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Evaluation of the effect of heterologous type I collagen on healing of bone defects
J Oral Maxillofac Surg 60:541-545, 2002
Evaluation of the Effect of Heterologous Type I Collagen on Healing of Bone Defects ¨ mer Kaya, DDS, PhD† Metin Gu ¨ ngo ¨ rmu ¨ ¸s, DDS, PhD,* and O Purpose:
The purpose of this investigation was to evaluate the effect of type I collagen on the healing of bone defects both experimentally and clinically. Materials and Methods: In the experimental study, 16 adult male rabbits were divided into 2 groups: a collagen group and a control group. After the induction of general anesthesia with intraperitoneal ketamine, the anterior surfaces of tibias of the rabbits were surgically exposed, and a hole 4 mm in diameter was made in each tibia. In the collagen group, the defects were filled with type I collagen. The unfilled defects of the other animals were used as controls. During the study, the serum alkaline phosphatase activity of the rabbits, and radiopacity changes in the radiographs of the tibias of the rabbits were evaluated. The rabbits were killed on the 35th day, and histologic sections of the tibias were prepared. The clinical study was carried out on periapical defects in a total of 15 patients who underwent apicoectomy. After the surgical procedure, the osseous defects in periapical regions of 7 patients were filled with type I collagen. The unfilled cavities of the other patients were used for control purposes. The patients were evaluated clinically and radiographically in the postoperative period. The data were analyzed by Student’s t-test and Mann-Whitney U test. Results: In the experimental study, there was an increase in radiopacity corresponding with the serum alkaline phosphatase activity, and there were statistically significant differences between the control and collagen groups both radiologically and biochemically on the 14th and 28th days of the study. In the clinical study, the control cavities filled with a tissue of normal bone density in about 5 months, but the collagen cavities filled in 3 months. Conclusions: It was determined that heterologous type I collagen provides a more rapid regeneration of bone defects. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:541-545, 2002 Collagen is an effective stimulator of osteogenesis.1,2 Therefore, collagens have been used in the treatment of various pathologic models, such as the closure of oral antral communications,3 alveolar augmentation (together with hydroxyapatite)4 guided-tissue regeneration,5 and healing of extraction wounds.6 Because collagen is a biocompatible material that can be tol-
erated by the body, the clinical use of heterologous collagens is becoming more widespread.7-11 On the other hand, it is not easy to determine, in terms of controlled clinical studies, the real effect of a biomaterial, even if there are potentially several parameters of clinical evaluation. Thus, to determine whether these materials are effective in tissue regeneration (especially in bone wound healing), additional studies are always needed. The purpose of this study was to evaluate the effects of type I collagen on the healing of bone defects in an experimental animal model and in humans.
Received from the Department of Oral and Maxillofacial Surgery, Atatu ¨ rk University, Erzurum, Turkey. *Assistant Professor. †Professor and Chief. Address correspondence and reprint requests to Dr Gu ¨ ngo ¨ rmu ¨¸s: Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Atatu ¨ rk University, Erzurum, 25240 Turkey; e-mail: gungormusm@ yahoo.com
Materials and Methods EXPERIMENTAL STUDY
Animals and Surgical Procedure In this study, 16 adult male rabbits were used, each weighing 1.5 to 2 kg. The rabbits were divided into 2
© 2002 American Association of Oral and Maxillofacial Surgeons
0278-2391/02/6005-0011$35.00/0 doi:10.1053/joms.2002.31852
541
542
THE EFFECT OF TYPE I COLLAGEN ON HEALING OF BONE DEFECTS
