Ultrastructural characterization of the implant/bone interface of immediately loaded dental implants

U. Meyer^{, , a}, U. Joos^a, J. Mythili^a, T. Stamm^b, A. Hohoff^b, T. Fillies^a, U. Stratmann^c and H. P. Wiesmann<sup>a

a</sup> Department of Cranio-Maxillofacial Surgery, University of Münster, Waldeyerstr. 30, D-48149, Münster, Germany
^b Department of Orthodontics, University of Münster, Germany
^c Department of Anatomy, University of Münster, Germany

Received 31 March 2003;  accepted 11 August 2003. ; Available online 5 November 2003.
Biomaterials
Volume 25, Issue 10 , May 2004, Pages 1959-1967

1. Abstract

Primary stability and an optimized load transfer are assumed to account for an undisturbed osseointegration process of implants. Immediate loaded newly designed titanium dental implants inserted in the mandible of minipigs were used for the characterization of the interfacial area between the implant surface and the surrounding bone tissue during the early healing phase. Histological and electron microscopical studies were performed from implant containing bone specimens. Two different load regimens were applied to investigate the load related tissue reaction. Histological and electron microscopical analysis revealed a direct bone apposition on the implant surfaces, as well as the attachment of cells and matrix proteins in the early loading phase. A striking finding of the ultrastructural immunocytochemical investigations was the synthesis and deposition of bone related proteins (osteonectin, fibronectin, fibronectin receptor) by osteoblasts from day one of bone/biomaterial interaction. Calcium-phosphate needle-like crystallites were newly synthesized in a time-related manner directly at the titanium surface. No difference in the ultrastructural appearance of the interface was found between the two loading groups. Our experimental data suggest that loading of specially designed implants can be performed immediately after insertion without disturbing the biological osseointegration process.

Author Keywords: Implants; Immediate loading; Interface; Osseointegration

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Material

2.2. Experimental animals

2.3. Surgical procedure

2.4. Transmission electron microscopy

2.5. Scanning electron microscopy

2.6. Immunolabeling

3. Results

4. Discussion

References

3. 1. Introduction

The use of immediately loaded implants has been introduced in clinical routine for many indications. Clinical and experimental studies demonstrate that osseointegration can be achieved when healing under load is allowed. The bone reaction around loaded implants has been described by various investigators [1, 2, 3, 4, 5, 6, 7 and 8] and Szmukler-Moncler et al. have given a literature review concerning the timing of loading and the effect of micromotion on the bone-implant interface [9]. Most studies on the interface reaction evaluate the bone reaction on a light microscopical level, whereas limited data are available on the reaction of osteoblasts at the interface on a cellular level. Mineral formation at the interface of loaded implants has been investigated predominantly by light microscopy (LM), and we know of no study that has evaluated on the relation between mineral deposition and mechanical loading by analytical ultrastructural methods [10 and 11].

It was a long time assumed that premature loading per-se induces fibrous tissue formation but recent research indicates that the extent of micromotions during the initial healing phase interferes with the concomitant bone reaction [9]. When a primary congruency between the implant and the bone is present, a direct transition of load from the implant to the surrounding tissue can be assumed. The micromotion at the implant/bone interface can have two principal effects on the cellular and extracellular components of bone. First, the micromotion can lead to a disruption of the bone-cell/implant contacts and therefore has the ability to disturb the cell reaction by a detachment; or second the micromotion can lead to a deformation of osteoblasts fixed at the surface in a strain-related manner.

That peri-implant tissue formation and mineralization by osteoblasts are dependent on the local mechanical environment in the interface zone is generally accepted [9 and 12]. Carter and Giori suggested that proliferation and differentiation of the osteoblasts responsible for peri-implant tissue formation are regulated by the local mechanical environment according to the tissue differentiation hypothesis proposed by Frost and his colleagues for callus formation [13 and 14]. The relationship of defined cell deformation and bone remodeling has been documented in various in vivo studies. Loading of intact bone after osteotomy, during growth, in fracture healing, and during distraction osteogenesis resulted in strain-related tissue responses [15 and 16]. Whereas physiological bone loading (500-3000 microstrains) leads to mature bone formation, higher peak strains result in immature bone mineral formation and fibroblastic cell pattern. Evaluation of the effects of implant loading on tissue ultrastructure in vivo will therefore aid in the understanding of the basic mechanisms of the formation of the bone/implant interface.

