Hydroxylapatite Implants with or without Collagen in the Zygomatic Arch of Rats. Histological Study


Marisa Aparecida Cabrini GABRIELLI1
Élcio MARCANTONIO JÚNIOR1
Gilberto GÓISSIS2
Tetuo OKAMOTO3
Mário Francisco Real GABRIELLI1
Eduardo Hochuli VIEIRA1

1Faculty of Dentistry of Araraquara, UNESP, Araraquara, SP, Brazil
2Institute of Chemistry, USP, São Carlos, SP, Brazil
3Faculty of Dentistry of Araçatuba, UNESP, Araçatuba, SP, Brazil


Correspondence: Marisa Aparecida Cabrini Gabrielli, Rua Humaitá, 1680, Faculdade de Odontologia de Araraquara, 14801-903 Araraquara, SP, Brasil. e-mail: secddc@black.foar.unesp.br


Braz Dent J (2001) 12(1): 9-15 ISSN 0103-6440

INTRODUCTION | MATERIAL AND METHODS | RESULTS | DISCUSSION | RESUMO | REFERENCES


The authors studied the behavior of calcium phosphate materials used as inlay implants into bone cavities prepared in the zygomatic arch of rats. Fifty male albino rats were divided into four groups as follows: group I - preparation of bone cavities which did not receive any implant material as controls; group II - implants of Interpore® 200; group III - implants of experimental hydroxylapatite; group IV - implants of experimental hydroxylapatite combined with collagen. The animals were sacrificed after 5, 15, 30, 60 and 120 days and the specimens were submitted to histological analysis. Results showed that the experimental hydroxylapatite used in group III presented better osteogenic properties compared to the other materials. All tested materials were biocompatible, although group IV presented a more intense inflammatory response.

Key Words: hydroxylapatite, biocompatibility, implants, calcium phosphate ceramics.


INTRODUCTION

Among other procedures, hydroxylapatite is presently used in the treatment of periodontal lesions, covering of metallic implants and orthognatic surgery. Several studies have shown that hydroxylapatite is an osteoconductive material (1,2) and that more bone is usually deposited in particulate ceramic implants in comparison to block implants (3). The material does not present systemic or local toxicity nor significant inflammatory or foreign body reaction (4).

One of the problems of calcium phosphate ceramics is stability in the implantation site. The granules may be dislocated and migrate locally. Lack of stability and alteration of implant anatomy is considered the greatest disadvantage of this kind of material (5). Biocompatible materials have been developed to confine hydroxylapatite granules to the site of implantation. Collagen has been used because it is biocompatible and
resorbable (6-9). This study was designed to evaluate a new calcium phosphate material, associated and not to bovine collagen.


MATERIAL AND METHODS

Fifty male albino rats (Rattus norvegicus, albinus, Wistar, 200-250 g body weight) were used in the present investigation. The animals were fed a balanced solid diet (Anderson Clayton S/A, São Paulo, SP, Brazil), except for the first 24 h postoperative, and water ad libitum.

Experimental Group

The animals were divided into 4 groups: I) bone cavity without implantation of any material, as negative control; II) Interpore® 200 (International, Irvine, CA, USA), as positive control; III) experimental hydroxyl apatite (HAe); IV) association of experimental hydroxylapatite with collagen (HAc). Both HAe and HAc were produced by the Chemistry Institute of the University of São Paulo at São Carlos (Dr. Gilbert Goissis, Av. Dr. Carlos Botelho, 1465, Caixa Postal 780, 13560-970 São Carlos, SP, Brasil. Fax: +55-16-274-9180; Tel: +55-16-274-9181).

Implantation of Materials

After induction of general anesthesia by intraperitoneal infiltration of 10% chloral hydrate (0.4 ml/100 g body weight; Quimibrás Indústria Química S/A, São Paulo, SP, Brazil), the right and left infraorbital regions were shaved. With the animal laying on its side, surgical access to the zygomatic arch was performed, after local antisepsis with alcohol-iodine solution. A skin incision was made with a number 15 blade, approximately 2 mm below the inferior orbital rim, following its contour, for an extension of 10 mm. After separation of the deeper tissues with small scissors, the periosteum was also incised and reflected and the tissues were retracted with iridectomy forceps.