groups: a collagen group (n ⫽ 8) and a control group (n ⫽ 8). After the induction of general anesthesia with intraperitoneal ketamine, the anterior surfaces of tibias of the rabbits were surgically exposed, and 1 hole of 4 mm in diameter was prepared on each tibia for the investigation. The defects of animals in the collagen group were filled with Condress (a type I collagen extracted from bovine Achilles’ tendon through a nondenaturing procedure; Euroresearch, Milano, Italy).11 Defects in the control group were unfilled, and the wounds were closed primarily. Biochemical Procedure Blood (2 mL) was collected from a marginal ear vein of the rabbits in both groups before surgery (day 0) and on postoperative days 14, 28, and 35. Serum alkaline phosphatase (ALP) activity was determined with an Hitachi 717 Autoanalyzer (Tokyo, Japan). Radiographic Procedure For radiographic investigation, the radiographs of the tibias of the rabbits were exposed soon after surgery (day 1) and on postoperative days 14, 28, and 35. A Siemens Nanodor 2-tip dental x-ray unit set (Pittsburgh, PA) was used for all exposures with a target-to-film distance of 82 cm and an exposure time of 0.10 second at 50 kVp and 1 mA. On a total of 64 of the postsurgical radiographs, the radiographic features of cavities were evaluated as following by 3 blinded independent investigators: grade 1, in which the border of cavity was unaltered and there was a complete radiolucent appearance; grade 2, in which the border of cavity was slightly changed, but there was still a radiolucent appearance; grade 3, in which there was a moderate radiopacity; and grade 4, in which there was entirely a radiopaque appearance in the cavity. Histopathologic Procedure For the histopathologic investigation, the animals were killed on day 35. Immediately after death, their tibias were removed. The tibias of rabbits were placed in 10% formalin. After decalcification in 5% formic acid, all specimens were serially sectioned longitudinally at 5-m intervals and stained with Masson’s trichrome. CLINICAL STUDY
Patients and Surgical Procedure The clinical study was carried out on a total of 15 patients (6 females and 9 males; age range, 35 to 47 years) who participated voluntarily and underwent apicoectomies on maxillary or mandibular incisors. After the surgical procedure, the osseous defects in periapical areas of 7 patients were filled with type I collagen. Other patients’ unfilled cavities were used for control purposes. Patients were given an appoint-
ment for their postoperative examinations. In every postoperative month, patients’ intraoral radiographs were taken with an Ardet Orix 70-kVp 8-mA unit (Buccinasco, Italy) fitted with a cylindrical collimeter (20-cm anode–film distance) and a Palmotime 100 timer (Ardel). When intraoral radiographs were taken, the patients’ heads were positioned in an occlusal plane parallel with the floor. The films of the maxillary incisors were taken from an angle of ⫹40°, and those of mandible incisors were taken from an angle of ⫺15°. In the periodical radiographs taken of patients, the films in which the first significant radiopacity change was observed by 3 blinded independent investigators and the films in which a radiopacity with the density of a normal bone was observed were determined. Statistical Analysis The data were analyzed with the computer program SPSS (Chicago, IL) for Microsoft Windows (Redmond, WA), Release 6.0. In the experimental study, the changes in ALP activity between collagen and control groups were analyzed by Student’s t-test, and radiographic results were analyzed by Mann-Whitney U test. In the clinical study, radiographic results were analyzed by Student’s t-test.
Results EXPERIMENTAL STUDY
Histopathologic Results On day 35, all cavities were filled with new bone tissue in both groups (Fig 1). Serum ALP Activity ALP activity changes in control and collagen groups are shown in Table 1. It was determined that there was no significant difference between both groups on days 0 and 35 (P ⬎ .05); however, there was statistically a significant difference between control and collagen groups on days 14 and 28 (P ⫽ .0001). Radiographic Results On a total of 64 postsurgical radiographs of animals, the radiographic features of cavities were evaluated by 3 independent investigators (2 specialist radiologists and 1 surgeon [M.G.]); each was blinded to when the radiograph was taken, and the results were recorded. Two weeks later, another evaluation was made by the same investigators using the same methods. Scores were compared with the previous ones. The differences between the evaluations were not statistically significant (P ⬎ .05). The postsurgical radiographic results for the cavities are shown in Table 2. It was determined that there was no statistically significant difference (P ⬎ .05) between the 2
543
¨ NGO ¨ RMU ¨ ¸S AND KAYA GU
Table 2. POSTSURGICAL RADIOGRAPHIC RESULTS FOR THE COLLAGEN AND CONTROL CAVITIES
Day
Control Group (n ⫽ 8)
Collagen Group (n ⫽ 8)
P
1 14 28 35
1.1250 ⫾ 0.354 2.5000 ⫾ 0.535 3.2500 ⫾ 0.463 3.6250 ⫾ 0.518
1.3750 ⫾ 0.518 3.0000 ⫾ 0.000 3.7500 ⫾ 0.463 3.8750 ⫾ 0.354
.2636 .0253 .0147 .2521
NOTE. Values for the 2 groups are given as mean ⫾ SD radiograph changes/d.
