Effects of Fibrin Adhesive Material (Tissucol®) on Alveolar Healing in Rats under Stress

 
Maria C.R. ALVES-REZENDE1
Tetuo OKAMOTO2
 
1Disciplina de Cirurgia e Traumatologia Buco-Maxilo-Facial
2Disciplina de Materiais Odontológicos e Prótese, Faculdade de Odontologia, UNESP, Araçatuba, SP, Brasil

Braz Dent J (1997) 8(1): 13-19 ISSN 0103-6440

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

The effects of Tissucol® on alveolar healing following stress were evaluated histologically, comparing three groups of 28 male albino rats each. Stress was applied and their right upper incisors were extracted. Group A served as an empty control site. In Group B, Tissucol® was applied into the alveolar cavity. Group C received local antifibrinolytic treatment (alveolar irrigation with epsilon-aminocaproic acid solution) before implant of Tissucol® into the tooth socket. Four animals in each group were killed at 1, 3, 6, 9, 15, 21 and 24 days after surgery. Results showed that: 1) Tissucol® did not interfere with connective and osseous tissue formation; 2) Tissucol® allowed new bone formation; 3) Tissucol® residues in Group B in sections of 24-day specimens did not impair healing; 4) Tissucol® was usually completely resorbed and healing was complete 24 days after surgery in Group C.

Key Words: fibrin sealing, alveolar healing, stress.

Introduction

Management of coagulation disorders of oral surgery patients under heavy stress require an efficient and practical method of treatment. Rapid hemostasis is an important goal because it shortens operating time, facilitates visibility and enhances patient comfort and safety. A material that not only produces hemostasis but also resorbs and allows bone regeneration would be most beneficial.

Studies evaluating the effects of the fibrin tissue adhesive Tissucol® as a local hemostatic agent in patients with abnormal hemostatic function have generally found that it serves the triple purpose of tissue sealing, hemostasis and promotion of wound healing (Wepner et al., 1982; Matras, 1982, 1985; Baldin et al., 1985; Bauldo et al., 1985; Palattella et al., 1985; Pini-Prato et al., 1985).

Staindl (1979) reported the importance of tissue rinse procedures with epsilon-aminocaproic acid (EACA) before using the fibrin tissue adhesive.

Therefore, the present study was designed to evaluate the effects of the fibrin tissue adhesive Tissucol® with and without EACA alveolar irrigation on dental extraction wound healing in rats under stress.

Material and Methods

Eighty-four male albino rats (Rattus norvegicus albinus, Wistar) weighing 300-350 g were used. They were divided into three groups, A, B and C, each consisting of 28 animals. Rats were fed a diet of standard Purina rat chow (except during the first 24 hours postoperatively) and water ad libitum. All rats underwent stress according the method of Alves and Okamoto (1989). They were inserted into a PVC (Tigre A.G., Brazil) restraining tube, 13.5 cm long with an internal diameter of 5.5 cm and four openings for the legs of the animals (Figure 1, top). The rats were left in the tube for 4 hours and then were removed. Each animal was anesthetized by inhalation ether for surgery. Their right upper incisors were extracted with an instrument designed by Okamoto and Russo (1973) (Figure 1, bottom). The alveolar cavities of Group A served as an empty control site. In Group B, Tissucol® (Immuno A.G., Vienna) was placed into the tooth socket after it was mixed according to manufacturer directions. Group C received alveolar irrigation with epsilon-aminocaproic acid (EACA; 5 ml of a 5% solution for 2 min) before insertion of the experimental material. All gingival edges were approximated and stabilized with 5.0 vicryl sutures (Johnson & Johnson A.G., Brazil). Four animals in each group were killed by ether overdose at 1, 3, 6, 9, 15, 21 and 24 days after surgery. The right maxillary region was dissected and placed in 10% formaldehyde. Following fixation, the specimens were decalcified in 15% formic acid and processed for routine paraffin embedding. Six-micrometer sections were cut and stained with hematoxylin and eosin. All sections were examined with a Zeiss universal microscope.

Figure 1 - Top, Restraining tube used to induce stress according to the method of Alves and Okamoto. Bottom, Instruments used to extract incisors.

Results

One day after surgery

Fibrin clot organization was more pronounced in the Tissucol® specimens (Group B) and Tissucol®/EACA specimens (Group C) than in the control specimens (Group A). There were neutrophils adjacent to the test material which occupied a larger portion of the alveolar cavity. Some macrophages were observed permeating the central region of the test material. In the Tissucol®/EACA specimens, more new budding capillaries and fibroblasts were observed in the vicinity of the remaining periodontal ligament. Gingival edges were discontinued in every group.

