Experimental study of the biointegration of dental implants fabricated by laser sintering In-Vivo

Currently, titanium remains the most sought-after material for dental implants [26]. There are several types of modern titanium implant surfaces, which are created by exposing titanium to various physical and physicochemical factors.

The most common methods of implant surface modification include sandblasting, acid etching, sandblasting and acidizing (SLA), anodizing (TiO2) and laser treatment of the implant surface. The effect of these factors on the titanium surface gives it a micro- and nano-roughness, which is represented as “Peaks and walleyes” (peaks and pits) under electron microscopy.

Such relief of the titanium surface allows mesenchymal osteogenic cells to be in direct contact with the inorganic implant surface and their further differentiation into osteoblasts, which facilitates the closest possible production of bone tissue to the surface of the placed implant. It has been repeatedly demonstrated experimentally that it is the micro- and nanostructure of the implant surface that forms a tight contact between the implant surface and the bone tissue (Bone-implant contact, BIC) without a soft tissue layer at an earlier date than in the case of the previous generation of implants [6]. It is important to mention that not only the micro- and macrostructure of the implant allows a more effective interaction with the bone tissue, but also the electrochemical modification of the surface, which strengthens the bond between the artificial material and the bone tissue.

Anodised implant surfaces have these properties, as shown experimentally (Albrektsson et al., 2000; Henry et al., 2000) [24]. That is why the main task of the various surface modifications of modern implants is to intensify the osseointegration process by modifying the 3D structure of the implant surface in a different way from that of the previous generation of implants [3].

However, although there is a significant difference between the results of experimental robotic studies (invivo and invitro) investigating the osseointegration of previous and current generation implants, the number of clinical research with long-term results is not enough to draw a definitive conclusion about the dominant effectiveness of one or another surface of modern implants in the osseointegration process [25]. As a consequence, the lack of research data (clinical and preclinical) is causing some controversies between specialists and often leaves the clinician at a loss regarding the type of implant to rehabilitate patients.

The research of implants with new surfaces or new architecture, with which it is hoped to optimize or even improve the osseointegration processes, follows certain phases: technical study of the implant surface, study of the reaction of cell cultures to the implant surface (InVitro), experimental study in animal models (InVivo) and then moving on to the clinical phase of the research. In this way a complete picture is obtained and conclusions drawn regarding the effectiveness of a particular material implanted into the human body. It is also important to mention that by using material that has already been researched in advance, it is acceptable to omit some of the research phases of new implants, given the availability of sufficient data from research studies that have already been carried out.

One of the most important fields of research in evidence-based medicine, however, is still experimental modeling in the animal body. In such scientific research, there is the possibility of finding new properties of various materials (including titanium) after they have been introduced into the animal body. As a striking classic example, we can offer the experimental study of Professor I. Brånemark's experimental research on intraosseous blood flow in rabbits, in which the phenomenon of titanium osseointegration was accidentally discovered and resulted in the active development of implantology as a discipline [1]. The importance of experimental research on animals is also determined by the closeness of the experimental model to the human body. Even InVitro researches with human cell culture do not fully simulate the reaction of all tissues and fluids with which the implant comes into contact (blood, tissue fluid, oral fluid and its components) as it is inserted into the formed bone bed (21).

Several different species can be used as experimental animals to study the osseointegration of implants: rats, rabbits, pigs, dogs and sheep. The PubMed biomedical literature base provides more than 2,000 results for animal experimental studies in the field of implantology[4]. However, considering the peculiarities of animal housing, bioethical regulations in different countries and regions, the availability of animals, the speed of regeneration processes and the reproductive potential of the animal, rabbits are one of the most popular experimental animals, which is determined by the relative easiness of animal maintenance, fast metabolism, high reproductive potential and low aggressiveness [27].

Moreover, among rodents, rabbits are the most suitable animals for modeling alveolar healing processes after removal of lower incisors; in other rodents, the size of the jaws and the anatomy of the tooth tissues do not allow efficient and complete alveolar surgery, particularly tooth extraction. The most common bones used for implants in rabbits are long bones, particularly the femur and tibia. But it is important to mention that the origin and regeneration characteristics of these bones are different from those of the jaws. Also, the operation of implant placement in the limb bone of rabbits is a highly sterile procedure, unlike in the jawbone (particularly for immediate implants after tooth extraction).