FIGURE 1. A collagen site at day 35 (Masson’s trichrome stain, original magnification ⫻100).
groups on days 1 and 35; however, there was a statistically significant difference between the control and collagen groups on day 14 (P ⫽ .0253) and on day 28 (P ⫽ .0147) (Figs 2, 3). CLINICAL STUDY
No infection was observed in the patients who had undergone apicoectomy in the postoperative period. The postoperative radiographs of these patients were evaluated by the same 3 independent investigators (each was blinded to the time interval at which the radiograph was taken after surgery) again, and the results were recorded. Two weeks later, another evaluation was made by the same investigators using the same methods. Scores were compared with the previous ones, and the differences between the evaluations were not statistically significant (P ⬎ .05). The postsurgical radiographic results for the patients are shown in Table 3. It was determined that unfilled cavities after apicoectomy showed an opacity in normal bone density at an average of 158.50 ⫾ 10.597 days, and that collagen cavities showed an opacity at an average of 83.57 ⫾ 18.483 days (Figs 4, 5). However, it was determined that there was a statistically significant difference between the control and collagen groups, both in the period in which the first significant radiopacity change was observed (P ⫽
Table 1. ALKALINE PHOSPHATASE ACTIVITIES IN THE CONTROL AND COLLAGEN GROUPS
Day
Control Group (n ⫽ 8)
Collagen Group (n ⫽ 8)
t
P
Preop 14 28 35
376.0 ⫾ 7.11 400.0 ⫾ 8.08 540.0 ⫾ 8.60 601.3 ⫾ 13.74
352.0 ⫾ 7.60 790.0 ⫾ 6.23 690.0 ⫾ 2.56 628.0 ⫾ 16.19
0.59 10.80 4.73 0.37
.567 .0001 .0001 .720
NOTE. Values for the 2 groups are given as mean ⫾ SD units/L.
FIGURE 2. Postoperative radiograph of an animal in the control group. A, day 1. B, day 14. C, day 28.
544
THE EFFECT OF TYPE I COLLAGEN ON HEALING OF BONE DEFECTS
FIGURE 4. Radiograph of a patient in the control group. A, Preoperative appearance. B, Postoperative day 62. C, Postoperative day 155.
.021) and in the period in which a radiopacity in normal bone density was observed (P ⫽ .0001).
Discussion
FIGURE 3. Postoperative radiograph of an animal in the collagen group. A, day 1. B, day 14. C, day 28.
In the animal experiments and clinical studies made previously, the histologic condition, biochemistry, and radiology of bone wound healing were thoroughly elucidated. In these investigations, it was established that ALP activity increased together with the new bone formation and osteogenesis, and that important radiopacity changes occurred.12-20 Thus, the activity of ALP is believed to indicate the presence of osteoblast cells and the formation of new bone. In the experimental study, we observed that serum ALP activity increased much more in the collagen group than in the control group, in accordance with the radiopacity changes. Previous investigations have supported our observations. In those investigations, it was determined that the collagen accelerates the healing process,7,8 when it is applied to the bone defects, and that ALP activity increases much more depending on the effect of collagen.21-23 However, ALP is produced in multiple tissues, including bone. For this reason, it might not be correct to say that there is a direct relation between the new bone formation and the increase in serum ALP level. Nevertheless, when the biochemical and radiologic changes that occurred in the control and collagen groups during the experimental study are taken into consideration, it may be said that the increase in serum ALP activity observed in our study can be related to new bone formation. Although there was no biochemically significant dif-
Table 3. POSTSURGICAL RADIOGRAPHIC RESULTS FOR THE PATIENTS
First significant radiopacity change Trabecular appearance
Control Group (n ⫽ 8)
Collagen Group (n ⫽ 7)
t
P
68.00 ⫾ 10.085 158.50 ⫾ 10.597*
51.40 ⫾ 5.413* 83.57 ⫾ 18.483
2.69 9.21
.021 .0001
NOTE. Values for the 2 groups are given as mean ⫾ SD days. *Missing one datum.
