Homogenous Implant in Rat Tibias of Matrix Preserved in 98% Glycerin: Histomorphologic Study

Marcos Eduardo KIRA1
Roberta OKAMOTO1
Idelmo Rangel GARCIA JUNIOR1
Walter Domingos NICCOLI FILHO2

1Disciplina de Cirurgia e Traumatologia Buco-Maxilo-Facial, Faculdade de Odontologia, UNESP, Araçatuba, SP, Brasil
2Disciplina de Estomatologia, Faculdade de Odontologia, UNESP, São José dos Campos, SP, Brasil

Braz Dent J (2000) 11(2): 79-87 ISSN 0103-6440

     Introduction | Material and Methods | Results | Discussion | Conclusions | Resumo | References

As a chemical medium for preservation of tissues, glycerin has shown good results because it maintains the cellular integrity despite the tissue dehydration it causes. Taking advantage of the osteoinducing properties of the osseous matrix and glycerin as a proper medium for tissue preservation, osseous matrix was implanted in rat tibias. Twenty-four rats were used, each receiving two surgical wounds. In one of the wounds an osseous matrix preserved in 98% glycerin was implanted and the other received a matrix without preservatives. Six animals were sacrificed on days 10, 20, 30 and 60 post-implant. After routine histological processing, the specimens were stained in hematoxylin-eosin and Masson's trichrome. The results showed that the matrixes preserved in glycerin presented faster resorption with replacement by newly formed tissue.

Key Words: osseous matrix, homogeneous implant, glycerin, conservation mediums.


Implants and grafts have been used to prevent or correct bone defects, thus improving the morphofunctional conditions of the tissue (Schwarz et al., 1991; Carvalho et al., 1993). Autologous and homogeneous grafts have shown the best results. The disadvantages of autologous grafts is the scarcity of donor areas and the need for an additional surgical procedure. However, this type of graft will be always the first choice in appropriate cases. In homogenous grafts, there is a need to obtain the graft as well as to preserve it.

Graft rejection is an important factor in these procedures, not only due to the immunogenic aspect, but also the quality of the tissue surrounding the graft, which determines its biocompatibility. There is an incorporation process by the inherent tissue into the receptor area and, in some cases, by the connective tissue without inflammatory infiltrate. Processing methods such as bone demineralization are currently under study in an attempt to utilize the demineralized osseous matrix as a substitute for autologous and homogeneous grafts (Tuli and Singh, 1978; Bernick et al., 1989; Nimni, 1989; Bessho et al., 1991). Van de Putie and Urist (1966) reported that in rats the replacement of the osseous matrix graft occurs more rapidly than the graft which uses non-decalcified osseous matrix.

After demineralization, the matrix is made up of morphogenetic proteins which induce the differentiation of mesenchymal cells into chondroblasts and the formation of osteoid tissue (Van de Putie and Urist, 1966). This can be modified depending on how the matrix is obtained. Many means for obtaining and conserving this tissue have been reported (Aspenberg and Andolf, 1989; Reddi and Cunnighan, 1991; Aspenberg et al., 1991; Dícesare et al., 1991; Ripamonti and Reddi, 1992).

Homogenous and heterogeneous tissue grafts require conservation media when not used immediately. Several conservation processes such as cooling, freezing, lyophilization, inclusions in paraffin, dehydration and autocleavage have been described elsewhere. Bone tissue freezing leads to intense fibrous formation and less osseous neoformation which does not occur at a temperature of -70ºC which favors rapid revascularization and replacement of these grafts (Boyne, 1971; Okamoto et al., 1990). A study comparing the use of frozen demineralized bone matrix and autologous graft in human dental alveolus concluded that the former looses its vitality and its osteoinduction properties while the latter is revascularized, maintaining cellular activities of the osteoblasts and osteoclasts (Becker et al., 1994). However in a study on human periodontal defects, Werbitt (1987) reported both clinically and radiographically that there was a gain in bone tissue and a decrease in pocket depth.

In addition to physical means of conservation, chemical solutions such as 70% alcohol, merthiolate and saline 1:4 have shown similar results for tissue freezing and dehydration (Boyne, 1971; Okamoto et al., 1990). Ninety-eight percent glycerin promotes intracellular dehydration without leading to an ionic imbalance and collagen denaturation. Thus, tissues such as the duramater, cornea and cartilage are kept in glycerin for future utilization as grafts, which induces their immunogenic properties (Pigossi, 1964; Carvalho, 1980).

