TO A QUESTION OF THE DIFFERENTIATED APPLICATION OF OSTEOPLASTIC MATERIAL S AT FILLING OF A DENTAL SOCKET

The current stage of implantology development is char­ acterized by an intensive search for new materials to expand the indications for this treatment method. The most common reasons that worsen the prognosis of implantation or make surgery impossible at all are insufficient bone volume for implant placement and its unsatisfactory quality [1, 6, 7, 10, 11, 12, 13, 14]. These problems are solved by the development of new methods of reconstructive surgeries using materials that replenish the lost bone volume and improve its reparative properties. According to their origin, these materials are divided into three large groups: autografts, allografts, and implants [2, 3, 4, 5, 8, 9, 12]. Despite the different composition, all these materials must meet certain requirements: not to cause toxic, allergic and inflammatory reactions; not to affect the gene apparatus, to integrate socket with body tissues and not to cause their resorption; to be easy to prepare and versatile in use; to be relatively cheap to produce. Thus, in spite of the fact that the literature contains a large amount of information on the use of various types of bone substituting materials, and a large number of their foreign and domestic analogues have appeared in the Russian market, the long-term results of their use both in the experiment and in the clinic have been insufficiently studied. There is still no clear systematization of their application. The use of these materials requires a strictly differentiated approach, depending on the tasks solved with their help. Objects of research were undemineralized spongiosa (can­ cellous bone), crystalline material of a mineral component of bone tissue the so-called «allogenic hydroxyapatite», received on technology «Lioplast»® (production of tissue bank CSRL SamSMU); synthetic hydroxyapatite (porous hydroxyapatite ceramics production «Fihimed», Stavropol). Methodology of comparative morphological study of bone tissue after osteoplastic surgeries In the clinical part of the study in 30 patients after ex­ traction of teeth for subsequent installation of implants, the alveolar cavities were filled with the investigated osteoreparative materials: synthetic hydroxyapatite (porous hydroxyapatite ceramic), 12 sockets; • allogeneic hydroxyapatite, 14 sockets; • non-demineralized spongiosa, 15 sockets. Biopsy material from tooth 1.1 socket without osteoreparative agents was examined as control material. Bone biopsy specimens were taken during early or de­ layed implantation in this area at 1, 3, 6 and 12 months. After standard processing of the morphological material, staining of the sections was made with hematoxylin and eosin, van Gieson microfuchsin; studied under a microscope at 100 and 200-x magnification.

The current stage of implantology development is char acterized by an intensive search for new materials to expand the indications for this treatment method. The most common reasons that worsen the prognosis of implantation or make surgery impossible at all are insufficient bone volume for implant placement and its unsatisfactory quality [1, 6,7,10,11,12,13,14].
These problems are solved by the development of new methods of reconstructive surgeries using m aterials that replenish the lost bone volume and improve its reparative properties. According to their origin, these materials are divided into three large groups: autografts, allografts, and implants [2,3,4,5,8,9,12].
Despite the different composition, all these materials must meet certain requirements: not to cause toxic, allergic and inflammatory reactions; not to affect the gene apparatus, to integrate socket with body tissues and not to cause their resorption; to be easy to prepare and versatile in use; to be relatively cheap to produce.
Thus, in spite of the fact that the literature contains a large amount of information on the use of various types of bone substituting materials, and a large number of their foreign and domestic analogues have appeared in the Russian market, the long-term results of their use both in the experiment and in the clinic have been insufficiently studied. There is still no clear systematization of their application. The use of these materials requires a strictly differentiated approach, depending on the tasks solved with their help.

Methodology of comparative morphological study of bone tissue after osteoplastic surgeries
In the clinical part of the study in 30 patients after ex traction of teeth for subsequent installation of implants, the alveolar cavities were filled with the investigated osteoreparative materials: synthetic hydroxyapatite (porous hydroxyapatite ceramic), 12 sockets; • allogeneic hydroxyapatite, 14 sockets; • non-demineralized spongiosa, 15 sockets.
Biopsy material from tooth 1.1 socket without osteoreparative agents was examined as control material.
Bone biopsy specimens were taken during early or de layed implantation in this area at 1, 3, 6 and 12 months. After standard processing of the morphological material, staining of the sections was made with hematoxylin and eosin, van Gieson microfuchsin; studied under a microscope at 100 and 200-x magnification.