¨ NGO ¨ RMU ¨ ¸S AND KAYA GU
FIGURE 5. Radiograph of a patient in the collagen group. A, Preoperative appearance. B, Postoperative day 33. C, Postoperative day 66.
ference between the control and collagen groups in the preoperative period, it was determined that there was a statistically significant difference between both groups on postoperative days 14 and 28. Furthermore, it was established that there was a radiographically significant difference between both groups in these periods again. On the other hand, in previous studies, it was pointed out that bone wound healing continued for 3 to 7 weeks in the study models, such as extraction wound and other osseous wounds,4,6,16,24,25 and for 3 to 4 months or longer in fracture healing and in cyst or tumor excisions.17,18 In the clinical study, we observed radiographically that the bone defects to which we applied type I collagen filled completely with tissue to normal bone density in about 3 months, whereas the defects in the control group filled in by about 5 months. Many authors who have corresponded with us regarding our findings determined that collagen applied to bone defects quickened the healing process and caused the bone defects to be filled with new bone tissue in a shorter time.7,8,10,21,22,26-28 In this present study, it has been determined that heterologous type I collagen provides a more rapid regeneration of bone defects.
References 1. Masi L, Franchi M, Santucci D, et al: Adhesion, growth, and matrix production by osteoblasts on collagen substrata. Calcif Tiss Int 51:202, 1992 2. Kinoshita S, Finnenga M, Bucholz RW, et al: Three-dimensional collagen gel culture promotes osteoblastic phenotype in bone marrow derived cells. Kobe J Med Sci 45:201, 1999 3. Nort AF, Gould AR, Means WR: Microfibrillar collagen and dehydrated dura xenografts for the closure of oroparanasal communications. Oral Surg Oral Med Oral Pathol 39:97, 1981 4. El Deeb M, Roszkowski MT, El Hakim I: Extracranial and mandibular augmentation with hydroxyapatite-collagen in induced diabetic and nondiabetic rats. J Oral Maxillofac Surg 49:165, 1991 5. Mundell RD, Mooney MP, Siegel MI, et al: Osseous guided tissue regeneration using a collagen barrier membrane. J Oral Maxillofac Surg 51:1004, 1993
545 6. Mannai C, Leake D, Pizzoferrato A, et al: Histologic evaluation of purified bovine tendon collagen sponge in tooth extraction sites in dogs. Oral Surg Oral Med Oral Pathol 61:314, 1986 7. Micheletti G, Onorato I, Micheletti L. Heterelogous, lyophilized, non-denatured type-1 collagen in dentistry. Int J Tiss Reac 14:39, 1992 ¨ , et al: Pleomorphic adenoma 8. Gu ¨ ngo ¨ rmu ¨¸s M, Savran A, Ertas¸ U (a case report). J Turkish Oral Maxillofac Surg 2:34, 1998 9. Mian M, Beghe F, Mian E: Collagen as a pharmacological approach in wound healing. Int J Tiss React 14:1, 1992 10. Mehlisch DR, Leider AS, Roberts WE: Histologic evaluation of the bone/graft interface after mandibular augmentation with hydroxylapatite/purified fibrillar collagen composite implants. Oral Surg Oral Med Oral Pathol 70:685, 1990 11. Beghe F, Menicagli O, Neggiani P, et al: Lyophilized nondenatured type-I collagen Condress extracted from bovine achilles tendon and suitable for clinical use. Int J Tiss React 14:11, 1992 12. Hatano K, Inoue H, Matsunaga T, et al: Effect of surface roughness on proliferation and alkaline phosphatase expression of rat calvarial cells cultured on polystyrene. Bone 25:439, 1999 13. Nishimoto SK, Chang CH, Gendler E, et al: The effect of aging on bone formation in rats: Biochemical and histological evidence for decreased bone formation capacity. Calcif Tiss Int 37:617, 1985 14. Inoue K, Ohgushi H, Yoshikawa T, et al: The effect of aging on bone formation in porous hydroxylapatite: Biochemical and histological analysis. J Bone Miner Res 12:989, 1997 15. Jamsa T, Koivukangas A, Kippo K, et al: Comparison of radiographic and pQCT analyses of healing rat tibial fractures. Calcif Tiss Int 66:288, 2000 16. Guglielmotti MB, Cabrini RL: Alveolar wound healing and ridge remodeling after tooth extraction in the rat: A histologic, radiologic, and histometric study. J Maxillofac Surg 43:359, 1985 17. Heppenstall RB: Fracture Treatment and Healing (ed 