Comparison of 3 bone substitutes in canine extraction sites

J Oral Maxillofac Surg 60:53-58, 2002

Comparison of 3 Bone Substitutes in Canine Extraction Sites Anthony Indovina, Jr, DDS,* and Michael S. Block, DMD† Purpose:

The purpose of this study was to evaluate the healing response with 3 different bone substitute materials in extraction sites in the dog. Materials and methods: Four dogs had their mandibular and maxillary premolars extracted atraumatically. The sites were immediately grafted with anorganic bovine bone (Bio-Oss, Osteohealth, Shirley, NY), Bone Source (Leibinger, Inc, Kalamazoo, MI), or Embarc (Lorenz Surgical, Jacksonville, FL), or left untreated as a control. After 8 weeks, the sites were removed for histologic evaluation of bone fill and the healing response. Results: All sites healed well without signs of infection. No significant differences were noted in the shape of the ridges between groups. The control sites had radiographic bone fill by 8 weeks. The Bio-Oss sites showed bone fill with a similar appearance to the control sites. The Bone Source and Embarc sites showed implant material taking up most of the extraction site. In all sites the control and Bio-Oss sites had significantly more bone formation than the Embarc and Bone Source sites (P ⬍ .05). The control sites contained woven bone. The Bio-Oss sites were similar to the control sites, but with remnants of Bio-Oss in the bone. The Bone Source and Embarc sites were filled predominantly with the graft material without evidence of resorption and replacement of the materials, and with minimal bone formation. Conclusions: Based on this study, the control and Bio-Oss sites were similar, with bone filling most of the extraction site. The other 2 materials did not show replacement with bone. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:53-58, 2002 The replacement of a tooth with an implant is dependent on available bone tissue to allow placement in a position that optimizes prosthetic replacement with a crown. Often, the patient requires a tooth to be extracted before implant placement. After tooth extraction, the socket becomes filled with bone to a variable degree. In addition, a portion of the facial bone may resorb or be absent after healing. In an effort to enhance the bone healing of the extraction socket to allow for optimal implant placement, various materials have been advocated to be placed into the extrac-

tion socket. This study compared 3 different materials used in extraction sites. Hydroxyapatite (HA) has been shown to enhance bony ingrowth into a bone defect.1 However, because HA particles are difficult to maintain in a specific location, HA cements have been designed to prevent particle migration. HA cements (HAC) are formed through a chemical reaction when tetracalcium phosphate and dicalcium phosphate dihydrate dissolve in water.2 HACs can be molded to fit a defect and then harden to remain in a desired position. The resultant hardened material is dense and solid in form.2 Previous use in calvarial defects indicates that HAC has the potential for use to fill bone defects; however, the amount of bone fill in the presence of the HAC is not clear.3,4 Particulate anorganic bovine bone can be placed directly into a bone defect and maintained in position by the blood clot and overlying soft tissue envelope. It has been used in extraction sites to prepare them for future implant placement as early as 8 weeks after tooth extraction based on the assumed bone fill in the presence of the material.5 Anorganic bovine bone is composed of the various forms of calcium phosphate that are commonly found in mineralized bone tissue and hence is believed to be resorbed and replaced with mineralized tissue.

Received from the Louisiana State University School of Dentistry, Department of Oral and Maxillofacial Surgery, New Orleans, LA. *Fellow. †Professor. Supported by AAOMS Foundation Student Research Award and LSU Peltier Chair in OMFS. The investigators acknowledge the donation of the materials from the manufacturers for use in this study. Address correspondence and reprint requests to Dr Block: LSU School of Dentistry, 1100 Florida Ave, New Orleans, LA 701192799; e-mail: [email protected] © 2002 American Association of Oral and Maxillofacial Surgeons

0278-2391/02/6001-0001$35.00/0 doi:10.1053/joms.2002.0000

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54 Our hypothesis is that particulate bone graft material permits more bone formation in extraction sites than a solid or dense implant material, such as an hydroxyapatite cement. The purpose of this study was to compare the healing of a fresh extraction site to the healing of extraction sites implanted with particulate or solid bone substitutes, using a dense, relatively inert HAC or particulate bovine bone.

