Hydroxylapatite and Tricalcium Phosphate Implants in the Dental Alveolus of Rats. A Histometric Study

 
Adalberto Luiz ROSA[1]
Luiz Guilherme BRENTEGANI[2]
Sylvestre Arnaldo GRANDINI[1]
 
[1]Departamento de Cirurgia,
[2]Departamento de Estomatologia, Faculdade de Odontologia de Ribeirão Preto, USP, Ribeirão Preto, SP, Brasil

Braz Dent J (1995) 6(2): 103-109 ISSN 0103-6440

| Introduction | Material/Methods | Results | Discussion | References |


The objective of the present study was to analyze histomorphometrically the biological behavior of microgranular hydroxylapatite (MIC), particulate hydroxylapatite (HA 40), and tricalcium phosphate (TCP) implanted in the dental alveolus of rats. All three materials retarded alveolar repair when compared to controls, since less bone was formed during all periods of study. Nevertheless, MIC and TCP showed higher compatibility than HA 40.


Key words:hydroxylapatite, tricalcium phosphate, alveolar repair.


Introduction

Calcium and phosphate ceramics have been widely used as substitutes for bone tissue, mainly because they have a composition similar to that of bone. Among these ceramics, the most frequently used ones are those with a Ca:P proportion of approximately 1.67 or 1.50, i.e., hydroxylapatite and tricalcium phosphate.

Clinical evaluations of these ceramics used to coat metal implants, in the treatment of periodontal lesions, in the prevention of alveolar ridge resorption and in the recovery of an atrophic alveolar ridge have shown conflicting results (Block and Kent, 1986; De Wijs et al., 1993).

These facts indicate the need to carry out thorough experimental and clinical investigations of new ceramics composed of hydroxylapatites or tricalcium phosphate, in order to determine which materials may be used in patients and in which clinical situations.

The objective of the present study was to evaluate histometrically the biological behavior of three calcium and phosphate ceramics differing in composition and/or in particle size, implanted into the dental alveoli of rats.


Material and Methods

A total of 80 male rats (Rattus norvegicus, Wistar strain) weighing 120 150 g were submitted to extraction of the right upper incisor. The animals were divided into 4 groups: control, no treatment; implanted with tricalcium phosphate (TCP) (S.R. Graft Center Implant, USA) whose granules ranged in size from 800 to 1200 µm; implanted with microgranular hydroxylapatite (MIC) (Biohapatita Odontec, São Paulo) whose granules ranged in size from 10 to 50 µm; implanted with particulate hydroxylapatite (HA 40) (H.A. 40 Homus, São Paulo) whose granules ranged in size from 1000 to 2000 µm.

During the experiment the animals were fed a solid diet for rodents and received water ad libitum, except during the first 24 h after extraction.

The animals were anesthetized intraperitoneally with 40 mg/kg thionembutal (Abbott, São Paulo). The right upper incisor was then extracted, irrigated with saline, filled with the materials and sutured with monofilament nylon. The animals received a bolus dose of Pentabiótico Veterinário (Fontoura Wyeth, São Bernardo do Campo, SP) intramuscularly.

Five rats were decapitated after each post extraction period, i.e., 1, 2, 3 and 6 weeks. Their mandibles were separated from the maxillae and the right maxilla was then separated from the left one with a lancet through a median sagittal incision along the intermaxillary suture. The specimen with the right dental alveolus was obtained by a straight scissors cut tangentially to the distal surface of the molars.

The specimens were immersed in 10% formalin and fixed for 24 h, decalcified, dehydrated, cleared, embedded in paraffin and oriented so as to permit 6-µm thick longitudinal sections which were stained with hematoxylin eosin. A total of 20 sections were obtained per block, each section separated from the other by an interval of 10 sections.

In order to permit a better comparison of the results, in the present study the middle third of the alveolus was chosen for quantitative analysis.

The histological sections were examined with a binocular light microscope (Jenamed, Zeiss Jena, Germany) fitted with an immersion objective (100X) and equipped with a light camera. In each alveolus 10 microscopic fields were analyzed with the use of a grid developed by Merz and Schenk (1970).

