Use of Biositall Grafts for Substitution of Bone Tissue Defects: Cytological and Cultural Experimental Substantiation

Orlov V.P.1, Dulaev A.K.1, Lysenok L.N.2, Pinaev G.P.3, Nikolaenko N.S.3

Medicomilitary Academy 1, State Technologic Institute 2, Cytology Institute of the Russian Academy of Sciences 3, Russia

The analysis of modern literature on biomaterials for substitution of bone tissue defects shows, that existing grafts, based on metals, alloys, polymers, glass, ceramics, do not possess sufficient reliability and osteoreparative properties. Moreover, grafts, made of metals and synthetic polymers, are foreigners for the human body and their chemical composition does not correspond to bone tissue [1, 2, 4-8, 12, 14-17].

There were several unsuccessful attempts to solve the problem of substitution of bone defects with wide-pore grafts, made of biodegrading materials and based on calcium phosphate and combinations of hydroxylapatite with glass, whose chemical composition was very close to that of natural bone tissue [3, 9, 10, 11]. These materials lacked good strength; besides, there was no synchronisms of biodegradation and osteoreparation processes (these grafts destroyed before formation of a reliable bone block). According to Hitchon P.W. et al [13] and Ryu K.S et al [18], grafts, made of dense and strong phosphate (hydroxyapatite) materials, biodegrading calcium, hamper osteointegration processes; it results in formation of a connective-tissue capsule just on a borderline with a graft, leading to its instability in skeletal structures.

The purpose of the present research was studying properties of glass ceramic substances, standing very close to a natural bone in their composition.

Material and Methods

Glass ceramic biositall grafts were made at the Chair of Glass Technology of the Saint Petersburg State Technologic Institute.

Porous biositalls (M-12 and M-31) were tested for determining toxicity of this class of substances and sensitivity of cellular structures to them in vitro. The procedure was based on extraction of ions and other microelements out of them, carried out in media, which were analogous to biological fluids, with subsequent control of their ability to support growth and functional activity of cells, cultivated in them. The next step was studying conditions under which osteoblasts could be cultivated on biositall and search for the most sensitive methods of control of their proliferation and differentiation. This research was carried out in Department of Cellular Cultures of the Cytology Institute. Cells of ovaries of a Chinese hamster (the CHO-K1 line) and osteosarcoma cells of a rat (the ROS 17/2 line) were subject to two tests. The first one dealt with clonal growth of the CHO-K1 cells in control and experimental media. The second test was aimed at studying mass growth and differentiation of the ROS 17/2 osteoblast-like cells in control and experimental media.

Biositalls were studied in vivo in the Experimental Laboratory of the Medicomilitary Academy. Experiments were carried out on 17 mongrels with a body mass of 10-15 kg (Fig.1).

Biositall grafts, whose dimensions varied from 0.2´0.3´0.7 cm up to 0.5´0.7´1.0 cm, were inserted into iliac crests (two grafts on each side). The material was taken in 1, 3, 6 and 12 months after operation. During this procedure new fragments of biositall were implanted into intact areas of the upper flaring portions of the ilia (Table 1).

Table 1

Distribution of Grafts under Study in Compliance with Duration of a Postoperative Period.

A Term of Observation months

-12

-31

N 216

Total

1

8

7

8

23

3

10

9

9

29

6

9

8

8

26

12

8

8

8

25

Total

35

32

33

103

A fragment of the material was resected together with an adjacent bone tissue. Then a macropreparation was fixed in 10% solution of neutral formalin. Decalcification was carried out in a solution of trilon-B with the help of standard methods. Histological microsections of 10 mm were stained with hematoxylin and eosin according to Van-Gizon. The Jenamed-2 microscope (Germany) and magnification of ´50; ´100; ´200 and ´400 were used for their estimation. Histological studies were carried out at the Chair of Pathoanatomy of the Medicomilitary Academy.

A biositall-bone contact zone was studied, using electron-probe X-ray microanalysis.