The aims of our investigations were to establish the features of osteoblast behavior and matrix mineralization during the early bone/biomaterial interaction and the reactions that were dependant on implant loading We therefore applied two different immediate load regimes on biomechanically designed titanium implants and examined the early cell behavior and matrix mineralization by histology and electron microscopy.

4. 2. Materials and methods

2.1. Material

The implants used in this study were newly developed conical screw-shaped implants with a length of 10 mm and a diameter of 4.1 mm at the shoulder of the implant (Fig. 1a). The implants were made from pure titanium with a characteristic progressive thread design in order to achieve a direct bone/biomaterial contact after insertion and to optimize the load related bone deformation in the vincinity of the implant interface.

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Fig. 1. (a) Scanning electron micrograph of the implant used in this study (length 10 mm, shoulder diameter 4.1 mm). (b) CT scan and (c) clinical picture of implant placement. The distal implant was placed under occlusal contact and the mesial implant under non-occlusal contact.

The gross morphology of the implants was designed on the basis of previous investigations with the help of finite element analysis (FEA) [17 and 18]. The relation between implant body and the thread as well as the curvature of the implant was calculated to exhibit a homogenous and physiological strain distribution (1500-3000 microstrains) over the whole implant surface under masticatory loading conditions.

2.2. Experimental animals

Six male Göttinger minipigs were used in this study. The experimental study was approved by the Animal Ethics Committee of the University of Münster under the reference number G 90/99. Minipigs were selected to ensure adequate alveolar ridge size and height for implant placement [19].

The animals were sedated with an intramuscular injection of ketamine (10 mg/kg), atropine (0.06 ml/kg), and stresnil (0.03 ml/kg). Second premolars of the porcine mandible were extracted bilaterally, and the extraction sites were allowed to heal for 3 months before implant placement (Fig. 1b). A total of 24 implants were inserted into the mandibles of the minipigs. In accordance with our experimental design, two treatment groups were tested in each animal: 2 immediately loaded implants placed in occlusal contact and 2 implants placed in a non-occlusal relation ( Fig. 1c).

2.3. Surgical procedure

The implants were carefully inserted by manual tapping of the screw-shaped implants until the implant bodies were fully embedded in bone. Single crowns were prepared at the day of surgery on a plaster model and were then inserted to the implants. On days 1, 3, and 14 of implant loading two animals each were sacrified and implant containing block specimens dissected. One implant per study group was embedded in Technovit and subsequent undecalcified histological sections were prepared to evaluate the gross histology. The other block samples containing the implants were divided and then each sample was further dissected, in order to obtain a sample containing the implant embedded in the alveolar bone and the corresponding bone sample detached from the implant. Samples containing the implant were used for scanning electron microscopical (SEM) investigations and immunostaining, whereas the bone sections without implant were prepared for transmission electron microscopy (TEM) and diffraction analysis.

2.4. Transmission electron microscopy

For TEM, samples were harvested adjacent to the shoulder, the body, and the tip of the implant (Fig. 2a). Tissue samples were fixed in 100 m
phosphate buffer containing 2.5% glutaraldehyde (pH 7.4). Specimens were dehydrated in a graded series of alcohol and embedded in Araldite resin. Ultrathin sections were stained for 1 h with 2% uranyl acetate followed by incubation in Reynold's lead citrat. Sections were examined under a Philips CM10 electron microscope.

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Fig. 2. (a) Microscopic view of the mandibular implant. Mature bone contacts the implant surface. (Specimen: occlusal load, 3 days of loading.) Boxes indicate sample positions for TEM (×10), (b) bone/implant relation at the implant threads (×20), (c) higher magnification of the interface at the implant body (×20) and (d) cells are present at the surface and in the bone area adjacent to the implant (×40).