Knowing the area covered by the grid, it was possible to estimate the volumetric density of bone and connective tissue and of the implanted materials in the alveolus by counting the points on these structures (1000 per alveolus analyzed, corresponding to 10 microscopic fields times 100 grid points). The evaluation was made on the projection and drawing of the structures on a 100-point grid printed on paper, with a final magnification of 1050X. The volumetric density of bone and connective tissue in the alveolus was estimated by the method of point counting.

Data were analyzed statistically by analysis of variance (ANOVA) and by the Tukey test.


Results

Figure 1 shows the mean percentages of the volumetric density of connective tissue in the middle third of the rat dental alveolus evaluated 1, 2, 3 and 6 weeks after the implant. ANOVA showed a significant reduction in the volumetric density of connective tissue along these periods of time (F = 246.33; d.f. = 3; P<0.001). The control group presented a larger amount of connective tissue than the other groups (F = 66.75; d.f. = 3; P<0.001). A significant difference was also detected for time x treatment interaction (F = 85.51; d.f. = 9; P<0.001). Tukey's test showed that the control group presented higher volumetric density of the connective tissue than the other groups at 1 and 2 weeks. At these times, the group implanted with MIC was not different from the group implanted with TCP, but both groups presented more connective tissue than the group implanted with HA 40. After 6 weeks, the group implanted with HA 40 showed higher volumetric density of connective tissue than the other groups.
 
 

Figure 1 - Mean volumetric density of connective tissue in the middle third of the rat dental alveolus.
 
 

Figure 2 shows the mean percentages of volumetric density of bone tissue, evaluated in the middle third of the rat dental alveolus 1, 2, 3 and 6 weeks after the implant. ANOVA showed a significant increase in the volumetric density of bone tissue along the observation periods (F = 1008.23; d.f. = 3; P<0.001). A significant difference was also detected between treatments (F = 109.23; d.f. = 3; P<0.001). The Tukey test showed a higher volumetric density in the control than in the other groups, as well as a higher density in the groups implanted with MIC and TCP than in the group implanted with HA 40. A significant difference was found for time x treatment interaction (F = 13.19; d.f. = 9; P<0.001). Tukey's test showed no difference between groups one week after the implant, but 2, 3, and 6 weeks after there was a higher volumetric density of bone in the control than in the other groups. No difference was detected between groups implanted with MIC and TCP during these periods, but both groups showed a higher bone volume than the group implanted with HA 40 3 and 6 weeks after the implant.
 
 

Figure 2 - Mean volumetric density of bone tissue in the middle third of the rat dental alveolus.
 
 

Figure 3 shows the mean percentages of volumetric density of the implanted material, evaluated in the middle third of the rat dental alveolus 1, 2, 3 and 6 weeks after the implant. ANOVA showed a significant reduction of the implanted materials along the observation periods (F = 35.73; d.f. = 3; P<0.001). The group implanted with HA 40 presented a larger amount of material than the other groups (F = 22.86; d.f. = 2; P<0.001). A significant difference was found for time x treatment interaction (F = 40.47; d.f. = 6; P<0.001). One and 2 weeks after the implant, the group implanted with HA 40 presented higher volumetric density than the other groups. After 3 weeks there was no difference between groups and after 6 weeks the group implanted with HA 40 showed a smaller volume of material than the other groups.
 
 

Figure 3 - Mean volumetric density of the material in the middle third of the rat dental alveolus in the three experimental groups.
 


Discussion

In the present study, analysis of the results for the groups that received MIC, TCP and HA 40 implants showed that these materials caused qualitative and/or quantitative alterations during the different phases of the process of alveolar repair. All materials caused a delay in the repair process and presented a distinct biological behavior. MIC and TCP showed a higher compatibility with the repair tissues than HA 40. During the first week, the amount of inflammatory infiltrate was similar in all groups. However, in the control group and in the groups implanted with MIC and TCP the inflammation decreased during the second week and disappeared during the third, while in the group implanted with HA 40 an inflammatory response was observed up to the third week.

The delay we observed in the repair is in accordance with the data reported in a series of studies recently reviewed by Carvalho and Okamoto (1987) in which the same findings were obtained after the intraalveolar implant of different materials such as root canal sealers, "synthetic bone", polyurethane, boplant, gelatin sponge, proplast, dentin and enamel fragments, and propolis. In general, placing any material inside the dental alveoli retards to some extent the repair process. This happens because the intraalveolar implants disturb clot organization and injure the remaining periodontal ligament (Carvalho and Okamoto, 1978). Implanting MIC, TCP and HA 40 into the alveoli resulted in the formation of a smaller amount of connective tissue during the initial phases (1 and 2 weeks) because these materials were occupying the space that would be initially filled by the clot.