Dynamics o f the healing o f the alveolar socket after extraction o f the tooth without osteoreparative agents (control material)
In 1 month after extraction the socket is filled with con nective tissue. On its surface there are remains of necrotic masses with a large number of leucocytes.
Under the leucocytic layer there is granulation tissue dif fusely infiltrated by leucocytes. Granulation tissue is formed by a large number of capillary-type vessels and plurality of cellular elements. Cellular elements of granulation tissue are presented by fibroblasts, lymphocytes, plasma cells and macrophages. Among capillaries and cellular elements thin delicate collagen fibers are defined.
Granulation tissue is diffusely infiltrated with leukocytes. Lymphocytes with small admixture of macrophages, plasma cells and neutrophils prevail among the infiltrate.
On the surface of granulation tissue areas of regeneration of multi-layered squamous epithelium are found. Epithelium crawls over the granulation surface and forms a layer of 2-3 rows of epithelial cells.
Under the young granulation tissue there is a layer of ma turing granulation tissue in which the number of vessels and cellular elements has significantly decreased and the number of fibrous structures has increased. Differentiation of cellular elements with a decrease in the number of hematogenous elements and an increase in the number of fibroblasts takes place. The synthesis of collagen by fibroblasts intensifies, and argyrophilic fibers and then collagenous fibers are formed. Collagen fibers are grouped into bundles. Synthesis of glycosaminglycans by fibroblasts with formation of the main substance of connective tissue is intensified.
In the maturing granulation tissue, foci of inflammation represented by focal lymphocytic infiltrates are determined, which contributes to the delay of granulation tissue maturation.
Mature connective tissue is found beneath the maturing layer. Thus, the healing of the hole after extraction of the tooth occurs according to the type of secondary tension. The lunette is filled with granulation tissue in which a superficial leuko cytic-necrotic layer, a superficial layer of young granulation tissue, a layer of m aturing granulation tissue and a layer of mature connective tissue are found. Inflammatory infil tration in the superficial layers remains, which slows down the processes of granulation tissue maturation.
Three months after extraction, mature connective tissue with predominantly fibrous structures is determined in the tooth socket. Vessels are differentiated into arteries and veins. Fibroblasts prevail among cellular elements. The surface of the scar was epithelialized. Signs of osteogenesis were not detected. No inflammatory changes were detected.
After six months the dental socket contains mature con nective tissue with the formation of a scar. The surface of the scar is covered by multi-layered squamous non-keratinized epithelium. No signs of osteogenesis were detected.
After twelve months there is a connective tissue scar with foci of hyalinosis in the dental socket. Osteogenesis is not detected.
Thus, the healing of the extraction site after a tooth extraction without the use of osteoreparative agents occurs by second ary tension. At first, necrotic masses are rejected, the cavity is cleaned, and then the hole is filled with granulation tissue with its subsequent maturation and scar formation. Epithelium regeneration and epithelialization of the scar surface take place.
After twelve months, hyaline deposits appear in the scar. There are no signs of osteogenesis in the scar. In some cases, the inflammatory reaction delays the maturation of granulation tissue and the formation of the scar is delayed.

Dynamics o f the healing o f the alveolar socket after tooth extraction with porous hydroxyapatite (PHAK)
After one month in a dental socket blocks of hydroxy apatite are found, which are surrounded by macrophages, phagocytosing particles of hydroxyapatite. Between the Thus, in a month after extraction of a tooth with the sub sequent application of synthetic hydroxyapatite in a hole around the hydroxyapatite granules growth of fibroreticular tissue with the formation of islands of young mesenchymal cells is noted.
In 3 months after tooth extraction differentiation of out growth mesenchymal cells into osteoblasts was observed. Osteoblasts were located on blocks of hydroxyapatite in the form of a chain. Active proliferation of fibroblasts, increased synthesis of collagen with formation of collagenic fibers was noted. There were clusters of osteoclasts.
Thus, 3 months after the extraction of the tooth in the hole there were determined primitive bone bulks, differentiation of the process mesenchymal cells into osteoblasts and osteocytes took place. Bone balloons were located haphazardly, between them there was a growth of mature connective tissue.
After 6 months, mature cancellous bone tissue was detect ed in the tooth socket. Calcification of the bone beams took place. Bony crossbars were formed between the bone beams and a wide network of bone beams and crossbars was formed. The bone-brain cavities filled with a mature connective tis sue with presence of blood vessels were formed. There was a final differentiation of osteoblasts and osteocytes, single osteoclasts were found.
Thus, after 6 months bone tissue was formed in the dental socket and the final differentiation of osteoblast and osteocyte bone cells took place.
After 12 months a mature bone tissue with an orderly arrangement of bone beams was detected in the tooth socket. The formation of bone tissue in the hole was based on porous hydroxyapatite by direct osteogenesis.