Three days after surgery

The alveolar cavity was filled with an organized (test groups) or disorganized (control group) fibrin clot which showed more evidence of macrophage invasion in the test groups than in the control group. The test implant was observed in the center of the alveolar cavity. Some neutrophils were noted surrounding the material. Fibroblast proliferation was more scattered throughout the implant in Tissucol®/EACA specimens. All three groups exhibited discontinuous gingival edges. Occasional epithelial proliferation from the basal layer was found in the test groups.

Six days after surgery

Tissucol® and Tissucol®/EACA implant sites showed residual masses of test material which were surrounded by fibrous connective tissue, numerous macrophages and occasional lymphocytes. Control specimens showed active bone formation adjacent to the alveolar bone. There was a substantial amount of fibrin clot remaining in the three groups. Gingival edges were discontinued in all groups.

Nine days after surgery

Tissucol® and Tissucol®/EACA residues and fibrin clot remnants were still observed in implant sites (Figure 2, middle and bottom). The material was surrounded by peripheral fibrous connective tissue with small amounts of new bone scattered around the periphery in Tissucol®/EACA specimens. In the control group, fibrous connective tissue and immature bony trabeculae were noted in a larger portion of the cavity (Figure 2, top). Remnants of fibrin clot were still evident.

Fifteen days after surgery

In the control specimens, fibrous connective tissue still occupied most of the alveolar cavity and trabeculae of immature bone were comparable to that seen 9 days after surgery. There was a substantial decrease in the amount of material remaining. In Tissucol® specimens there was a fibrous connective tissue layer surrounding the material and the post-extraction site was filled by fibrous connective tissue. In Tissucol®/EACA specimens the residual material was surrounded by dense fibrous connective tissue peripheral to which was immature and mature bone. Osseous regeneration was advanced in the remnants of the cavity.

Twenty-one days after surgery

In the control group, there was a substantial amount of fibrous connective tissue remaining. All specimens showed masses of disorganized fibrin clot (Figure 3, top). In the Tissucol® group, occasional material remnants were observed which were surrounded by immature bony trabeculae or fibrous connective tissue. The alveolar cavity was filled with immature bone (Figure 3, middle). In Tissucol®/EACA specimens, dense trabeculae of bone were observed in most of the cavity (Figure 3, bottom). Some specimens showed residual masses of material while other specimens showed no material.

Twenty-four days after surgery

Specimens from the control group showed fibrin clot remaining. The alveolar cavity was filled with immature bony trabeculae. In the Tissucol® group, the specimens showed more bone repair compared to 21 days. Residual masses of material were still present. Tissucol®/EACA specimens showed a substantial increase in the amount of mature bone with no material in any of the specimens.

Figure 2 - Top, Nine-day control specimen with fibrous connective tissue and immature bony trabeculae filling the cavity (H&E, original magnification, 63X). Middle, Nine-day Tissucol® specimen with material residues (M) and fibrin clot remnants (FC) (H&E, original magnification, 63X). Bottom, Nine-day Tissucol®/EACA specimen with a small amount of material (M) and fibrin clot remnants (FC) (H&E, original magnification, 63X).

Figure 3 - Top, In the 21-day specimen of the control group, there were masses of disorganized fibrin (FC) (H&E, original magnification, 63X). Middle, In the 21-day specimen of the Tissucol® group, the alveolar cavity was filled with immature bony trabeculae (H&E, original magnification, 63X). Bottom, In the 21-day specimen of the Tissucol®/EACA group, dense bony trabeculae filled most of the cavity (H&E, original magnification, 63X).

Discussion

In this study, stress was used to create a situation in which some deleterious effects of coagulation disorders would be present.

The healing of the control site was disturbed by disorganization of the fibrin clot provoked by stress. Twenty-four hours after surgery, a fibrin clot showing no signs of organization filled the alveolar cavity. A small amount of osteogenesis was evident at 9 days. Bone regeneration was not complete 24 days after surgery and remnants of fibrin clot were interpreted as indicating that the disorganization of the fibrin clot prevented healing. This view is in agreement with Alves and Okamoto (1989) who studied the influence of stress on dental extraction wound healing in rats. They observed some disturbances in the formation of the fibrin clot and these were assumed to result from an increase of plasminogen activator levels.

Histological examination of the sites containing test material showed that distinct osseous reactions occurred depending on the fibrinolytic treatment (alveolar irrigation with epsilon-aminocaproic acid (EACA) solution) utilized. Healing of the Tissucol®/EACA implant sites (Group C) occurred more rapidly than the Tissucol® sites (Group B). However, in the Tissucol® specimens the tissue reactions were qualitatively similar to those observed in Tissucol®/EACA sites.

This finding may be regarded as a beneficial action of alveolar irrigation EACA before implanting Tissucol®. This beneficial effect may be due to: 1) an increase of phagocytosis by Tissucol®, 2) inhibition of plasminogen activation, and 3) an increase of resistance of the fibrin clot.