Considering the histologic properties of demineralized osseous matrices and the proven quality of 98% glycerin as a conservation medium, the authors analyzed histologically the behavior of the demineralized osseous matrix obtained through slow decalcification by a solution of equal parts of formic acid and sodium citrate, conserved or not in 98% glycerin.

Material and Methods

Twenty-four male rats (Rattus norvegicus, albinus, Wistar, 150-200 g) were used. They were fed a commercial balanced diet (Anderson and Clayton S.A., São Paulo, SP, Brazil). The osseous matrix was obtained from rat tibias which were fixed in 10% formaldehyde for 24 h and then decalcified in a solution of equal parts of sodium citrate and formic acid for 60 days. Twelve pieces (approximately 3 mm in diameter) were washed in NaCl for 48 h and then placed in 98% glycerin in a sterile container for 20 days and twelve pieces were used for immediate grafts.

All animals received general anesthesia with thionembutal sodium (Abbott Laboratories Ltda, São Paulo, SP, Brazil). A trichotomy was performed on the anterior surface of the right posterior limb, a 20-mm linear incision was made and a flap was raised to expose the tibia. A 701 fissure dental bur mounted on a micromotor with constant saline irrigation was used to prepare two surgical cavities of approximately 3 mm in diameter, reaching the marrow (Figure 1). The distance between the two cavities was 10 mm. The lower wound received the matrix without preservatives (Group I) and the upper wound was filled with the matrix preserved in 98% glycerin (Group II). The grafts filled the cavities completely and the flap was repositioned and sutured with 910 polyglactine resorbable sutures (Sutupack-Ethicon, Johnson & Johnson, São Paulo, SP, Brazil).

Six animals were sacrificed on days 10, 20, 30 and 60 post-implant. The tibias were removed, decalcified and placed in paraffin to facilitate the microtomy. The six micrometer pieces were stained with hematoxylin-eosin and Masson's trichrome. The resorption of the matrix and the formation of new bone was evaluated in this study.


10 days

Group I (matrix without preservatives): All implants were intact. However, three specimens showed small areas with discreet superficial resorption and the presence of some multinuclei cells. Adjacent to the implanted matrix, there was newly formed well-vascularized connective tissue rich in fibroblasts (Figure 1). In some places there was a moderate number of lymphocytes and macrophages and some polymorphonuclear neutrophils. Thin collagen fibers, parallel to the implant, could be seen.

Group II (matrix in glycerin): The graft showed some resorption areas with many multinuclei cells. Connective tissue with a small number of lymphocytes and macrophages was seen in the area. In some places adjacent to the implant borders there was discreet new bone formation (Figure 2). Collagen fibers were similar to Group I.

20 days

Group I: The implant showed some resorption with a few multinuclei cells present in most cases. There was well-vascularized connective tissue with a small number of lymphocytes surrounding the implant (Figure 3). At a distance, there was small newly formed bone trabeculae with many osteoblasts.

Group II: There was a decrease in the volume of the implanted matrix caused by resorption. Close to the implant borders, there were several areas with immature bone tissue (Figure 4) and many osteoblasts were evident. In others areas, there was newly formed well-vascularized connective tissue rich in fibroblasts. A discreet number of lymphocytes was seen and there were collagen fiber bands parallel to the long axis.

30 days

Group I: The implant resorption was more accentuated with the presence of multinuclei cells. Close to the implant borders there was connective tissue with a moderate number of fibroblasts with collagen fiber bands. Few lymphocytes and macrophages were present. In some areas, there was new bone tissue adjacent to the implant.

Figure 1 - Group I - 10 days. Implant (M) practically intact. (HE, magnification: 160X).

Figure 2 - Group II - 10 days. Implant (M) showing newly formed bone (arrow). (HE, magnification: 160X).

Group II: There was an absence of active resorption with approximately 2/3 of the original volume remaining. Almost all of the implant was incorporated into the new well-differentiated bone tissue. In some cases there were small areas of active resorption with few multinuclei cells. In these areas a thin band of connective tissue separated the implant from the new bone tissue.

60 days

Figure 3 - Group I - 20 days. Implant (M) showing connective tissue (CT) with small number of lymphocytes. (HE, magnification: 160X).

Figure 4 - Group II - 20 days. Implant (M) showing well-formed adjacent bone tissue (OT). (HE, magnification: 160X).

Group I: Most of the specimens showed moderate reduction of the matrix when compared to the previous stage, with 2/3 of the implant remaining. In certain areas there was resorption of the matrix with multinuclei cells as well as new bone tissue. Close to the graft, there was new bone tissue. However, between the implant and the new tissue, there was a thin band of connective tissue with fibroblasts parallel to the matrix surface (Figure 5). The volume of newly formed bone around the matrix was moderate.