Materials and Methods STUDY DESIGN

The dog extraction site model6 was used for this study. In each animal, the mandibular and maxillary second, third, and fourth premolars were extracted and the sockets were filled with either Bio-Oss (Osteohealth, Shirley, NY), Bone Source (Leibinger, Inc, Kalamazoo, MI), Embarc (Lorenz Surgical Jacksonville, FL), or left unfilled according to a randomization schedule. After healing, the animals were followed by clinical and radiographic examinations, and filled after 8 weeks of healing. Eight weeks were chosen because this is a similar period of waiting for implant placement after tooth extraction.5 STUDY SAMPLE

Four adult female mongrel dogs approximately 30 kg in weight were chosen for this study. General anesthesia was induced in each dog and maintained by 4 mg/kg intramuscular injection of ketamine. Approximately 5 mL of 2% lidocaine with 1:100,000 epinephrine was infiltrated into the mandibular and maxillary tissues around the premolar teeth bilaterally. Each dog then had the right and left maxillary and mandibular second, third, and fourth premolars atraumatically extracted.6 An incision was made around the necks of the teeth, and a full-thickness flap was reflected to expose the crestal bone. A thin fissure bur was used with copious irrigation to section each premolar tooth. Gentle subluxation was performed, and each root was delivered separately with forceps, taking great care to avoid root and cortical bone fracture. Each site was irrigated with sterile saline, and any sharp bone edges were minimally smoothed to avoid trauma during healing. In a preplanned randomized schedule, the extraction sites were either filled with one of the 3 experimental materials or were left unfilled as a control. The materials were distributed among each of the 4 dogs to eliminate site and dog bias. EXPERIMENTAL MATERIALS

Embarc Embarc is a synthetic calcium phosphate material that has been approved by the Food and Drug Admin-

BONE SUBSTITUTES IN EXTRACTION SITES

istration for human use in extraction sockets. It is a collection of calcium phosphate materials in various forms, including hydroxyapatites in an amorphous and crystalline configuration. Embarc was available for this study in prepackaged sterile delivery systems in a unit dose of 1.0 gram. As supplied, Embarc is a sterile, nonpyrogenic, hydrophilic white powder contained within an elastomeric mixing bulb that allows addition of sterile saline as a diluent. The powder in the bulb is sterilized in a protective blister by gamma radiation. For placement into the extraction sites, the Embarc powder and the appropriate amount of sterile saline were thoroughly mixed under aseptic conditions until the mix was homogenous. The resultant putty-like material was placed into a sterile 1.0 mL syringe and injected into the socket. Then the mucosa was gently held over the extraction site for 10 minutes until the Embarc had hardened. Resorbable 4-0 polyglactin sutures were used to completely close the extraction sites. Bone Source This material is also a collection of calcium phosphates in various forms, which is mixed at the operating table using 2.5 mL of sterile water per gram of Bone Source powder. It was mixed for 2 to 4 minutes and then kneaded for 1 to 2 minutes to achieve a uniform distribution of sterile water throughout the powder. This thorough mixing created a uniform paste, which was then placed into the extraction site. Placement was performed gently using a blunt instrument. The implanted material reached a firm state within 20 minutes. Resorbable 4-0 polyglactin sutures were placed to completely close the extraction sites. Bio-Oss Bio-Oss is supplied in particulate form (200 to 500 ␮m) in a glass vial. The material is derived from bovine bone that has been treated to remove all organic elements, hence the term “anorganic bone.” This leaves a collection of hydroxyapatites in varying crystalline forms. The material is slowly resorbed over time and is replaced with bone.5,8 After the teeth had been extracted, the Bio-Oss material was wetted with sterile saline and placed into a 1.0 mL syringe. It was then injected directly into the extraction socket, and gentle pressure was placed to ensure adequate fill. Resorbable 4-0 polyglactin sutures were placed to completely close the extraction sites. Unfilled Socket as a Control After tooth extraction, the sockets were irrigated with sterile saline, and the gingiva was approximated with 4-0 polyglactin sutures to completely close the extraction site. POSTOPERATIVE PROTOCOL

The dogs were given 2 million U of procaine penicillin intramuscularly and pain medication (buprenor-

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INDOVINA AND BLOCK

FIGURE 1. Radiographs taken immediately after placement of materials and at time of killing. A, Sockets immediately postoperatively. B, Sockets at 8 weeks, just after killing. C, Sockets immediately postoperatively. D, Sockets seen in C at 8 weeks, just after killing.

phine, 0.01 mg/kg, intramuscularly, every 8 hours, for 5 days) as needed. They were maintained on a mush diet for the entire 8 week study. Periapical radiographs were taken immediately postoperatively and then biweekly until they were killed at 8 weeks. KILLING PROTOCOL

At the time of killing, photographs were taken, and the animals were then euthanized with intracardiac pentobarbital. The mandibles and maxillas were retrieved en bloc, sectioned buccolingually into small tissue blocks isolating each extraction site, fixed in 10% neutral buffered formalin for 10 days, decalcified, and stained with hematoxylin and eosin. EVALUATION PROTOCOL

Clinical Evaluation The dogs were examined every 2 weeks under general anesthesia. The areas were qualitatively evaluated for presence of extravasated implant material and inflammation. Radiographic Evaluation The periapical radiographs were qualitatively examined for the presence of residual material and general appearance of the socket (Fig 1).

