10.18019/1028-4427-2019-25-3-318-323 Morphological assessment of osteointegration of various implants for management of long bone defects (experimental

Federation Purpose To assess osseointegration of implants made of various osteoconductive materials in an animal experiment by management of a long-bone diaphysis defect complicated with chronic osteomyelitis. Materials and methods Experimental studies were conducted on 24 healthy outbred adult rabbits for six weeks. Diaphyseal defects were managed with allo-, ceramic, and carbon implants. Results and discussion With carbon material, an adhesive interaction between the implant and the maternal bone tissue was of intercalation type. Osteointegration was evidenced by the presence of microfragments of an implant in bone tissue extracted for morphological study from the peri-implant zone, with areas of numerous osteoid islets. With a ceramic implant in the defect, no signs of remodeling and osteointegration in the implant extracted for morphological examination of bone tissue were detected. In the experiment with allograft, the area of implantation was isolated by a fibrous capsule, in which individual osteo-osteoid areas were seen without typical bone structure formation, and complete bone fusion was not achieved. Conclusion Morphological data on the regeneration processes prove the advantages of a carbon nanostructured implant relative to the other osteoplastic materials used in this animal


INTRODUCTION
Regeneration of organs and tissues which were lost due to the pathological process is one of the challenging problems of the contemporary medicine. Introduction of synthesized materials into the affected body area leads to disruption of homeostasis, the consequence of which is poor integration of the explant and even its rejection, as well as inability of its long-term functioning [1,2].
Currently, various types of grafts have been widely used to manage bone defects. In particular, a demineralized allogenic material with the ability to stimulate osteogenesis, carbon composite materials and implants based on ceramics have been used [3,4,5]. The literature contains data on the study of osseointegration of carbon implants in an experiment on a native preparation. However, data on the integration of various implants and their morphological characteristics have been described insufficiently [6,7] The results of applying different options for surgical treatment of patients with bone defects complicated with chronic osteomyelitis are controversial; therefore the search for criteria for evaluating the effectiveness of using different types of bone grafting remains an objective of medical practice [8,9].
Purpose Evaluate osteointegration of implants fabricated of various osteoconductive materials in an experiment using a model of chronic osteomyelitis and bone diaphysis defect.

MATERIAL AND METHODS
Experimental studies were carried out on 24 outbred adult rabbits, chosen according to the principle of analogues. The studies complied with the principles of humanity directives of the European Community (86/609 / EEC) and the Helsinki Declaration.
At the first stage, primary chronic fistulous osteomyelitis of the diaphysis of the radial bone was modelled in all the animals [1]. After two weeks, segmental necrsequestrectomy was performed to debride the inflammatory nidus, and then bone defects were replaced with various types of implants.
To study the processes of reparative bone regeneration in the resulting defect, the animals were randomly organized into groups ( Table 1).
Evaluation of bone strength was performed on healthy intact animals of the control group.
Reparative regeneration was evaluated correlating the results of histological studies with X-ray data and the strength properties of the regenerate at the boneto-implant interface. After six weeks, animals were sacrificed by intravenous administration of sodium thiopental at a dose of 200 mg/kg.

Original Article
Due to inability to have a solid bone microscopic preparation with the carbon implant, we carried out a preliminary extraction of the implant, after which we performed a morphological study of the remaining bone with ingrown elements. The implant was extracted from the bone tissue using a tensile testing machine R-05 UHL 4.2. The results of the experiment were analyzed from the point of view of the mechanical strength of the biological union formed at the bone-to-implant interface in Ns [4].
For morphological analysis, slices of 10-12 microns thick were prepared from pieces of bone tissue with a sledge microtome (Reichard, Germany) after decalcification in a 7 % solution of nitric acid and celloidinous filling. Next, the celloidin sections were stained with hematoxylin and eosin, using Masson's trichrome method, Schmorl thionin-picric acid. Later they were studied under a magnifying glass. Microphotographs were also taken under magnification using a lens 10 and eyepiece 12.5 (× 125). For microscopic examination, an AxioScope A1 stereomicroscope and an AxioCam ICc 5 digital camera were used, completed with Zen blue software (Carl Zeiss MicroImaging GmbH, Germany).

RESULTS
With allogenic bone material in the defect (group 2), histological assessment of the reparative process detected the presence of staged morphogenetic manifestations resulting from the formation of a bone-implant conglomerate. The bone allograft underwent resorption with the formation of a cavity in which fragments of detritus were located (Fig. 1a). Fibrous and osseo-fibrous areas around the resorption cavity and the fibrous layer of the interfragmental regenerate were combined into a common regeneration complex. Alongside, pronounced restructuring processes were detected in the maternal bone adjacent to the implant with spongiosis areas, rarefaction of the dense bone matrix and formation of osteoid fields and sections of rough fibrous bone (Fig. 1b). When the material was stained with the Masson's trichromic method, fragments were obtained that showed predominantly immature rough fibrous bone fields with non-mineralized fields of chondroosteoid type, which were characterized by high cellularity. In red mineralized fields, the density of cellular elements was noticeably lower, and in some areas small fields were revealed featuring the initial lamellar structuring (Fig. 1c). In group 3 with a ceramic implant in bone defect, it was found that in axial projections from the location of this material there was a clutch-like outgrowth of spongy bone tissue included in the bone regenerate, which was formed according to the hyperplastic type. Bone-osteoid trabeculae of the regenerate were at the stage of unfolding mineralization (Fig. 2a). Single areas of the newly formed tissue were a network of bone-osteoid trabeculae, visualized as mineralized and non-mineralized bone-osteoid structures in the area of contact between the mother bone and ceramics. There were no signs of osteogenesis processes at the bone-to-implant interface (Fig. 2b). In all samples, the newly formed structures featured rough fibrous bone tissue of a spongy structure with areas of immature bone tissue in the trabeculae of the forming regenerate. Lamellar bone structures were not detected either. The prevalence of nonmineralized structures (blue) over mineralized ones (red) was convincingly proven by the preparations with the Masson trichromic staining (Fig. 2c).
Unlike the above two experimental groups, the formation of a single conglomerate was macroscopically observed in the experiments with the carbon implant (group 1) due to the envelope of the maternal bone on the implant periphery (Fig. 3a). After extraction of the implant from the conglomerate formed as a result of the regeneration process, the remaining bone contained carbon implant particles that maintained contact with the mother bone and were detected at bone-to-implant interface (Fig. 3b).

