COMPARATIVE ASSESSMENT OF THE METHODS OF CHEMICAL STERILISATION OF DEMINERALISED BONE MATRIX

The osteoinductive properties of DBM sterilised by various chemicals (chlorhexidine, catamine AB, antiseptic complex, and performic acid) were studied. Experiments were conducted on sexually mature rats aged 3–4 months. Thigh bones were removed from the animals, cleared of soft tissues, sterilised, and decalcified. The resulting DBM was freed from the bone marrow and washed with running water for 2–2.5 hours to pH 7.2, and then sterilised by various chemical agents. The sterilised DBM was implanted intramuscularly in the anterior thigh surface of recipient rats aged 3–4 months. Results. Demineralised bone matrix sterilised with chlorhexidine and catamine AB has no osteoinductive activity, therefore these substances in the studied dosages cannot be used for its preparation. Antiseptic complex (polymyxin, furazolidone, sorbic acid) and performic acid reduce the osteoinductive properties of DBM in the sterilisation process, and antiseptic complex reduces them to a greater extent. The most suitable chemical agent for DBM sterilisation is performic acid. Conclusions. Allogeneic unsterilised DBM of rats has pronounced osteoinductive properties. Various methods of chemical DBM sterilisation usually reduce the osteoinductive properties of the graft. Performic acid has the least ability to reduce the osteoinductive properties of DBM and can be recommended for sterilisation of demineralised bone matrix.


INTRODUCTION.
Wide implementation of osteoplastic surgeries in clinical practice is aimed to obtain such grafts that would exert a constant osteogenesis effect in a relatively short time. There is an acute necessity in the development of better methods of preservation of graft tissues for transplantation, especially those that have a locomotor or mechanically framework structure. The search for an inductive basic structure brought researchers to the organic part of the bonebone matrix [1,2].
Long-term studies successfully finished with the discovery of the inducing osteogenesis factor revealed in decalcified bone matrix (DBM), that is, bone morphogenetic protein. It was known long before its discovery that this factor was thermolabile and degraded after the demineralization of bones with nitric, trichloroacetic, chromic, osmic acids, alkalis, UV rays, and gamma irradiation.
The high osteoinductive activity of the demineralized matrix was also confirmed by other researchers. DBM is more immune inert than allograft bone [3]. Experimental transfer of DBM for bone defect repair showed its practical equivalence to autografts [4]. Application of DBM in clinical practice for repairing the defects of the skull and maxillofacial bones, different injuries and diseases of the locomotor apparatus allowed the specialists to recommend it for the implementation in practice. However, the issues of mass preparation of DBM are still acute, especially, when it comes to the development of methods of sterilization of demineralized bones. The application of physical methods of sterilization of DBM is excluded because they destruct the osteoinductive factor. For this reason, chemical sterilization is the only possible option. The methods of chemical sterilization should be tested thoroughly because, under the influence of certain chemical substances, the osteoinductive activity of DBM can be also affected [5].
The choice of chemical sterilization of DBM that would preserve its properties is a complicated task. Some studies showed that a 0.5% solution of formalin for the sterilization and conservation of DBM slowed down the osteogenesis [6]. Other authors proposed to sterilize DBM with an antiseptic complex that included kanamycin, sulphosalicylic acid, honey, and formalin [7].
The authors presented clinical results of the application of DBM sterilized with this antiseptic complex but they did not report its influence on the osteoinductive properties of DBM [7,8].
Significant experimental material showed that nonsterile DBM treated with silver salts at a concentration of 10 -4 M became sterile and antiseptic and preserved these properties without a reduction of osteoinductive properties.
The study was aimed to compare several methods of chemical sterilization of the demineralized bone matrix.

MATERIALS AND METHODS.
The present study focused on the osteoinductive properties of DBM sterilized with different In all the cases, sterilization was performed at +2 -+4 °С.
The transfer of sterilized DBM to 3-4-month-old recipient rats was performed intramuscularly from the anterior surface of the thigh.
Totally, 4 series of tests were conducted. The authors transferred DBM sterilized with chlorhexidine, catamine AB, antiseptic complex, and performic acid. In the control series, DBM prepared in sterile condition was transferred (K). Each series included 20 operated animals. In 30, 60, 90 and 120 days after the surgery, 4-5 rats from each series were euthanized. On these days, Xray was performed and processes of osteoinduction were studied by histological methods (van Gieson and hematoxylin-eosin staining).

RESULTS.
The study results showed that after the sterilization by the studied methods, DBM was 100% sterile. In the control series, the histological study revealed the areas of absorption of DBM on Day 30. Along the edge of the formed cavities, osteoblast chains and newly formed bone were seen. The cavities primarily contained myeloid bone marrow. In 60 days, the areas of absorptions and the volume of induced bone increased. Medullary cavities contained myeloid and fatty bone marrow.
By the 3rd month, the process of resorption of DBM continued increasing and areas of newly formed bone tissue calcified. In 120 days, DBM was fully absorbed and substituted with a newly formed bone. On Day 30, X-ray images revealed a weak shadow of the graft with distinct boundaries. On Day 60, the intensity of shadow increased and its structure was inconsistent. In 90-120 days, the graft shadow acquired a consistent structure with distinct boundaries. However, the shadow intensity was weaker than in the normal bone of the recipient.  The intensity of shadows increased, graft boundaries were distinct but did not reach the shadow intensity of the bone recipient Induced osteogenic tissue was observed in separate areas The intensity of shadows increased, graft boundaries were distinct but did not reach the intensity of the bone recipient The formed cavities contained myeloid and fatty marrow. The samples obtained after a 4month observation had more expressed absorption and focal calcificates along the edges of the formed cavities. Some areas contained induced osteogenic tissue. X-ray images taken 30 days after the surgery did not show the graft. By Day 60, a weak shadow of the graft with indistinct boundaries and uneven structure appeared. In 90-120 days, the intensity of the shadow increased, the boundaries of the graft were distinct but did not reach the intensity of the bone in the recipient (Table 1).
It should be noted that non-sterilized DBM (control test series) underwent intensive resorption and provided the osteoinductive effect after the ectopic implantation into the muscle. In 90 days, the appearance of fatty bone marrow was observed. The volume of the induced tissue was larger than in the previous test series but lower than in the control group. Calcificates were also detected during all the periods of observation.

CONCLUSIONS.
The results of the present study showed that allogenous non-sterilized DBM in rats had osteoinductive properties. Connective tissue elements that penetrate the graft during the process of resorption differentiate for ontogenesis. DBM sterilized with performic acid preserved the osteoinductive properties but they were reduced. DBM that was sterilized with an antiseptic complex was characterized by an even lower degree of osteoinduction. When it comes to the technique of the surgery, it should be noted that precise apposition of the donor and recipient bone parts is required because a larger area of contact and the firmness of fixation of the graft provide the quick formation of osteotylus and the restoration of blood circulation in the graft, which significantly influences the transformation of the donor bone. It was shown that DBM sterilized with chlorhexidine and catamine AB did not exert osteoinductive activity, so these substances in the

FINANCIAL SUPPORT AND SPONSORSHIP
Nil.