1). Philadelphia, PA, Saunders, 1980, p 41, 80, 98 18. Goaz PW, White SC: Oral Radiology: Principles and Interpretation (ed 2). St Louis, MO, Mosby, 1987, p 742 19. Kawai T, Murakami S, Hiranuma H, et al: Healing after removal of benign cysts and tumors of the jaws: A radiologic appraisal. Oral Surg Oral Med Oral Pathol 79:517, 1995 20. Bodner L, Kaffe I, Littner MM, et al: Extraction site healing in rats: A radiologic densitometric study. Oral Surg Oral Med Oral Pathol 75:367, 1993 21. Strates BS, Connolly JF: Osteogenesis in cranial defects and diffusion chambers: Comparison in rabbits of bone matrix, marrow, and collagen implants. Acta Orthop Scand 60:200, 1989 22. Horisaka Y, Okamoto Y, Matsumoto N, et al: Histological changes of implanted collagen material during bone induction. J Biomed Mater Res 28:97, 1994 23. Shi S, Kirk M, Kahn AJ: The role of type I collagen in the regulation of the osteoblast phenotype. J Bone Miner Res 11:1139, 1996 24. Laskin JL, Lucas WJ, Davis WM: The effects of a granular gelatin preparation on the healing of experimental bone defects. Oral Surg Oral Med Oral Pathol 52:23, 1981 25. Olson RAJ, Roberts DL, Osban DB: A comparative study of polylactic acid, Gelfoam, and Surgicel in healing extraction sites. Oral Surg Oral Med Oral Pathol 53:441, 1982 26. De Vore DT, Baltimore D: Collagen xenografts for bone replacement: The effects of aldehyde-induced cross-linking on degradation rate. Oral Surg Oral Med Oral Pathol 43:677, 1977 27. Sakano S, Murata Y, Miura T, et al: Collagen and alkaline phosphatase gene expression during bone morphogenetic protein (BMP) induced cartilage and bone differentiation. Clin Orthop 292:337, 1993 ¨ , Gu 28. Gu ¨ ngo ¨ rmu ¨¸s M, Kaya O ¨ ndog˘du C, et al: The effect heterologous type I collagen on the osteoblastic activity during bone defects regeneration: A histopathologic evaluation. J Turkish Oral Maxillofac Surg 4:30, 2000
Evaluation of the Effect of Heterologous Type I Collagen on Healing of Bone Defects ¨ mer Kaya, DDS, PhD† Metin Gu ¨ ngo ¨ rmu ¨ ¸s, DDS, PhD,* and O Purpose:
The purpose of this investigation was to evaluate the effect of type I collagen on the healing of bone defects both experimentally and clinically. Materials and Methods: In the experimental study, 16 adult male rabbits were divided into 2 groups: a collagen group and a control group. After the induction of general anesthesia with intraperitoneal ketamine, the anterior surfaces of tibias of the rabbits were surgically exposed, and a hole 4 mm in diameter was made in each tibia. In the collagen group, the defects were filled with type I collagen. The unfilled defects of the other animals were used as controls. During the study, the serum alkaline phosphatase activity of the rabbits, and radiopacity changes in the radiographs of the tibias of the rabbits were evaluated. The rabbits were killed on the 35th day, and histologic sections of the tibias were prepared. The clinical study was carried out on periapical defects in a total of 15 patients who underwent apicoectomy. After the surgical procedure, the osseous defects in periapical regions of 7 patients were filled with type I collagen. The unfilled cavities of the other patients were used for control purposes. The patients were evaluated clinically and radiographically in the postoperative period. The data were analyzed by Student’s t-test and Mann-Whitney U test. Results: In the experimental study, there was an increase in radiopacity corresponding with the serum alkaline phosphatase activity, and there were statistically significant differences between the control and collagen groups both radiologically and biochemically on the 14th and 28th days of the study. In the clinical study, the control cavities filled with a tissue of normal bone density in about 5 months, but the collagen cavities filled in 3 months. Conclusions: It was determined that heterologous type I collagen provides a more rapid regeneration of bone defects. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:541-545, 2002 Collagen is an effective stimulator of osteogenesis.1,2 Therefore, collagens have been used in the treatment of various pathologic models, such as the closure of oral antral communications,3 alveolar augmentation (together with hydroxyapatite)4 guided-tissue regeneration,5 and healing of extraction wounds.6 Because collagen is a biocompatible material that can be tol-