Results

Experimental Study of Toxic Properties and Cultural Substantiation of Applying Biositall Grafts. The analysis of toxicity allowed to conclude, that clonal growth of cells of ovaries of the CHO-K1 Chinese hamster and osteosarcoma cells of the ROS 17/2 rat in an experimental medium (with a powder of tested biositalls) was practically identical to growth of these cells in a control medium. Thus, a chemical composition of tested biositall grafts is not toxic in relation to sensitive cellular systems in vitro.

Studying proliferation and differentiation of cultivated cells of osteosarcoma of the ROS 17/2 rat and cells, obtained from a skull of new-born rats, showed, that both normal and transformed osteogenic cells stick to M-31 biositall plates rather well and proliferated on their surface with a proliferation index, reaching 5.8-8.5 during 5-15 days.

Obtained data were indicative of the fact, that the ROS transformed cells migrated from biositall quite easily and formed a monolayer on plastic after 10 days of cultivation, which was very close to control cells in its character (Fig.2).

Besides, it was noted, that good attachment of diploid bone cells to biositall was watched even after 2 weeks of cultivation; they did not migrate to plastic in contrast to control cells, cultivated on glass (Fig.3).

Differentiation of normal and transformed cells, cultivated on biositall was estimated by changes of a level of alkaline phosphatase activity. Transformed cells on biositall had alkaline phosphatase activity close to that of control cells. This enzyme activity in normal cells, cultivated on biositall, was higher, than control indices.

An effect of a conditioned medium on splitting, proliferation, formation of a borderline layer and functional activity of normal cells, cultivated on biositall, was studied with the purpose of obtaining a complex source of components of extracellular matrix and growth factors. It was found out, that a conditioned medium, received at a stationary stage of cellular growth reduced proliferation of cells and their number in a borderline layer. This medium increased alkaline phosphatase activity and accelerated mineralization of a cellular layer. Data of scanning electron microscopy confirmed, that biositall as well as a conditioned medium promoted formation of mineralized bone matrix (Fig.4).

The analysis of results allows to come to the following conclusions: transformed osteoblast-like cells of osteosarcoma of the rat and normal cells, isolated from a skull of new-born rats, can proliferate and preserve signs of osteoblast differentiation during their long-term cultivation (more than 2 weeks) on biositall M-31 and M-12; a conditioned medium, obtained at a stationary stage of cultivating normal bone cells in vitro, reduces proliferative activity of these cells at a logarithmic stage of growth; biositall and a conditioned medium accelerate mineralization of a cellular layer; biositall M-31 and M-12 ensure fixing and proliferation of osteobalst-like and normal bone cells on their surface, i.e. they are an osteoconductor of bone tissue.

Peculiarities of Formation of Biositall-Bone Contact Areas in Experimental Animals. Macroscopic study of a character of a bone tissue response to biositall grafts (N 216, n=8 with volumetric porosity of 10-15% and a pore size of 40-50 mm) showed, that in a month a graft was surrounded by a tender connective-tissue capsule on the side, facing soft tissues; there was no response on the part of a wound and tissues. Studying macropreparations revealed a thin layer of connective tissue, separating a graft and soft tissues and a bone; thickness of this layer was different. Microscopic examination of a capsule was indicative of developing collagen fibers. Biositall particles were surrounded by tissues, containing macrophages, single polymorphonuclear leukocytes, plasmocytes, lymphocytes (Fig.5).

There appeared a dense fibrous capsule around biositall grafts (N 216, n=9) three months later. A reliable fibrous block was formed thanks to it. Histological examination revealed no signs of osteogenesis (presence of osteoblasts and immature bone trabeculae) in perifocal connective tissue. Intertrabecular spaces were covered with fibrous connective tissue along a defect margin (Fig.6).

Microscopic examination, carried out in 6 months, showed, that grafts (N 216, n=8) were surrounded by a dense connective capsule with an adjacent spongy bone with thickened trabeculae and narrowed medullary spaces, containing mainly fatty bone marrow. A capsule wall microstructure changed and consisted of bundles of mature collagen fibers; fixation of a graft in a bone bed was rather reliable.