Histologic Evaluation The serial sections were examined by 2 evaluators who were calibrated at 1 session. The calibration session allowed each to observe a series of slides until their grades were similar. The serial sections were then graded for the presence of bone (0 ⫽ no bone, 1 ⫽ minimal [less than 50%] bone formation in the socket, 3 ⫽ moderate [greater than 50% but not complete] bone formation in the socket, and 4 ⫽ complete bone fill in socket). One grade was recorded for each site after agreement of the 2 examiners. DATA ANALYSES

Analysis of variance and Duncan’s Multiple Range tests were used to compare the sites, dogs, and extraction site treatment, with bone fill as the variable.

Results All of the animals tolerated the procedures well. There were no postoperative complications. Data collection was complete regarding all of the clinical, radiographic, and histologic variables.

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BONE SUBSTITUTES IN EXTRACTION SITES

Table 1. AVERAGE BONE FILL FOR EXTRACTION SITES

All sites grouped together Maxillary sites Mandibular sites

Control Site

Bio-Oss

Bone Source

Embarc

3.4 ⫾ 0.5 3.0 ⫾ 0 3.6 ⫾ 0.5

2.6 ⫾ 1.0 2.7 ⫾ 0.9 2.4 ⫾ 1.3

0.75 ⫾ 1.1 1.2 ⫾ 1.3 0.75 ⫾ 1.2

1.5 ⫾ 1.5 1.0 ⫾ 1.7 1.9 ⫾ 1.3

IMMEDIATELY POSTOPERATIVELY

Clinical Primary wound closure was obtained and radiographs were taken of each quadrant in all 4 dogs. No significant differences were noted in the shape of the ridges among groups. Radiographic Adequate condensation of the material in each extraction site was evident, and the unfilled sites were clear of debris. The radiographic density of the BioOss sites was less than with the other 2 materials (Fig 1A, C).

borders becoming less smooth at the interface with the surrounding bone. EIGHT WEEKS

Clinical The gingival tissue remained healthy and free of infection. Radiographic The control and the Bio-Oss sites showed complete bone fill. The Bone Source and Embarc implant sites showed the material taking up most of the extraction site, with minimal changes from immediately preoperatively (Fig 1B, D).

TWO WEEKS

Clinical All sites were healing well, with no signs of infection. There was no extravasated material present in the mouth. Radiographic The unfilled sites showed increasing density, presumably because of the generation of bone. The BioOss sites were still visible. The Bone Source and Embarc implants were intact, with little change from the immediate postoperative radiographs. FOUR WEEKS

HISTOLOGIC EVALUATION

The bone fill scores are shown in Table 1. Using analysis of variance and Duncan’s Multiple Range test, the bone fill in the control and Bio-Oss sites was found to be significantly different than in the Embarc and Bone Source sites (P ⬍ .05). There were no significant differences for sockets with the same material among dogs or sites. Histologically, the control sites were filled with woven bone (Fig 2). The Bio-Oss sites had a similar appearance, except that there were still remnants of Bio-Oss particles intertwined within the new bone (Fig 3). The Embarc (Fig 4) and Bone Source (Fig 5) sites were filled predominantly with

Clinical Tissue healing was complete in all sites, with no sign of infection. Radiographic The control sites continued to increase in radiopacity. The Bio-Oss sites were quite similar in radiodensity to the control sites. The Bone Source and Embarc implants were still intact, showing little change from previous radiographs. SIX WEEKS

Clinical The gingival tissues were smooth and healthy, with no sign of infection. Radiographic The control and the Bio-Oss sites continued to show progressive healing with bone. The Bone Source and the Embarc implants were intact, with the

FIGURE 2. Photomicrograph of control site after 8 weeks. Note woven bone (b) and active bone formation in the extraction site, without the presence of inflammation (hematoxylin and eosin stain, original magnification ⫻25).