erated by the body, the clinical use of heterologous collagens is becoming more widespread.7-11 On the other hand, it is not easy to determine, in terms of controlled clinical studies, the real effect of a biomaterial, even if there are potentially several parameters of clinical evaluation. Thus, to determine whether these materials are effective in tissue regeneration (especially in bone wound healing), additional studies are always needed. The purpose of this study was to evaluate the effects of type I collagen on the healing of bone defects in an experimental animal model and in humans.
Received from the Department of Oral and Maxillofacial Surgery, Atatu ¨ rk University, Erzurum, Turkey. *Assistant Professor. †Professor and Chief. Address correspondence and reprint requests to Dr Gu ¨ ngo ¨ rmu ¨¸s: Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Atatu ¨ rk University, Erzurum, 25240 Turkey; e-mail: gungormusm@ yahoo.com
Materials and Methods EXPERIMENTAL STUDY
Animals and Surgical Procedure In this study, 16 adult male rabbits were used, each weighing 1.5 to 2 kg. The rabbits were divided into 2
© 2002 American Association of Oral and Maxillofacial Surgeons
0278-2391/02/6005-0011$35.00/0 doi:10.1053/joms.2002.31852
541
542
THE EFFECT OF TYPE I COLLAGEN ON HEALING OF BONE DEFECTS
groups: a collagen group (n ⫽ 8) and a control group (n ⫽ 8). After the induction of general anesthesia with intraperitoneal ketamine, the anterior surfaces of tibias of the rabbits were surgically exposed, and 1 hole of 4 mm in diameter was prepared on each tibia for the investigation. The defects of animals in the collagen group were filled with Condress (a type I collagen extracted from bovine Achilles’ tendon through a nondenaturing procedure; Euroresearch, Milano, Italy).11 Defects in the control group were unfilled, and the wounds were closed primarily. Biochemical Procedure Blood (2 mL) was collected from a marginal ear vein of the rabbits in both groups before surgery (day 0) and on postoperative days 14, 28, and 35. Serum alkaline phosphatase (ALP) activity was determined with an Hitachi 717 Autoanalyzer (Tokyo, Japan). Radiographic Procedure For radiographic investigation, the radiographs of the tibias of the rabbits were exposed soon after surgery (day 1) and on postoperative days 14, 28, and 35. A Siemens Nanodor 2-tip dental x-ray unit set (Pittsburgh, PA) was used for all exposures with a target-to-film distance of 82 cm and an exposure time of 0.10 second at 50 kVp and 1 mA. On a total of 64 of the postsurgical radiographs, the radiographic features of cavities were evaluated as following by 3 blinded independent investigators: grade 1, in which the border of cavity was unaltered and there was a complete radiolucent appearance; grade 2, in which the border of cavity was slightly changed, but there was still a radiolucent appearance; grade 3, in which there was a moderate radiopacity; and grade 4, in which there was entirely a radiopaque appearance in the cavity. Histopathologic Procedure For the histopathologic investigation, the animals were killed on day 35. Immediately after death, their tibias were removed. The tibias of rabbits were placed in 10% formalin. After decalcification in 5% formic acid, all specimens were serially sectioned longitudinally at 5-m intervals and stained with Masson’s trichrome. CLINICAL STUDY
Patients and Surgical Procedure The clinical study was carried out on a total of 15 patients (6 females and 9 males; age range, 35 to 47 years) who participated voluntarily and underwent apicoectomies on maxillary or mandibular incisors. After the surgical procedure, the osseous defects in periapical areas of 7 patients were filled with type I collagen. Other patients’ unfilled cavities were used for control purposes. Patients were given an appoint-