This characteristic does not allow to display the closest to reality conditions using the long bone model and does not provide for the possibility of different amounts and composition of oral specific microorganisms entering the implanted area, what can subsequently seriously affect the difference in the study results between implants placed in the jaw and the extraction site from the same implants placed in the sterile bone tissue of the limbs. Few osseointegration researches are known that have been performed on the jaws of rabbits [22, 23]. As for the content of the research results in recent years, we should highlight those focused on comparing the effectiveness of implants with different surfaces for the osseointegration process. As a criterion, most of the authors used twist tests, RFA (resonance frequency analysis) values and morphometric examination of bone preparations with implants placed at different times. In the experimental research by Jung-Woo Koh at al. (Seol, 2009), the author compared the data on the osseointegration of the implants made by milling (without modification) with the modified surfaces: SLA, anodized and Ca-P implant surfaces.

As a criterion of osseointegration the values of RFA and implant unscrewing test were used, as well as the examination of the bone macrostructure in the area of inserted implants. This research showed a significant difference between the osseointegration data in favour of implants with modified surfaces, although no significant difference was found between implants with anodised and SLA surfaces [6].

There is disagreement about the effectiveness of the macrostructure of the implant. In an experimental research on mini-pigs, Yee-Seo Kwon et al. (Seoul, 2013) and Osstem Implant Co (Busan, 2013) found that implant neck micro whorls designed for increased stability in the cortical bone layer did not show a significant difference in stability and osseointegration data at different times (from 2 to 14 weeks). These results should also be taken into consideration in the search for a more effective implant surface macro-relief [8].

It should be mentioned that experimental research of the effectiveness of the implants surface treated by the laser (Nd:YAG laser) has been carried out. This modification gives the implant surface a specific micro-roughening, which according to the experimental study on rabbits considerably increases morphometric and mechanical indices of integration of the implants so modified [10].

In the same way, experimental research has been carried out regarding the hydrophilic properties of the implant surface. In the experiment 32 implants with different hydrophilic surface properties and degrees of hydrophilicity Neoporos and Acqua (Titamax CM; Neodent, Curitiba, PR, Brazil) were placed on 16 rabbits. As the result of the morphometric research performed there was no significant difference between the rates of osseointegration of the implants with different degrees of hydrophilicity in the early period (2-4 weeks). The author concludes that the most important factor for osseointegration is the surface microroughness and the quality of the osteotomy of the receiving implant bed [14].

2. Goal

To experimentally compare the qualitative and quantitative indices of osseointegration of implants made by titanium rod milling and laser sintering of titanium powder.

3. Materials and methods

A total of 12 rabbits of the .... breed were taken for the experiment (Pic. 1, Pic. 2).  The plan and structure of the experiment were discussed by the bioethics committee of N.I. Pirogov ENMU and an opinion was received, as well as permission to carry out this study.

The animals were divided into 2 groups: a control group (n=6) and a main group (n=6). In the control group, implants with an anodized surface made by milling from Cell Implants titanium rod (Germany) were placed. In the main group, we used anodized implants made by sintering the titanium powder Cell Implants? (Germany) (Pic. 3). For the implantation procedure, the animals underwent combined intramuscular anesthesia with ketamine solution (10 mg/kg of animal weight) and local anesthesia with Ultracaine DS-Forte. After anesthesia, the surgical field was treated with soapy water and Betadine. The anesthetized animal underwent a typical mandibular incisor extraction and curettage of the extraction socket (Pic. 4), after which a skin incision was made along the alveolar process of the lower jaw in the submandibular area on the side of the extracted tooth (Pic. 5) and the soft tissue was bluntly and acutely prepared to expose the external compact plate of the extracted tooth socket (Pic. 6). This access was chosen because of the lack of space in the animal's mouth and the inability to insert the dental instrument needed for the implantation with the classic intraoral approach. Then, according to the classic protocol, an osteotomy of the implant socket was performed perpendicular to the alveolar process through the external cortical plate of the extraction socket until the lingual surface, which has a spongy substance layer, is resting on it. The implant was inserted manually into the socket and was inserted to the cortical layer using a torque wrench (Pic. 6). At the end of the operation, the wound was irrigated with 0.9% NaCl and sutured layer by layer with Vicryl (Jonson&Jonson) atraumatic suture. The animals were removed from the experiment by overdosing with Thiopental sodium solution (60 mg/kg animal weight) on 15/30/45/60 days in 2 individuals in each group. After cardiac and respiratory arrest and signs of biological death, the jaw preparations with implants were dissected and removed (Pic. 7). At the same time, cone beam computed tomography (CBCT) was performed with a Gendex CB-500 (KaVo) with the following interpretation of the tomography data in the i-CATVisionTM (Imaging Science International) software interface (Pic. 8). The jaw preparations were fixed with 10% isotonic formalin solution, followed by dehydration in increasing concentrations of 70%-96% ethanol (Pic. 9). Morphological research was carried out using electron microscopy of a series of slides of prepared preparations…

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6. Other product research