During the periods of study we observed that in the alveoli implanted with MIC and TCP the amount of connective tissue and implanted material decreased with increasing amount of bone tissue. From the second to the third week a two fold increase in the amount of bone was noted and during this period the decrease in the amount of connective tissue was more marked than the decrease of the implanted materials. Thus, the materials were incorporated into the formed bone, as confirmed by the histological aspect of the trabecular bone surrounding MIC and TCP particles, with no foreign body reaction and without any intense inflammatory infiltrate.

An increase in the amount of connective tissue was detected in the alveoli implanted with HA 40 during the first three weeks, followed by a period of stabilization. The implanted material was present in large amounts at the beginning and decreased progressively up to the sixth week, when it virtually disappeared from the middle third of the alveoli. The bone tissue increased along the six week period of the study, but the final amount was much smaller than in the other implanted or control groups. Although it was possible to observe trabecular bone in contact with HA 40, no particle completely surrounded by trabeculae was found. Furthermore, the amounts of connective and bone tissues observed after six weeks, as well as the absence of HA 40 in the middle third, indicate that the material is expelled from the alveolus before repair can begin. This fact was confirmed by the histological finding of large amounts of HA 40 in the cervical third. Even after 6 weeks the cervical third of the alveoli was open, i.e., filled only with material and connective tissue in maturation. When the animals were sacrificed, it was possible to find fragments of the material that projected outward from the alveoli.

Although the same method for alveolar filling was employed, different amounts of material were detected in the alveoli during the first week, a fact that interfered directly with the amount of connective tissue formed. The groups implanted with MIC and TCP presented less material than the group implanted with HA 40 and this may be a consequence of the differences in the properties of the materials. Although it was implanted in the particulate form, HA 40 appeared after one week as a compact block of material, which hampered the growth of connective tissue between the particles. Physicochemical analysis of this material showed that each particle of HA 40 is formed by aggregated smaller particles that are covered with some type of agglutinant (Granjeiro et al., 1992), and it is possible that, when immersed in biological fluids, they join to form a block.

This difference between materials may also be responsible, at least in part, for the differences in biological behavior. As both MIC and TCP remain in particulate form, they may be more rapidly stabilized by the infiltration of connective tissue between the particles, and this seems to contribute to a reduction in the intensity of the inflammatory reaction evoked by the materials and, consequently, to better compatibility (Jarcho et al., 1977).

As to HA 40, it is possible that it did not reach an initial stabilization because it became a block inside the alveoli, and the growth of connective tissue may have led to exposure of the implants, with their consequent expulsion from the alveoli. Such facts have been considered to be the cause of the failure of hydroxylapatite implanted as blocks (Cranin et al., 1988). Furthermore, it has been demonstrated that HA 40 has contaminants such as titanium, strontium, iron, sulfur and potassium (Granjeiro et al., 1992), and this must have contributed to the expulsion of the material from the alveoli and to the presence of a longer-lasting inflammatory infiltrate than in the other implanted groups.

In the initial phases from implantation up to the third week, the main mechanism responsible for the disappearance of MIC and TCP must have been the extrusion of particles through alveolar openings. This has been frequently reported in the initial periods when an intraalveolar hydroxylapatite implant is used (Sherer et al., 1987; Cranin et al., 1988). However, during the third week trabecular bone almost closing the alveolar openings was observed, preventing the extrusion of more particles thereafter. Thus, it seems that the disappearance of materials between the third and sixth weeks occurred as a consequence of their biodegradation. Considering that the stereological analysis permitted the quantification of the materials, it was possible to verify that the volumetric density of MIC changed from 25.64 to 10.98 and the volumetric density of TCP changed from 29.22 to 20.96. It was then possible to estimate the absorption rates of MIC and TCP that were 26% and 28%, respectively, during a 3 week period. This high absorption rate of MIC is quite different from those of other hydroxylapatites that generally are nonabsorbable materials (Jarcho, 1981).