Electron diffraction analysis was carried out on ultrathin sections with a Philips CM10 TEM. Three positions from each implant were used to investigate the mineral formation.

2.5. Scanning electron microscopy

For SEM, glutaraldehyde fixed specimens were critical-point dried. Additionally, samples were fixed in liquid nitrogen-cooled propane and cryodried at −80°C. Samples were sputter-coated with gold for histological analysis. Specimens were examined under a field emission scanning electron microscope (LEO 1530 VP, Oberkochem, Germany).

2.6. Immunolabeling

After the various time intervals, the probes containing the implants were fixed with 3% paraformaldehyde, followed by immunogold labeling for the localization of fibronectin, fibronectin receptor, and osteonectin on the surface. Cells were incubated with primary antibodies, anti-fibronectin, anti-fibronectin receptor, and anti-osteonectin (Takara Chemicals, Japan), at a dilution of 1:200. This was followed by secondary 20 nm gold conjugated antibody incubation (British Bio-Cell International). After washing a silver enhancement was done for the gold particles using a Silver Enhancement kit (British Bio-Cell International). The samples were then dehydrated, critical point dried, carbon coated and viewed also through the LEO 1530 VP scanning electron microscope. Samples without first antibodies were used as controls.

5. 3. Results

The histology confirmed the clinical finding that all implants were well integrated. There was lamellar and in some areas spongiosal bone along the implant surfaces. A direct contact between the biomaterial and the bone over most of the length of the implant was present at a light microscopical level (Fig. 2a). The histological picture of the bone adjacent to the implant surface of the occlusal loaded implants resembled that of the non-occlusally loaded implants during the experimental period. Histological analysis of the bone/implant interface revealed an intimate contact between the titanium surface and the bony implantation bed ( Fig. 2b). At the bone-titanium interface, a thin tissue layer stained with Toluidine blue was seen in some areas coming into direct contact with the titanium, using LM ( Fig. 2c). Higher magnification demonstrated the presence of bone cells directly adjacent to the titanium surface ( Fig. 2d) and in the surrounding bone tissue. Application of occlusal loads did not alter the features of the implant/bone contact with direct contact areas at the implant surface.

SEM probe preparation by fracturing the implant containing the bone sample indicated that the strength of the implant/bone bond is comparable to the intercellular bond in the bone tissue adjacent to the implant (Fig. 3a). The fractured specimens demonstrated a direct bond between osteoblasts and the titanium surface. A striking feature of the SEM specimens was the cell and matrix coating of the titanium surface in some areas of the removed half of the bulky bone. Adhesion of cells and extracellular matrix proteins was present from day one of the implant/bone contact and was found throughout the experimental period independent on the kind of load application.

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Fig. 3. Scanning electron micrographs at different magnifications. Bone implant interface after 7 days of occlusal loading: (a) Specimens showing the artificially fractured bone area. A direct contact between lamellar bone and the implant is visible. (b) The surface of the implant is covered by cells and extracellular matrix proteins. Cells display phenotypic signs of osteoblastic-cells in a manner comparable to those found under cell culture conditions (c,d). (Primary osteoblast-like cells, 7 days of culture time, experimental conditions as described by Meyer et al. [33]). Bars indicate magnification.

Cells displayed at higher magnifications all phenotypic signs of osteoblastic cells. The attachment of cells was mediated in part by extracellular matrix proteins (Fig. 3b). Flattened cells adhered to the surface in a manner that was also found under cell-culture conditions ( Fig. 3c and d), indicating a focal contact dependant adhesion. Scanning microscopical observation of the interface matrix revealed a time-dependant mineralization process. Small crystals were found in the protein layer adjacent to the titanium surface. Over the time period of 14 days, the minerals coalesced in an attempt to form bone-like nodules ( Fig. 4a-c). Electron dense, needle-like structures, regarded as early crystallites, were found early after implant insertion. Over time, crystals grow along newly synthesized collagen fibers and minerals were hardly to distinguish from pre-existing minerals after 14 days of implant loading. Mineral formation was found to be collagen-associated (Fig. 4d).