INDOVINA AND BLOCK

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FIGURE 3. Photomicrograph of Bio-Oss site after 8 weeks. Note presence of bone (b), Bio-Oss (BO) particles, and active bone formation. There is no inflammatory response around (hematoxylin and eosin stain, original magnification ⫻25).

implant material, with little evidence of resorption and replacement. A foreign body reaction with multinucleated cells was occasionally observed with the Bone Source (Fig 5B) and Embarc materials.

Discussion The hypothesis of this study was that a particulate material will allow more bone formation in an extraction site than a solid, dense material, such as HAC. This hypothesis was proven correct, because the bone fill in the particulate anorganic bone sites was greater than in the solid, relatively inert HAC sites. An interesting finding was the minimal difference between the control sites and the anorganic bone sites. The Bone Source and Embarc materials did not resorb significantly over the time of this study. This is a different result than that reported in the literature

FIGURE 4. Photomicrograph of Embarc site after 8 weeks. The implant is the nonstaining “empty” region on the right side of this photograph. There was minimal inflammation and minimal bone formation seen in the extraction sites (hematoxylin and eosin stain, original magnification ⫻40).

FIGURE 5. A, Photomicrograph of Bone Source site after 8 weeks. There is minimal bone formation and minimal inflammation (hematoxylin and eosin stain, original magnification ⫻25). B, Higher power of A showing a small number of multinucleated cells in the implant material (arrow) (hematoxylin and eosin stain, original magnification ⫻40).

for different defects.1-4 These materials would not be useful in preparing the extraction site for the placement of an implant at 8 weeks because there was minimal resorption of the material and minimal bone formation in the extraction site. In fact, the presence of the material inhibited bone formation. Similar results were found in a related study using HAC repair periodontal osseous defects in humans.7 The cements were exfoliated in 6 months and failed to improve clinical attachment levels and reduce pocketing. However, the Bio-Oss and the control sites showed consistent bone formation. The Bio-Oss particles were slowly resorbing but were still present at 8 weeks, as expected.8 The control sites had the most bone present when comparing the groups. Therefore, the natural question is whether it is necessary to add any material into the extraction site. The defects in this dog study had 4 walls of dense cortical bone, without a labial defect. The study did not address the clinical situation where a portion of the facial cor-

58 tex in the extraction site is missing or when the overlying cortex is thin. In the dog, facial bone resorption was not observed in these sites and thus measurements made of the width of the alveolar crest were not different among the groups. Because the model does not lend itself to meaningful extrapolation to the clinical situation of thin bone around an extraction site, the data on ridge width measurements were not included. If the presence of the Bio-Oss particles does decrease resorption of the facial cortex of bone in an extraction site and if the presence of Bio-Oss particles enhances bone formation to create a new facial cortex when it is missing, then its use in 3-wall extraction defects or in the presence of thin labial bone may be indicated. However, further study is necessary. Acknowledgment We would like to thank Dr Diana Gardiner for performing the statistical analysis.

References 1. Salata L, Craig G, Brook I: Bone healing following use of hydroxylapatite or ionomeric bone substitutes alone or combined with a guide bone regeneration technique: An animal study. Int J Oral Maxillofac Implants 13:44, 1998 2. Kveton J, Friedman CD, Costantino PD, et al: Indication for hydroxylapatite cement reconstruction in lateral based skull surgery. Am J Otol 16:456, 1995 3. Kamerer D, Hirsch BE, Snyderman CH, et al: Hydroxylapatite cement: A new method for achieving watertight closure in transtemporal surgery. Am J Otol 15:47, 1994 4. Maniker A, Cantrell S, Valcys C: Failure of hydroxylapatite cement to set in repair of a cranial defect: Case report. Neurosurgery 43:953, 1998 5. Sclar AG: Preserving alveolar ridge anatomy following tooth removal in conjunction with delayed implant placement. Atlas Oral Maxillofac Surg Clin North Am 7:39, 1999 6. Block MS, Kent JN: A comparison of dense and particulate forms of hydroxylapatite in dog extraction sites. J Oral Maxillofac Surg 44:89, 1986 7. Brown GD, Mealey BL, Nummikoski PV, et al: Hydroxylapatite cement implants for regeneration of periodontal osseous defects in humans. J Periodontol 69:146, 1998 8. Berglundh T, Linde J: Healing around implants placed in bone defects treated with Bio-Oss: An experimental study in the dog. Clin Oral Implants Res 8:117, 1997