A number 700 bur (Mailleffer, Ballaigues, Switzerland) mounted in a low-speed handpiece was used to prepare a 4-mm long and 2-mm deep bone cavity. The width was determined by the diameter of the bur. The osteotomy was performed with saline irrigation. The cavity was prepared in the anterior portion of the zygomatic arch, in the temporal process of the malar bone, without exceeding the thickness of the arch. The preparation was then thoroughly irrigated with saline to remove debris.

The material to be implanted was hydrated with saline solution in a sterile Dappen flask prior to implantation. The control animals had a non-implanted cavity in the left side and an implant of Interpore® 200 on the right side. The experimental animals received implants of hydroxylapatite in the right arch and hydroxylapatite associated with collagen in the left arch.
 
 
 
 

After adaptation of the implant to the cavity with iridectomy forceps the periosteum was brought back to its original position and the wound closed with 4-0 silk (Ethicon, Johnson & Johnson, São José dos Campos, SP, Brazil) interrupted sutures. The surgical sites were again disinfected with alcohol-iodine solution. Each animal received a single im dose of penicillin G (0.2 ml; Fountoura Wyeth, São Paulo, SP, Brazil).

Histological Processing

Five animals in each group were sacrificed by sulfuric ether inhalation, after 5, 15, 30, 60 and 120
days. Specimens were obtained by removal of the whole zygomatic arch, which was then freed from the remains of surrounding soft tissue. The specimens were fixed in 10% formalin and decalcified in 50% sodium citrate and formic acid solution, following routine laboratory procedures for hematoxylin/eosin (H&E) staining and histological examination of the sections.


RESULTS

Non-Implanted Control Cavities

After 5 days, the surgical site showed newly formed well developed connective tissue, close to the bottom and lateral walls, with newly formed bone spicules and osteoblasts (Figure 1). After 15 days, the cavity was filled with immature bone trabeculae and ample medullary spaces (Figure 2). After 30 days, the cavity was filled with well developed bone (Figure 3) and after 60 and 120 days, it was totally repaired with well developed new bone (Figure 4).
 


Figure 1. Control (group I): 5 days. Newly formed connective tissue to the bone walls. H&E. Magnification, X160.

Figure 2. Control (group I): 15 days. Surgical cavity showing immature bone trabeculae. H&E. Magnification, X160.

Figure 3. Control (group I): 30 days. Surgical cavity filled with well developed bone. H&E. Magnification, X160.

Figure 4. Control (group I): 120 days. Surgical cavity totally repaired. H&E. Magnification, X160.
 
 
 
 
 

Interpore® 200

After 5 days, blood clot infiltrate by macrophages was found close to the implant (Figure 5). After 15 days, the material partially filled the surgical cavity and was surrounded by organized connective tissue (Figure 6), a moderate number of fibroblasts and numerous macrophages. At 30 days, the material was
surrounded by poorly organized connective tissue, some collagen fiber bundles could be found parallel to the surface of the implanted material (Figure 7). At 60 days, there was poorly organized connective tissue around the material with macrophages and multinucleated giant cells. After 120 days the material was sometimes surrounded by defined bone, however, there were areas of connective tissue at the interface between bone and the implant (Figure 8).
 


Figure 5. Interpore® (group II): 5 days. Implanted material partially filling the surgical site (M) with blood clot in proximity. H&E. Magnification, X160.

Figure 6. Interpore® (group II): 15 days. Implanted material (M) showing poorly organized connective tissue in proximity. H&E. Magnification, X160.

Figure 7. Interpore® (group II): 30 days. Collagenous fibers parallel to the implant surface (M). H&E. Magnification,
X160.

Figure 8. Interpore® (group II): 120 days. Newly formed bone surrounding the implant. Areas of connective tissue without bone differentiation are seen. H&E. Magnification, X160.
 