In 12 months there appeared newly-formed bone plates of an osteoid character with calcification areas and thickened bone trabeculae in collagen tissue on a graft-bone borderline (N 216, n=8). A spongy bone, adjacent to collagen tissue, had marked thickening of bone trabeculae (Fig.7).

Studying a character of response of bone tissue to porous biositall grafts (M-12, M-31, volumetric porosity of 40%, a pore size of 75-120 mm) revealed the following peculiarities. There were no wound suppuration and response of tissues in a place of implantation in a month after it; all animals were active. Macroscopic findings were indicative of presence of a thin connective-tissue layer, separating a graft and soft tissues, there was no connective tissue in the area of a contact with a bone.

Microscopic examination was indicative of presence of granulation tissue between a graft and a bone, growing into biositall, and signs of a graft fragmentation and macrophagal response in the form of biositall resorption. A cellular proliferative response was marked weakly. It was possible to separate a graft from a bone with a small effort.

In 3 months after implantation grafts (M-12, n=10 and M-31, n=9) were covered with a capsule of connective fibrous tissue in a zone of a contact with soft tissues; there was no cellular response. A capsule was represented by collagen fibers with newly-formed bone trabeculae and calcification areas in a zone of a contact with a bone. Intertrabecular spaces along a borderline were overlapped by bone plates; fixation of a graft was rather strong (Fig.8).

Formation of a strong bone-biositall block was watched in 6 months. Its stability increased due to growing of bone tissue into a graft. When 12 months passed, macroscopic changes in soft tissues were identical to those watched after 6 months; a graft was covered with a dense layer of newly-formed bone tissue in a zone of its contact with a bone (Fig.9).

Results of Studying a Bone-Biositall Contact Zone with Electron-Probe X-ray Microanalysis. Examination of a bone-biositall (M-12 and M-31) contact zone, carried out in 3 months after implantation, revealed a good contact area with a width of 100 mm. There were no intermediate structures, cavities and other signs of a conflict between a graft and a bone. A graft was surrounded by a dense layer of newly-formed bone tissue with preservation of connective tissue in some areas (Fig.10).

Distribution of ions of silicon, calcium and magnesium was as follows: silicon ions were detected mainly in a graft. Their number in a borderline layer was great; it was connected with a graft resorption and proved, that a graft under discussion belonged to bioactive glass ceramics (Fig.11).

The analysis of graft samples was carried out in 12 months after implantaton. The analysis of obtained images with taking into account their scale showed, that there was a good contact between a graft (M-12, volumetric porosity of 305, a pore size of 80 mm) and a bone bed; there were no cavities, intermediate structures, collagen and fibrous tissue. Macroscopic study was indicative of formation of a bone-biositall block.

A process of gradual replacement of a graft by bone tissue is presented in Figure 12. One can see a borderline between a graft and a bone bed in microphotoes. A bone-graft contact zone is widened; there are areas of connective osteogenetic tissue with elements of formed bone tissue, growing into a graft. Raster patterns of distribution of silicon ions repeat outlines of a bone and osteogenesis foci in a graft rather precisely. There are marked changes of the content of different ions in a graft-bone bed contact zone and in the center of biositall.

The analysis of results of cytological and experimental-morphologic studies demonstrated optimum strength and osteoconductive properties of glass ceramic grafts (biositalls), their osteocompatibility and absence of a negative effect on a process of bone tissue regeneration. Formation of a strong bone-biositall block demanded to ensure stability of a graft during not less than 3 months.

A biological experiment confirmed good tolerance of grafts (M-12, M-31, N 216) by bone tissue. Formation of a strong bone-biositall block, osteoconductive properties and strength of these grafts make it possible to use them in clinical practice for replacement of defects of vertebral bodies, caused by traumas and spinal diseases.

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