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Fig. 4. Scanning electron micrograph of time-related mineralization at the implant surface. Minerals coalesce over time and form bone-like nodules directly at the implant surface (a) 3 days, (b) 7 days, (c,d) 14 days. Bars indicate magnification.

Immunostaining against fibronectin, fibronectin receptor, and osteonectin demonstrated that cells at the titanium surface remained the ability to behave in a bone-cell manner from day one of implant insertion. Fibronectin and fibronectin receptor were found preferentially at the contact sites between osteoblasts and the titanium surface (Figs. 5a and b). Additionally, abundant osteonectin was visible in the surrounding of cells at the implant surface (Fig. 5c), indicating that the cells remain in their viable and differentiated state ( Fig. 5d). For osteonectin immunoreactivity, a diffuse homogenous extracellular staining pattern was observed over the whole implant length. There was no difference in fibronectin, fibronectin receptor, or osteonectin appearance between the differentially loaded implants.

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Fig. 5. Immunolabeling of proteins on day 3 of load application in porcine mandibular bone. (a,b) Extracellular fibronectin deposition at the titanium surface at different magnifications. The bond between cells and the implant seems to be mediated by fibronectin molecules. (c) For osteonectin immunoreactivity, a diffuse homogenous extracellular staining pattern is visible. Osteonectin was found in colocalization to the mineralized extracellular matrix. Synthesis of osteonectin seemed to be done by osteoblasts at the titanium surface (d). Bars indicate magnification.

In TEM, a sharp interface was found due to a direct apposition of calcified bone tissue at the titanium surface. Some areas exhibited in semithin sections a nanometer sized layer of unmineralized matrix right at the interface. Transmission electron microscopical pictures confirmed the observations of viable cells in the interface near the implant (Fig. 6a). Signs of necrosis or apoptosis were only rarely detected in the samples adjacent to the implant surface (Fig. 6b). No difference in the morphology of the cells was found in the various explant regions (shoulder, body, tip of implant) and between the various load applications. Mineral formation was found in association to collagen molecules. Fig. 6c shows characteristic TEM features of mineral formation directly at the implant surface and in the adjacent tissue layer ( Fig. 6d). Transmission electron microscopical and analytical (EDA) ( Fig. 6c, inlet) assessment of the interfacial area reveals the apatitic nature of the bone crystals. Mineralization of the collagen fibers in direct contact to the titanium surface seemed to be time-dependent.

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Fig. 6. (a) Ultrastructure of viable osteoblast in the layer adjacent to the implant surface (magnification 6300×). (b) Apoptotic cells were only seldom present in the bone layer adjacent to the implant (magnification 1500×). (c) Unstained section of collagen mineralization at the implant (I) surface (magnification 11000×) and (d) in the microenvironment of the implant (magnification 6300×). (c, inlet) Electron diffraction pattern of mineral formation adjacent to the implant surface.

6. 4. Discussion

The long-term success of bone-interfacing implants for load-bearing orthopaedic and dental applications requires rigid fixation of the implant within the host bone site. With dental implants, where axial symmetry is possible, threaded screw-shaped implants have proved effective for achieving secure implant fixation within bone. The threads are representative of macroscopic surface features that allow mechanical interlocking of implant within bone. The results of various studies suggest that the quality of implant osseointegration and stability is dependent to a large extent on the geometric implant design. However, the role of implant geometry in affecting early tissue healing and implant stability cannot be determined directly from observations made several weeks post-implantation after osseointegration and bone remodeling have occurred. The issue of immediate healing response next to implants and the ultrastructural characterization of the tissues formed within the implant/bone interface has not yet been addressed.

Inspection of the bone reaction around oral implants is conventionally performed and histomorphometrically evaluated either on ground sections of methyl-metacrylate-embedded materials or at the cellular level by histologic analysis of the peri-implant tissue by LM. Alternatively, the architecture of the tissue/implant interface is visualized by electron microscopy (SEM/TEM). The two approaches exclude each other because of the sample preparation. Therefore, a combined probe sampling and preparation was done in this study to give a better insight into the morphological and functional features of interfacial tissue formation.