Group II: There was an absence of active resorption in the organic matrix with 2/3 of the original volume remaining. The whole matrix was incorporated into the new bone tissue filling up the surgical wound completely (Figure 6).

Figure 5 - Group I - 60 days. Connective tissue separating the implant (M) from the immature newly formed bone tissue (arrow). (HE, magnification: 160X).

Figure 6 - Group II - 60 days. Remaining matrix (M) incorporated into the newly formed bone tissue (OT). (Masson's trichrome, magnification: 250X).


The best methods used to obtain osseous matrices are decalcifications using hydrochloric acid, ethylenediaminetetra acetic acid (EDTA), and nitric acid which induce neoformation of bone tissue in many cases (Van de Putie and Urist, 1966; Tulli and Singh, 1978).

In the present study, we used the decalcification process described by Morse (1945) for histological examination and to evaluate the demineralized osseous matrix obtained. This process is routinely used in our experimental surgery center. We were not concerned about maintaining the osteoinducing properties that some matrices have. However, we wanted to observe the biological behavior that this tissue could present after a long demineralization process. We used glycerin for conservation because it is efficacious and simple, has high bactericidal properties and causes less immunogenicity of the grafts (Pigossi, 1964). The tibia was the bone donor for the matrices and the receiving bed as well because it presents high osteogenic activity and it is easy to handle. In other studies, the tibia showed intense activity where in surgical wounds, such as in this study, 10 days post-surgery immature bone tissue filled all the wounds and at 30 days there was complete repair with well-organized trabeculated bone (Carvalho et al., 1993).

The results of this study show that the tissue reaction of the implant groups differ. In the group preserved in glycerin (Group II), the material had an earlier and more intense resorption which was observed by the 10th day. New bone tissue was also formed more rapidly in that stage. In Group I, the ossification was seen on the 30th day. These results are similar to those of the cartilage graft in which there was a faster resorption and replacement of the graft conserved in glycerin as reported by Okamoto et al. (1990).

If we consider that in the present study the implant was performed in an area of active osteogenesis, it is likely that the matrix did not trigger osteogenesis but there might have been stimuli to the formation of new bone tissue. On the other hand, the faster and more intense ossification in the glycerin group shows that the conservation method participated in the degree of graft resorption and replacement. Although it is not common to use formaldehyde solution and conserve transplant tissues, studies using bovine pericardium conserved in this solution or in glutaraldehyde showed good results, considering not only the graft biocompatibility but also its texture. An experimental study on bone surgical wounds showed evidence that the grafts conserved in formaldehyde led to similar levels of ossification as occurs with autologous grafts, although repair was delayed in the former group (Mehra et al., 1993). Other similar results were evident in this experiment with demineralized osseous matrices without the conservation media.

Another important aspect is the matrix volume. Although the differences between the two matrices are evident, in the last stage we observed that approximately 2/3 of the original volumes remained. This can be taken as a discreet resorption if compared to other types of implants such as cartilage preserved in glycerin.

The results of this study are not good if one has the goal of graft resorption and replacement by the receptor bed tissue. However, the primary objective was to evaluate the demineralized osseous matrix regarding its biocompatibility and the behavior of the bone tissue in its presence. It is likely that the resorption observed in this study was related to the morphologic structure of the bone tissue that was used to obtain the demineralized matrix, in this case, the compact bone.

One of the characteristics of the resorption seen in this study was that it was limited to the borders of the graft, denoting the intention of the local tissue to lead to smooth contours for the incorporation process. Thus, at 60 days post-surgery the conserved graft is completely incorporated into the newly formed bone tissue. On the other hand, in group I, this was not evident.


We can conclude that the osseous matrix treated with 98% glycerin undergoes earlier and more intense resorption than osseous implants not treated with glycerin. In addition, the resorbed areas are more quickly replaced by newly formed bone tissue and the remaining matrix is incorporated into this newly formed bone.


Okamoto T, Kira ME, Okamoto R, Garcia Junior IR, Niccoli Filho WD: Implante homógeno de matriz óssea preservada em glicerina a 98% em lojas cirúrgicas preparadas em tíbias de ratos. Estudo histomorfológico. Braz Dent J 11(2): 79-87, 2000.