Original Article
Histological study of the bone-to-implant border found periosteal osteogenesis around the implant. Adhesive cords and carbon microparticles, scattered in the bone structures, were located at the boneto-implant border and at a distance from this zone (Fig. 4a); sections of the cortical plate reconstruction were seen as cortex rarefaction with expansion of the intraosseous spaces. We also observed a change in the orientation of the direction of accumulations of bone plates as they acquired a wavy configuration. In the thickness of the compact bone of the maternal bone cortical layer, the presence of young osteoid nonmineralized structures of an irregular configuration was revealed in the process of restructuring. They testified to the insertion or intercalation growth of bone structures in this experiment (Fig. 4b). When the samples were stained with thionine-picric acid according to Schmorl, osteocyte lacunae and blood vessels could be traced in a remodeling bone matrix (Fig. 4c).

DISCUSSION
Surgical activity in chronic osteomyelitis is over 70 % [10]. In various types of the process, surgical debridement of the osteomyelitis nidus is only performed, or it is supplemented by soft tissue and bone defect management [11,12]. It should be noted that the problem of bone defect management is rather complicated and means, first of all, that the infected bone defect cavity is bridged [13].
The most rational technique for compensating segmental bone defects is distraction regenerate using the Ilizarov method which is based on the idea of inducing and maintaining osteogenesis by creating tension stress in the osteogenic tissue under conditions of rigid fixation of bone fragments [10,14].
The literature describes methods of combined techniques for treating long bone defects using transosseous and intramedullary osteosynthesis [15].
Autogenic bone is often used as a material for defect coverage [13,16,17]. In order to prevent local infection in the plasty area, antibiotics can be added to a free bone autograft [18].
Currently, the use of allografts has become widespread [8]. All materials used for defect filling can be divided into three types: designed for rejection or removal in the future, designed for resorption, and designed for integration and subsequent encapsulation. To non-resorbable fillings currently used, one can refer bone cement, which is of a complex polymer composition based on polyacrylate. It is possible to add an antibiotic to this composition in order to prevent the development of local infectious processes [8,19].
Currently, the use of carbon composite materials and ceramic-based implants has become widespread [21,22]. Hydroxyapatite has a porous structure comparable to the spongy bone, and functions as an effective osteoconductive matrix [7].

Original Article
During the experiment, we obtained convincing data on the advantages of osseointegration when using a nanostructured carbon implant in comparison with allogeneic bone material and ceramic implant; it correlates with published data.
In the presence of a noticeable reparative reaction, complete bone fusion with the use of an allobone implant was not achieved. The resorption cavity at the implantation site was isolated by the emerging fibrous capsule, in which individual fragmentary bone-osteoid sites were determined without the formation of typical bone structures.
In observations using ceramic implants in the area of the bone defect, the absence of fusion of the maternal bone with ceramic material was noted. The periphery of the implant revealed the formation of connective tissue encapsulation. Newly-formed immature bone-osteoid structures of reactive bone formation were adjacent to the periphery of the forming connective tissue capsule. No signs of organotypic bone remodeling were observed in this group.
Morphological analysis in the experiment with the nano-carbon implant indicates formation of a conglomerate consisting of an implant and recipient bone tissue which type is envelope cover. Moreover, the portions of the connective tissue layer formed during the repair process were replaced by bone tissue. The presence of small angular fragments in the adjacent maternal bone tissue, including at a certain distance, indicates the processes of osseointegration. Numerous sites with osteiod islets were found in the matrix of the rarefied maternal bone, which indicated bone formation of the intercalation type. At the same time, we did not reveal a full-fledged "creeping" replacement according to Andersson of the newly formed bone tissue of the entire volume of the carbon implant.
In our earlier studies [4], it was proven that the maximum bone-implant conglomerate strength (0.097 ± 0.013 N/m) was reached by the end of six weeks in this experiment with carbon implants. It turned out to be comparable with the strength characteristics of a healthy bone (0.095 ± 0.008 N/m). According to the results of the X-ray examination in animals of the group with the implantation of carbon material, the segmental defect was bridged by this time due to the formation of a periosteal bone-to-implant union in comparison with animals of other groups, where an unbridged bone defect was preserved on the radiographs.
Thus, the results of the morphological study correlate with the available literature data, as well as the previously obtained results of biomechanical and radiological studies [4]. CONCLUSION 1. When assessing the condition of the implantation zone, the carbon implant was enveloped with bone tissue, and after its extraction, signs of osseointegration, including the presence of carbon microparticles in the maternal bone tissue were determined at the bone-to-implant border.
2. Morphological study confirmed the presence of rearrangement of the cortical layer area at the border with the carbon implant that featured formation of lacunae of osteocytes and blood vessels in the remodeling bone matrix.
3. Morphological data on the regeneration processes prove the advantages of a carbon nanostructured implant relative to the other osteoplastic materials used in this animal experiment, and also indicate the possibility of optimizing bone remodeling processes in the implantation zone.