Of the two mechanisms involved in the biodegradation of these materials, chemical dissolution and phagocytosis (Jarcho, 1981), it seems that the former was the main factor responsible for MIC and TCP absorption during the period from 3 to 6 weeks. The amount of phagocytic cells observed from 3 to 6 weeks near these materials was too small to explain such a rapid absorption by means of phagocytosis. The presence of a small number of these cells could be due to the fact that both MIC and TCP are dense materials, thus presenting a smoother surface. This hypothesis is supported by the results of Gomi et al. (1993) who demonstrated that the rougher the surface of hydroxylapatites and tricalcium phosphate, which occurs when these materials are porous, the higher is the number of multinucleated giant cells in contact with these surfaces. This occurs because the roughness of the surface has a positive influence on the fusion of mononuclear precursors.

This process of chemical dissolution that these materials seem to present is important because it may result in an increase of the amount of phosphate and calcium ions in the medium. These ions, in turn, may be absorbed by the cells, mainly by means of endocytosis, leading to an increase in the mitogenic activity of the fibroblasts (Cheung and McCarty, 1985) and stimulating RNA transcription and protein synthesis in osteoblasts (Gregoire et al., 1990), thus contributing to the high compatibility of the materials.

Bonachela et al. (1992) suggested the use of MIC for alveolar filling immediately after extraction, in order to keep the height and contour of the alveolar ridge. This happens either because the implants prevent the collapse of the bone walls (Denissen et al., 1989) or because they occupy the place where bone should be (Block and Kent, 1986). Thus, when hydroxylapatites are used with this objective it is desirable that the material act as a permanent implant; considering its high absorption rate, this seems not to occur with MIC.

Since both MIC and TCP are absorbable materials, they should be used in those situations in which a temporary filling material is desired, which is initially incorporated into the tissue and later replaced by it. Thus, it would be of interest to investigate clinically the validity of employing these materials in situations such as those of periodontal or surgical bone defects.


References

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Bonachela WC, Molo Jr FA, Taga EM, Granjeiro JM: Manutenção do rebordo alveolar com hidroxiapatita microgranular. Rev Gaúcha Odont 40: 212-213, 1992

Carvalho ACP, Okamoto T: Implantes intra-alveolares: considerações sobre estudos experimentais. Rev Assoc Paul Cirurg Dent 32: 273-279, 1978

Carvalho ACP, Okamoto T: Reparação do alvéolo dental. In: Cirurgia bucal: fundamentos experimentais aplicados à clínica. 55-80, Panamericana, São Paulo, 1987

Cheung HS & McCarty DJ: Mitogenesis induced by calcium containing crystals: role of intracellular dissolution. Exp Cell Res 157: 63-70, 1985

Cranin AN, Ronen E, Shpuntoff R, Tobin G, Dibling JB: Hydroxylapatite particulate versus cones as post-extraction implants in humans. J Biomed Mater Res 22: 1165-1180, 1988

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Granjeiro JM, Taga EM, Fonseca M, Maeda L, Taga MSL, Trebachetti CR, Negrato MLAB: Hidroxiapatita para uso clínico: caracterização físico-química. Rev Gaúcha Odont 40: 130-134, 1992

Gregoire M, Orly I, Menateau J: The influence of calcium phosphate biomaterials on human bone cell activities. J Biomed Mater Res 24: 165-177, 1990

Jarcho M, Kay JF, Gumaer KI, Doremus RH, Drobeck HP: Tissue, cellular and subcellular events at a bone-ceramic hydroxylapatite interface. J Bioeng 1: 79-92, 1977

Jarcho M: Calcium phophate ceramics as hard tissue prosthetics. Clin Orthop 157: 259-278, 1981

Merz WA, Schenk RK: Quantitative structural analysis of human cancellous bone. Acta Anat 75: 54-66, 1970

Sherer AD, Slighter RG, Rothstein SS, Drobeck HP: Evaluation of implant Durapatite particles in fresh extraction sockets to maintain the alveolar ridge in beagle dogs. J Prosthet Dent 57: 331-337, 1987


Correspondence: Adalberto Luiz Rosa, Departamento de Cirurgia, Faculdade de Odontologia de Ribeirão Preto, USP, Av. do Café s/n, 14040-904 Ribeirão Preto, SP, Brasil.


Accepted September 28, 1995
Electronic publication: March, 1996


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