Experimental Hydroxylapatite (HAe)

After 5 days the material was surrounded by connective tissue. Close to the limits of the implanted material osteoblast-like cells were found (Figure 9).


Figure 9. HAe (group III): 5 days. Small fragments of material (M) covered by osteoblast-like cells. H&E. Magnification,
X250.
 

After 15 days there was less implanted material, which was surrounded by well developed connective tissue. At the interface, osteoblasts were found. At some areas, new bone formation was seen proximal to the implant (Figure 10). After 30 days, bone was seen in close contact with the implanted material (Figure 11) and after 60 days, the implant was incorporated by newly formed bone. The surgical cavity was totally filled by well developed bone. The material was incorporated by well developed bone (Figure 12) after 120 days.
 


Figure 10. HAe (group III): 15 days. Surgical site showing the implant and newly formed bone trabeculae close to the walls. H&E. Magnification, X160.

Figure 11. HAe (group III): 30 days. Fragment of the material (M) covered by newly formed bone. H&E. Magnification, X160.

Figure 12. HAe (group III): 120 days. Small amount of material incorporated by bone. H&E. Magnification, X160.
 

Experimental Hydroxylapatite with Collagen (HAc)

After 5 days, the material partially filled the surgical cavity. Neutrophils, macrophages and lymphocytes (Figure 13) were seen. Small bands of poorly organized connective tissue were found close to the bone walls. After 15 days, poorly organized connective tissue with great numbers of macrophages, lymphocytes and multinucleated giant cells were observed (Figure 14). At 30 days, the cavities were filled with connective tissue without new bone formation (Figure 15), and after 60 days, there was still no bone formation. The connective tissue was infiltrated by a moderate number of fibroblasts and macrophages. After 120 days, the implants were absent and the cavity showed areas without complete healing with irregular and poorly defined bone trabeculae (Figure 16).
 


Figure 13. HAc (group IV): 5 days. Implanted material and neutrophil infiltration. H&E. Magnification, X160.

Figure 14. HAc (group IV): 15 days. Small fragments of material and multinucleated giant cells. Magnification, X160.

Figure 15. HAc (group IV): 30 days. Surgical site filled with connective tissue without bone differentiation. H&E. Magnification, X160.

Figure 16. HAc (group IV): 120 days. Surgical site showing bone areas without complete healing. H&E. Magnification, X160.
 


DISCUSSION

Interpore® 200, a hydroxylapatite with a porous microstructure resembling bone and a porosity size of 150 µm, considered adequate for bone ingrowth (10), was used as positive control. Other materials, such as collagen (11), may be associated with hydroxylapatite to provide stability. Thus, we evaluated the association of hydroxylapatite and bovine collagen and the same experimental hydroxylapatite without collagen. The tested hydroxylapatite was formulated so that the calcium/phosphorus proportion was 1:55. This makes the material similar to immature bone, possibly favoring osteoconduction. Empty cavities were used as negative control.

After 5 days, the control cavities, Interpore® and HAe presented similar findings. However, in group IV (HAc), a greater inflammatory reaction was found, with more intense macrophagic reaction. Vargas Neto (personal communication, 1992) used an association of hydroxylapatite and collagen, as a paste, with a mild inflammatory reaction. The different results may be due to the fact that lyophilized granules were used in the present study, resulting in greater surface area, with greater inflammatory infiltrate and macrophagic reaction, as demonstrated by Van Blitterwi Jr. et al. (12). Heterogeneous collagen, associated or not to ceramics, is considered immunogenic (13). On the other hand, previous studies used similar collagen and foreign body reactions were not seen (14). Variations in preparation of experimental materials may be responsible for these differences.

After 15 days, group III (HAe) presented a significant increase in osteoblastic activity. At some areas the material was already surrounded by bone. For group II (Interpore® 200), findings resembled those of Bell and Bernie (8), who observed hydroxylapatite involved by fibrous tissue, with macrophage activity adjacent to the particles. There continued to be inflammatory infiltrate, with foreign body giant cells for group IV (HAc).