ment for their postoperative examinations. In every postoperative month, patients’ intraoral radiographs were taken with an Ardet Orix 70-kVp 8-mA unit (Buccinasco, Italy) fitted with a cylindrical collimeter (20-cm anode–film distance) and a Palmotime 100 timer (Ardel). When intraoral radiographs were taken, the patients’ heads were positioned in an occlusal plane parallel with the floor. The films of the maxillary incisors were taken from an angle of ⫹40°, and those of mandible incisors were taken from an angle of ⫺15°. In the periodical radiographs taken of patients, the films in which the first significant radiopacity change was observed by 3 blinded independent investigators and the films in which a radiopacity with the density of a normal bone was observed were determined. Statistical Analysis The data were analyzed with the computer program SPSS (Chicago, IL) for Microsoft Windows (Redmond, WA), Release 6.0. In the experimental study, the changes in ALP activity between collagen and control groups were analyzed by Student’s t-test, and radiographic results were analyzed by Mann-Whitney U test. In the clinical study, radiographic results were analyzed by Student’s t-test.
Results EXPERIMENTAL STUDY
Histopathologic Results On day 35, all cavities were filled with new bone tissue in both groups (Fig 1). Serum ALP Activity ALP activity changes in control and collagen groups are shown in Table 1. It was determined that there was no significant difference between both groups on days 0 and 35 (P ⬎ .05); however, there was statistically a significant difference between control and collagen groups on days 14 and 28 (P ⫽ .0001). Radiographic Results On a total of 64 postsurgical radiographs of animals, the radiographic features of cavities were evaluated by 3 independent investigators (2 specialist radiologists and 1 surgeon [M.G.]); each was blinded to when the radiograph was taken, and the results were recorded. Two weeks later, another evaluation was made by the same investigators using the same methods. Scores were compared with the previous ones. The differences between the evaluations were not statistically significant (P ⬎ .05). The postsurgical radiographic results for the cavities are shown in Table 2. It was determined that there was no statistically significant difference (P ⬎ .05) between the 2
543
¨ NGO ¨ RMU ¨ ¸S AND KAYA GU
Table 2. POSTSURGICAL RADIOGRAPHIC RESULTS FOR THE COLLAGEN AND CONTROL CAVITIES
Day
Control Group (n ⫽ 8)
Collagen Group (n ⫽ 8)
P
1 14 28 35
1.1250 ⫾ 0.354 2.5000 ⫾ 0.535 3.2500 ⫾ 0.463 3.6250 ⫾ 0.518
1.3750 ⫾ 0.518 3.0000 ⫾ 0.000 3.7500 ⫾ 0.463 3.8750 ⫾ 0.354
.2636 .0253 .0147 .2521
NOTE. Values for the 2 groups are given as mean ⫾ SD radiograph changes/d.
FIGURE 1. A collagen site at day 35 (Masson’s trichrome stain, original magnification ⫻100).
groups on days 1 and 35; however, there was a statistically significant difference between the control and collagen groups on day 14 (P ⫽ .0253) and on day 28 (P ⫽ .0147) (Figs 2, 3). CLINICAL STUDY
No infection was observed in the patients who had undergone apicoectomy in the postoperative period. The postoperative radiographs of these patients were evaluated by the same 3 independent investigators (each was blinded to the time interval at which the radiograph was taken after surgery) again, and the results were recorded. Two weeks later, another evaluation was made by the same investigators using the same methods. Scores were compared with the previous ones, and the differences between the evaluations were not statistically significant (P ⬎ .05). The postsurgical radiographic results for the patients are shown in Table 3. It was determined that unfilled cavities after apicoectomy showed an opacity in normal bone density at an average of 158.50 ⫾ 10.597 days, and that collagen cavities showed an opacity at an average of 83.57 ⫾ 18.483 days (Figs 4, 5). However, it was determined that there was a statistically significant difference between the control and collagen groups, both in the period in which the first significant radiopacity change was observed (P ⫽
Table 1. ALKALINE PHOSPHATASE ACTIVITIES IN THE CONTROL AND COLLAGEN GROUPS
Day
Control Group (n ⫽ 8)
Collagen Group (n ⫽ 8)
t
P
Preop 14 28 35
376.0 ⫾ 7.11 400.0 ⫾ 8.08 540.0 ⫾ 8.60 601.3 ⫾ 13.74
352.0 ⫾ 7.60 790.0 ⫾ 6.23 690.0 ⫾ 2.56 628.0 ⫾ 16.19
0.59 10.80 4.73 0.37
.567 .0001 .0001 .720
NOTE. Values for the 2 groups are given as mean ⫾ SD units/L.