In Tissucol®/EACA specimens (Group C) the healing was evident at 21 days after surgery and complete at 24 days. There were no signs of any residual Tissucol® 24 days after surgery. However, in the Tissucol® group (Group B), small fragments of the material were observed in sections of 24-day specimens. They did not induce a foreign body reaction and did not limit the formation of new bone.

As noted by other investigators (Wepner et al., 1982; Matras, 1982, 1985; Baldin et al., 1985; Bauldo et al., 1985; Palattella et al., 1985; Pini-Prato et al., 1985; Stajicic et al., 1985; Alves-Rezende and Okamoto, 1992), Tissucol® appears to meet the need for an agent to control bleeding at oral sites during surgery. The ideal topical hemostatic material has been described as one that has high hemostatic action, minimal tissue reactivity, nonantigenicity and in vivo absorbability (Silverstein and Chvapil, 1981).

In this study, it was shown that Tissucol® controlled bleeding, did not elicit foreign body reactions and was subject to lysis. As described by Staindl et al. (1981), the material facilitates the growth of new fibroblasts in the wound area. Since it is in the fibroblast where collagen is synthesized, sufficient fibroblast proliferation is therefore a precondition for undisturbed wound healing. Consequently, the effects of this material are of decisive importance during the exudative and proliferative stages of wound healing.

In addition, as reported by previous studies, resorption of the material is desirable and constitutes one of its main advantages (Matras, 1982, 1985; Baldin et al., 1985; Bauldo et al., 1985). It is probable that complete resorption of the implant in Tissucol® specimens would be seen with further postoperative observation.

It would appear from the findings of this study that both Tissucol® and Tissucol®/EACA are biocompatible. The biological properties of Tissucol®, increased in association with EACA, may have contributed to the dental extraction wound healing in rats under stress. Therefore, we believe that Tissucol® in association with EACA would be useful for hemostatic procedures.

Conclusions

Both Tissucol® and Tissucol®/EACA were effective hemostatic agents in this study. Tissucol® did not elicit foreign body reaction and did not prevent healing of alveolar cavities. Alveolar irrigation with epsilon-aminocaproic acid (EACA) before implanting Tissucol® facilitated the resorption of the material.

Acknowledgments

This work was supported by FAPESP.

References

Alves MCR, Okamoto T: Influência do “stress” no processo de reparo em feridas de extração dental. Estudo histológico em ratos. Rev Odont UNESP 18: 119-130, 1989

Alves-Rezende MCR, Okamoto T: Implante de “Tissucol” em alveolos dentais. Estudo histológico em ratos. Rev Odont UNESP 21: 161-170, 1992

Baldin C, Bedeschi G, Beltrame M, Storti E: Sull’impiego di colla di fibrina umana (Tissucol) in Odontoestomatologia. Giornale di Stomatol i di Ortognati 4: 69-75, 1985

Bauldo F, Cataldo F, Gatti R, Landonio G, Mutti G: Local hemostasis after tooth extraction in patients with abnormal hemostatic function. Hemostasis 15: 402-404, 1985

Matras H: The use of fibrin sealant in oral and maxillofacial sugery. J Oral Maxillofac Surg 40: 617-622, 1982

Matras H. Fibrin sealant in maxillofacial sugery. Development and indications. A review of the past 12 years. Facial Plastic Surg 2: 297-313, 1985

Okamoto T, Russo MC: Wound healing following tooth extraction. Histochemical study in rats. Rev Fac Odont Araçatuba 2: 153-169, 1973

Palattella G, Massi C, Corlelli V, Ruggeri B: Use of a lyophilized human fibrin glue “Tissucol” in oral sugery. Dental Cadmos 53: 65-68, 1985

Pini-Prato GP, de Paoli S, Cortellini P, Zerosi C: On the use of a biological sealing system (Tissucol) in periodontal therapy. Histological evaluation. Int J Periodontics Restorative Dent 5: 32-41, 1985

Silverstein ME, Chvapil M: Experimental and clinical experiences with collagen fleece as a hemostatic agent. J Trauma 21: 388, 1981

Staindl O: Tissue adhesion with highly concentrated human fibrinogen in otolaringology. Am Otol 88: 413-418, 1979

Staindl O, Galvan G, Macher M: The influence of fibrin stabilization and fibrinolysis on the fibrin-adhesive system. Arch Otorhinolaryngol 233: 105-116, 1981

Stajicic Z, Todorovic LJ, Petrovic V: Tissucol in closure of oroantral communication. A pilot study. Int J Oral Surg 14: 444-446, 1985

Wepner F, Fries R, Platz H: The use of the fibrin adhesive system for local hemostasis in oral sugery. J Oral Maxillofac Surg 40: 555-558, 1982

Correspondence: Tetuo Okamoto, Disciplina de Materiais Odontológicos e Prótese, Faculdade de Odontologia, UNESP, Araçatuba, SP, Brasil.

Accepted November 17, 1996
Electronic publication: September, 1997

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