Dynamics o f the healing o f the alveolar socket after extraction o f the tooth with the use o f allogenic hydroxyapatite
1 month after extraction of the tooth with filling the hole with allogenic hydroxyapatite there is an expansion of fibrore ticular tissue. In the center of the socket clusters of allogenic hydroxyapatite are visible. Fibrous structures and groups of sprouted mesenchymal cells, many fibroblasts are located between the lumps. There was an increased proliferation of fibroblasts, which adhere to the blocks of hydroxyapatite. There are single small lymphocytic infiltrates on the surface. Signs of osteogenesis in these terms are not found, but between islets of outgrowth mesenchymal cells formed amorphous intercellular substance.
As compared to group II, more intensive proliferation of fibroblasts, more intensive formation of amorphous in tercellular substance, less pronounced inflammatory reaction were observed.
At 3 months after the tooth extraction, little known prim itive bone globules are detected among the osteogenic fi broreticular tissue.
Osteoblasts are located on the surface of the bone beams in 1 row. The differentiation of osteocytes is more intense compared to group II. The number of bone beams is signifi cantly greater than in group II of observations. Bone beams are larger and arranged randomly. The central part of the bone beams is weakly mineralized. Osteocytes are found in small numbers. Mature connective tissue is determined between the bone beams. Inflammatory changes were not detected in the socket.
At 6 months after extraction, mature bone tissue is visible in the extraction socket.
The newly formed bone tissue in the dental socket corre sponds to the structure of normal bone in terms of histological picture. Bone beams and bone crossbones are completely calcified, bone cells osteoblasts and osteocytes are finally differentiated, bone marrow cavities are formed. Compared with group II of observations, bone formation and differen tiation were more intense.
After 12 months, mature bone tissue with final restructur ing of the main structures is determined in the dental socket.

Dynamics o f the healing o f the alveolar socket after tooth extraction with the use o f nondemyelinated spongiosis
1 month after extraction of a tooth with spongiosis the socket contains spongiosis granules surrounded by fibroreticular tissue. The fibroreticular tissue contains numerous islands of outgrown m esenchym al cells. In com parison with group II and III of observations with spongiosa the num ber o f cell islets is significantly higher. The prolif eration o f fibroblasts and outgrowth m esenchym al cells is more intensive. Inflammatory changes were not detect ed. Osteogenesis is not fully developed, but am orphous basophilic deposits are found among fibroreticular tissue. The described deposits are probably the germs of future bone beams.
At 3 months after tooth extraction, numerous bone beams are identified in the osteogenic fibre-reticular tissue.
Bone beam s are large, partially calcified, arranged haphazardly. On the surface of bone beams there are os teoblasts in one layer. There are processes of bone cells differentiation. When spongiosis is applied, more intensive bone formation and faster differentiation of bone structures take place.
After 6 months mature bone tissue is detected in the socket. After 12 months, differentiated bone tissue is detected in the tooth socket.

Summary
Thus, after the tooth extraction and filling with osteoreparative agents, bone formation occurred after 3 months and the final differentiation of the bone tissue by 6 months. The formation of bone tissue occurred as a primary osteo genesis, i.e., bypassing the chondrogenesis stage. In the control group, healing w ith the form ation o f a connec tive tissue scar occurred in the socket. We found no signs of osteogenesis. Filling the socket with osteoreparative agents stimulated osteogenesis, proliferation and outgrowth of mesenchymal cells, their differentiation into bone cells: osteoblasts and osteocytes. The mechanism of osteogenesis in all osteoreparative agents is the same -prim ary osteo genesis. The term s o f bone tissue form ation are alm ost the same, but differ in the degree of intensity of formation of bone structures, in the degree of differentiation of bone cells, intercellular substance. Spongiosa had the greatest osteoreparative properties and porous hydroxyapatite had the least. The use of spongiosa and allogenic hydroxyapatite significantly accelerates the formation of bone tissue and its further differentiation. Allogenic hydroxyapatite has a moderate anti-inflammatory effect. When it is used faster stops the inflam m atory process, which also accelerates reparative reactions.
Under the influence of allogenic HAP a moderate stimu lation of fibroblast proliferation in culture is observed.
Undemineralized spongiosis, which is the basis of b i oplast preparation, has the most pronounced osteoinduc tive and osteoconductive properties, stim ulates growth, development and differentiation of bone tissue progenitor cells. The material enhances mitotic process of fibroblasts and osteoid cells, simultaneously creating conditions for long-term deposition o f the m ain m ineralizing calcium and phosphorus ions required for m ineralization o f the induced bone matrix and compactization of the main bone substance.
The obtained data allow us to arrange the studied ma terials depending on the degree o f osteogenic potential expression in the following sequence. Undem ineralized spongiosa (series «Lioplast») has a pronounced osteoplastic potential, allogeneic HAP (series «Lioplast») -a weakly pronounced one, predominantly bioinert material -PGAC (f. Fichimed).

Conclusions
1. The use of nondemineralized spongiosis of allogenic origin and allogenic hAP («Lioplast»®) accelerates the processes of proliferation of connective-tissue cellular elements in the experiment; porous hydroxyapatite ceramics is a bioinert material and does not affect the proliferation of fibroblasts. 2. According to the data of dynamic morphological study for 12 months filling of the hole with osteoreparative agents stimulates osteogenesis, proliferation of sponged m es enchymal cells, and their differentiation into bone cells: osteoblasts and osteocytes. The greatest osteoreparative properties have allogenic spongiosa, the least -porous hydroxyapatite. 3. Allogenic non-mineralized spongiosa is the most effective in bone augmentation in the clinic of dental implantology; allogenic HAP for implantation purposes is less effective. The use of porous hydroxyapatite ceramics often results in complications and implant removal (0.8 %, 7.5 % and 13.2 %, respectively).