The histological overview of the bone/implant relation in the present study demonstrates a congruency between the implant and the surrounding bone tissue. A direct contact between titanium and bone was visible over the whole surface area of the implant directly after insertion and during the experimental period. Excellent adaptation of the host bone to titanium surfaces was observed on an ultrastructural level in a comparable manner as reported after insertion of self-tapping screws in calvaria bone by Sowden and Schmitz [20]. It was demonstrated that when self-tapping screws were placed in loading or non-loading positions the long-term histology showed that the bone tissue around the implants was maintained in both situations [21].

SEM observations demonstrate that not only mineralized bone tissue contacts the surface but that osteoblast are also attached firmly to the titanium surface. The ultrastructural data indicate that cells are attached on the implant surface from day one of implant insertion by adhesion sites, comparable to those found under in vitro cell culture conditions. Cells displayed the typical appearance of well-differentiated bone cells. No difference in the ultrastructural appearance of the interfacial cells was found in the two loading groups. Our results are in agreement with findings of Lavos-Valereto et al. and Simmons et al. who demonstrated also an intimate contact between bone cells and titanium implants on an SEM level in the early and late post-implantation time [22 and 23]. In addition to the present findings, Murai et al. demonstrated a thin 20-50-nm sized layer in some places at the implant surface. This layer consisted of a slender cell layer and slight mineralized zone between the bone and titanium [24].

Fibronectin, fibronectin receptor, and osteonectin was synthesized by cells attached to the surface, indicating that the insertion process does not disturb the viability of osteoblasts. The presence of fibronectin and fibronectin reseptor at the cell protrusions indicate a stable attachment between cells and the biomaterial, as revealed by other studies [25]. Immunohistochemistry demonstrated also the presence of the bone specific matrix protein osteonectin in the interfacial layer. The distribution of this calcium-binding protein indicates that it may be functionally involved in the load-related tissue remodeling [26]. It appeared that the expression of osteonectin was closely related to osteoblastic differentiation and osteoid mineralization. The present ultrastructural results are in accordance with other immunohistochemical in vivo findings [27 and 28]. Bone cell differentiation and bone specific protein synthesis was also observed around immediate or early loaded implants on a light microscopical level [6 and 7]. In addition, this study indicates that even cells in direct contact to the implant keep the ability to synthesize these proteins in the early healing phase.

For the stability of implants under load it is of major importance for the bone-forming osteoblast to promote matrix mineralization in the vincinity of the implant. When early or immediate implant loading is considered an accelerated bone healing around implants should result in an interfacial matrix with a composition, structure and biomechanical characteristic of normal bone. In line with the present immunohistochemical data the study revealed a time-related bone-like apatite mineralization. The early formation of a bone-specific extracellular matrix was concomitant with apatite crystal formation at the extracellular matrix proteins. Ultrastructural data of Futami et al. indicate that the mineral formation in the nanosized layer adjacent to the titanium surface increases over time [29]. Simmons et al. demonstrated also an early healing and mineralization of the bone tissue in the peri-implant region (the interface zone) [23]. A mature mineralized matrix even in the early osseointegration phase as found in various dental and orthopedic investigations is assumed to secure mechanical stability of the implant in order to prevent peak load related implant loss [28].

As the local mechanical environment around implants is dependent on the forces imposed and the implant/bone surface interaction, certain implant designs may promote osseointegration by providing a favorable local mechanical environment for bone formation [30 and 31]. Based on this reasoning, we assume that the undisturbed osseointegration process observed also with the occlusally loaded implants was the result of the makro-design with its characteristic three-dimensional form, providing a local mechanical environment in the early interfacial tissue that prevented peak strains at the interfacial layer [32].

The outcome found allow a faster recuperation for the patient with stable fixation between bone and implant that is the basis for immediate loading protocols. This latter point has great significance, in terms of decreased patient morbidity, improved patient comfort, and decreased health care costs.

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