surfaces

Brief comments of the studies described here are found in Table 2 . A cell culture study was performed to compare epithelial adhesion and spreading of the following surfaces titanium, titanium alloy (Ti 6 Al 4 V), dental gold alloy (Au 74.5%, Ag 12.0%, Cu 9.0%, Pb 3.5%, Zn 1.0%, Ru <1.0%), alumina, dental porcelain, and glass (used as control). Therefore, epithelial cells adhered more avidly to metallic surfaces than to ceramic surfaces. The authors suggested that based on these findings the collar of the implants and abutments should be as smooth as possible .

Table 2
Findings of comparative studies developed in vivo to compare differences among titanium surfaces.
Authors Surface treatment Findings Details of the study
Raisanen et al. (2000) (1) Titanium
(2) Titanium alloy
(3) Dental gold alloy
(4) Dental porcelain
(5) Alumina
(6) Glass (control)
Epithelial cells adhere more avidly to all metallic surfaces evaluated than to ceramic surfaces (dental porcelain and alumina). Cell culture
Abrahamsson et al. (1998) (1) Titanium
(2) Alumina
(3) Gold
Sites where abutments made of gold alloy or dental porcelain were used, no proper attachment was formed at the abutment level, but the soft tissue margin receded and bone resorption occurred. Histological analysis in dogs
Kohal et al. (2004) (1) Zirconia
(2) Sandblasted and acid etched titanium
Qualitative and quantitative analysis of periimplantar soft tissues were not able to detect differences between the surfaces. Histological analysis in monkeys
Degidi et al. (2006) (1) Zirconia
(2) Titanium
Titanium sites resulted in a higher rate of inflammation-associated processes than zirconia. Histological analysis in humans

The biological response of abutments composed by different material was investigated in dogs: titanium, alumina, and gold alloy (Au 60%, Pt 19%, Pd 20%, Ir 1%) Six months after abutment connection, those ones made of gold or alumina presented no proper attachment at the abutment level, moreover, the soft tissue margin receded and bone resorption occurred. The authors suggest that the material used influences the location and quality of attachment between soft tissues and implant .

In a monkey model, a comparison between transmucosal implants custom-made of zirconia or titanium was performed. Titanium surfaces were sandblasted (Al 2 O 3 particles) and acid-etched (H 2 O 2 /HF). Qualitative and quantitative analysis concerning the peri-implant soft tissues were not able to detect differences between the surfaces .

A comparative evaluation of soft tissue formed around titanium ( R a = 0.73 μm) and zirconia ( R a = 0.75 μm) healing caps was performed in humans. The inflammatory infiltrate was mostly present, and the extension of infiltrate was much larger in the titanium specimens. Titanium sites resulted in a higher rate of inflammation-associated processes represented for example higher values of microvessels density, in comparison to zirconia sites .

Response to plaque

The similarities between epithelium formed around teeth and implants are evident not only in the morphologically , but also, with respect to the homeostasis and the defense mechanisms . However, some differences are reported, and one of these concerns the vascularization. The supracrestal connective tissue lateral to the teeth is richly vascularized, with vasculature derived from supraperiosteal vessels and the vessels of the periodontal ligament. The corresponding site in the peri-implant tissue is almost devoid of vascular supply, and blood vessels are found to be terminal branches of larger vessels originating from the periosteum of the bone of the implant site .

The supracrestal peri-implant soft tissues have been reported as a significant factor for long-term success of implant, since it works as a barrier against bacterial invasion , and the rupture of this barrier can lead to implant failure .

The soft tissue around implant reacts to plaque forming an inflammatory lesion, where size and composition has many features in common with that formed around teeth . Also similarly to periodontium, under pathologic conditions peri-implant tissues may react with an apical epithelialization , and the osseointegration loss process is similar to that observed in aggressive periodontitis according to the number of T lymphocytes, but not to the vascular proliferation .

The interface between implant and soft tissue presents an epithelial cell attached zone, with a greater bond strength, that plays an important role in the prevention of bacterial invasion . However, this mechanism seems to be more permeable around implants than around teeth . Connective tissue barrier was described in an experimental study, in which the area between the keratinized mucosa and dental implant was investigated in two distinct areas nearby the implant. The first one, close to the implant surface up to 40 μm apart, was characterized by abundant fibroblasts interposed between collagen fibers, and absence of blood vessels. The second area, continuous laterally to the first one, consequently further from the implant, contained fewer fibroblast but more collagen fibers and blood vessels. The authors suggest that this fibroblast rich barrier play a role in the maintaining a sealing between oral environment and peri-implant bone .

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Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Histologic studies on osseointegration: Soft tissues response to implant surfaces and components. A review
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