Entre os meios químicos de preservação de tecidos, a glicerina tem oferecido bons resultados conservando a integridade celular mesmo provocando a desidratação tecidual. Tendo em vista as propriedades osteoindutoras da matriz óssea e a boa qualidade da glicerina como meio de preservação dos tecidos, foi realizado o implante de matriz óssea em tíbias de ratos. Foram utilizados vinte e quatro ratos e, em cada animal foram realizadas duas cavidades. Numa das cavidades foi implantada a matriz óssea preservada em glicerina a 98% e na outra, a matriz óssea sem preservação. Seis animais foram sacrificados aos 10, 20, 30 e 60 dias após o implante. As peças obtidas após processamento histológico de rotina foram corados em hematoxilina e eosina e tricrômico de Masson. Os resultados obtidos mostram que a matriz preservada em glicerina sofre reabsorção mais rápida e substituição por tecido ósseo neoformado.

Unitermos: matriz óssea, implante homógeno, glicerina, meios de conservação.


Aspenberg P, Andolf E: Bone induction by fetal and adult human bone matrix in thymic rats. Acta Orthop Scand 60: 195-199, 1989
Aspenberg P, Thorngren KG, Lohmander LS: Dose dependent stimulation of bone induction by basic fibroblast growth factor in rats. Acta Orthop Scand 62: 481-484, 1991
Becker W, Becker BE, Cafesse R: A comparison of demineralized freeze-dried bone and autologous bone to induce bone formation in human extraction sockets. J Periodontal 12: 175-178, 1994
Bernick S, Paule W, Ertel D, Nishimoto SK: Cellular events associated with the induction of bone by demineralized bone. J Orthop Res 7: 1-11, 1989
Bessho K, Tagawa T, Murata M: Analysis of bone morphogenetic protein (BMP) derived from human and bovine bone matrix. Clin Orthop 269: 226-234, 1991
Boyne PJ: Transplantation, implantation and grafts. Dent Clin North Am 15: 433-453, 1971
Carvalho ACP: Applications of homologous duramater in preprotetic surgery. Rev Ass Paul Cirurg Dent 34: 304-307, 1980
Carvalho PSP, Garcia Junior IR, Sanches MG: Comparative study between two hydroxyapatite: Osteosynt and HA-40. Rev G Odontol 41: 330-332, 1993
Dícesare PE, Nimni ME, Peng L: Effects of indomethacin on demineralized bone-induced heterotopic ossification in rat. J Orthop Res 9: 855-861, 1991
Mehra V, Giee SS, Dhillon MS, Bhusnurmath SR, Nagi ON: Comparison of fresh autogenous with formalin preserved allogenic bone grafts in rabbits: An experimental study. Int Orthop 17: 330-334, 1993
Morse A: Formic acid-sodium citrate decalcification and butyl alcohol dehydration of teeth and bone for sectioning in paraffin. J Dent Res 24: 143-145, 1945
Nimni ME: Dystrophic calcification and mineralization during bone induction: Biochemical differences. Nippon Seikeig Sasshi 63: 630-642, 1989
Okamoto T, Gabrielli MAC, Oliveira J: Autogenous transplantation of rib cartilage preserved in glycerol after removal of the perichondrium to the malar process of rats. A histological study (Part I). J Nihon Univ Sch Dent 32: 116-126, 1990
Pigossi N: Implantação de duramater homogena conservada em glicerina. Estudo experimental em cães. Arg Cir Exper 27: 213-247, 1964
Reddi AH, Cunnighan NS: Recent progress in bone induction by osteogenin and bone morphogenetic proteins: challenges for biomechanical and tissue engineering. J Biom Eng 113: 189-190, 1991
Ripamonti U, Reddi AH: Growth and morphogenetic factors in bone induction: role of osteogenin and related bone morphogenetic proteins in craniofacial and periodontal bone repair. Crit Rev Oral Biol Med 3: 1-14, 1992
Schwarz N, Schlag G, Thuenher M, Eschherger J, Zeng L: Decalcified and undecalcified cancellous bone block implants do not heal diaphyseal defects in dogs. Arch Orthop Trauma Surg 1: 47-50, 1991
Tulli SM, Singh A: The osteoinductive property of decalcified bone matrix. J Bone Joint Surg 60: 116-123, 1978
Van de Putie K, Urist MR: Osteogenesis in the interior of intramuscular implants of decalcification bone. Clin Orthop 43: 257-270, 1966
Werbitt M: Decalcified freeze-dried bone allografts: A successful procedure in the reduction of intrabony defects. Int J Periodon Restor Dent 5: 57-65, 1987

Correspondence: Tetuo Okamoto, Disciplina de Cirurgia e Traumatologia Buco-Maxilo-Facial, Faculdade de Odontologia de Araçatuba, UNESP, Rua José Bonifácio, 1193, 16015-050 Araçatuba, SP, Brasil.

Accepted November 29, 1999
Eletronic Publication october, 2000