After 30 days, the fibrous capsule Interpore® 200 was evident (Group II). The HAe presented a more intense new bone formation. Zaner and Yukna (15) measured ceramic particles and concluded that smaller sizes (less than 1 mm) result in improved new bone formation. Frank et al. (16) saw more intense new bone formation for particles of smaller diameters. Differences in diameter, porosity and form may be associated with the results observed in this period. In the control group healing was well advanced.

After 60 days, the control cavities were totally repaired. The incorporation of the material by bone in group III (HAe) is more consistent, compared to group II (Interpore® 200), presenting bone in close contact with the implant particles. Reaction to Interpore® 200, with the presence of giant cells, may be related to delay or impossibility of incorporation of the material by bone, resulting in an attempt to eliminate it. In group IV (hydroxylapatite/collagen), healing was delayed and the material absent. Elimination through skin or abcess formation were not observed in the experimental animals. Thus, one might speculate that the material was eliminated by resorption or phagocytosis. The histological reaction observed suggests that phagocytosis occurred due to particle size (15).

After 120 days, the cavities were totally filled with bone, except for group IV (HAc). Although results suggest that all tested materials delay bone healing, the experimental association of HA/collagen presented greater interference with healing. The inflammatory infiltrate and foreign body reaction were more intense for this group at all periods and clearly relates to delayed healing. The material was absent and may have undergone biodegradation by phagocytosis and/or chemical dissolution (17-18). It is even possible that the collagen may have stimulated absorption of the material. After 120 days, the experimental hydroxylapatite (group II) was present in smaller amounts, suggesting biodegradation or phagocytosis. HA particles of 50 µm or less undergo phagocytosis in osteoblast cultures (19). Also, there are traces of beta-tricalcium-phosphate present in the experimental material, according to the manufacturers. The particles were incorporated by bone in direct contact with the mateiral.

For group II, (Interpore® 200) in the same period, results showed involvement of the ceramic by newly formed bone. However, connective tissue was consistently found at the interface. As stated by Cobb et al. (4), even when the same hydroxylapatite is tested, different integration of the material may be observed.

El Deeb et al. (20) showed that porosity size is an important factor in new bone formation. Greater porosity results in better vascularization and new bone formation.

The combination HA/collagen (HAc) presented the worst results at all time periods, with intense and prolonged foreign body reaction. Also the particles of the ceramic were never found. Since the material is composed of HA/collagen in a 7:1 proportion, those particles should be seen. Since expulsion through skin was not found, the material may have undergone alter ations in the production process. The best results were shown for group III (HAe), where direct apposition of bone over the material was found. Since this is an experimental material, further testing is necessary.


RESUMO

Gabrielli MAC, Marcantonio Júnior E, Góissis G, Okamoto T, Gabrielli MFR, Hochuli-Vieira EH. Implantes de hidroxiapatita associados ou não a colágeno, em arco zigomático de ratos. Estudo histológico. Braz Dent J 2001:12(1):9-15.

Os autores realizaram estudo histológico sobre implantes à base de fosfato de cálcio em cavidades ósseas confeccionadas no arco zigomático de ratos. Para tanto 50 ratos albinos, machos, foram divididos em 4 grupos que receberam: grupo I - cavidades ósseas sem implantação de material; grupo II - implantes de Interpore® 200; grupo III - implantes de hidroxiapatita experimental; grupo IV - implantes de hidroxiapatita experimental associada a colágeno. Os animais foram sacrificados aos 5, 15, 30, 60 e 120 dias de pós-operatório, sendo os espécimes submetidos a análise histológica. Os resultados mostraram que a hidroxiapatita experimental (grupo III) apresentou propriedades superiores, em relação à neoformação óssea, em comparação aos outros materiais testados. No grupo IV (hidroxiapatita associada ao colágeno), houve reabsorção do material. Todos os materiais testados foram biocompatíveis com resposta inflamatória mais intensa para o grupo IV.

Unitermos: hidroxiapatita, biocompatibilidade, implantes, cerâmica à base de fosfato de cálcio.


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Accepted July 17, 2000
Braz Dent J 12(1) 2001


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