FIGURE 2. Postoperative radiograph of an animal in the control group. A, day 1. B, day 14. C, day 28.
544
THE EFFECT OF TYPE I COLLAGEN ON HEALING OF BONE DEFECTS
FIGURE 4. Radiograph of a patient in the control group. A, Preoperative appearance. B, Postoperative day 62. C, Postoperative day 155.
.021) and in the period in which a radiopacity in normal bone density was observed (P ⫽ .0001).
Discussion
FIGURE 3. Postoperative radiograph of an animal in the collagen group. A, day 1. B, day 14. C, day 28.
In the animal experiments and clinical studies made previously, the histologic condition, biochemistry, and radiology of bone wound healing were thoroughly elucidated. In these investigations, it was established that ALP activity increased together with the new bone formation and osteogenesis, and that important radiopacity changes occurred.12-20 Thus, the activity of ALP is believed to indicate the presence of osteoblast cells and the formation of new bone. In the experimental study, we observed that serum ALP activity increased much more in the collagen group than in the control group, in accordance with the radiopacity changes. Previous investigations have supported our observations. In those investigations, it was determined that the collagen accelerates the healing process,7,8 when it is applied to the bone defects, and that ALP activity increases much more depending on the effect of collagen.21-23 However, ALP is produced in multiple tissues, including bone. For this reason, it might not be correct to say that there is a direct relation between the new bone formation and the increase in serum ALP level. Nevertheless, when the biochemical and radiologic changes that occurred in the control and collagen groups during the experimental study are taken into consideration, it may be said that the increase in serum ALP activity observed in our study can be related to new bone formation. Although there was no biochemically significant dif-
Table 3. POSTSURGICAL RADIOGRAPHIC RESULTS FOR THE PATIENTS
First significant radiopacity change Trabecular appearance
Control Group (n ⫽ 8)
Collagen Group (n ⫽ 7)
t
P
68.00 ⫾ 10.085 158.50 ⫾ 10.597*
51.40 ⫾ 5.413* 83.57 ⫾ 18.483
2.69 9.21
.021 .0001
NOTE. Values for the 2 groups are given as mean ⫾ SD days. *Missing one datum.
¨ NGO ¨ RMU ¨ ¸S AND KAYA GU
FIGURE 5. Radiograph of a patient in the collagen group. A, Preoperative appearance. B, Postoperative day 33. C, Postoperative day 66.
ference between the control and collagen groups in the preoperative period, it was determined that there was a statistically significant difference between both groups on postoperative days 14 and 28. Furthermore, it was established that there was a radiographically significant difference between both groups in these periods again. On the other hand, in previous studies, it was pointed out that bone wound healing continued for 3 to 7 weeks in the study models, such as extraction wound and other osseous wounds,4,6,16,24,25 and for 3 to 4 months or longer in fracture healing and in cyst or tumor excisions.17,18 In the clinical study, we observed radiographically that the bone defects to which we applied type I collagen filled completely with tissue to normal bone density in about 3 months, whereas the defects in the control group filled in by about 5 months. Many authors who have corresponded with us regarding our findings determined that collagen applied to bone defects quickened the healing process and caused the bone defects to be filled with new bone tissue in a shorter time.7,8,10,21,22,26-28 In this present study, it has been determined that heterologous type I collagen provides a more rapid regeneration of bone defects.
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