Historically, augmentation or “regeneration” of alveolar bone lost as a result of tooth extraction, periodontal disease, and/or trauma has presented a significant challenge. In this context, many different materials and techniques have been developed and evaluated [
Emdogain is a mixture of enamel matrix derivatives (EMDs) that can be used as an osteopromotive agent for the aforementioned bone augmentation/regeneration treatments [
Bioactive ceramics such as hydroxyapatite (HA), tricalcium phosphate (TCP), or a combination of them (into biphasic calcium phosphate) are biocompatible materials that provoke no or little inflammatory response and have been accepted as bioactive osteoconductive scaffolds for new bone formation and the ingrowth of osteoprogenitor cells [
Therefore, the aim of this study is to propose a new method for improving bone regeneration using a combination of Emdogain and osteoconductive BC.
Three male canines of a mixed Iranian breed and nearly identical weights (25 kg) were selected for the study. Animal selection, management, and surgical protocols were accomplished in accordance with the guidelines of the animal and human experimentation committee of Isfahan University of Medical Sciences.
General anesthesia was induced by injecting acepromazine 1% (Neurotranq, Alfasan, Woerden, The Netherlands; 0.02 mL/kg) and ketamine hydrochloride 10% (Ketamine, Alfasan; 0.04 mg/kg) and was completed with inhalation anesthetic (Halothane BP, Nicholas Piramal India Limited, India). The oral area was rinsed with chlorhexidine 0.2% and then placed under local anesthesia (Persocaine E, Lidocaine HCl 2% + epinephrine 1/80,000; Daru Pakhsh Pharmaceutical Mfg Co, Tehran, Iran). All mandibular premolars were then extracted.
Postoperative antibiotics, including penicillin and streptomycin (Nasr Pharmaceutical Co, Fariman, Iran; 40,000 IU/kg), were prescribed for 7 days, and tramadol (Tehran Chemic Pharmaceutical Co, Tehran, Iran; 5 mg/kg) was applied for pain control. After 3 months of healing, the animals were anesthetized as before; then, under local anesthesia, full-thickness flaps were created bilaterally by making a crestal incision on the mandibular alveolar processes 3 mm from the canines to 2 mm from the first molars. With a trephine bur (No. 6, Meisinger, Neuss, Germany), four standardized cylindrical cavities with a depth and diameter of 6 mm were created on each side of the mandible (3 mm from each other) (Figure EMD/BC with membrane: the defect was filled with a combination of Emdogain (Institut Straumann, Basel, Switzerland) and BC (OSTEON, Geonosis, Suwan-si, Korea) and covered with a nonresorbable membrane (Osteo-Mesh TM-300, CYTOPLAST, Osteogenic Biomedical Inc, Lubbock, TX, USA) that was fixed with tacks (FRIOS, Dentsply/Friadent, Mannheim, Germany). EMD/BC without membrane: the defect was filled with a combination of Emdogain and BC. Membrane only: the defect was covered only with a nonresorbable membrane that was held in place with tacks. Control: the defect was left untreated.
(a) Four cylindrical defects, 6 mm deep and 6 mm in diameter, were prepared on each side of the mandible. (b) The four types of treated defects.
The flaps were repositioned and closed with resorbable polyglycolic acid sutures (PGA, TEB Keyhan, Eshtehard, Iran). At the end of surgery, all animals received intramuscular injections of antibiotics and analgesics, as with the first surgery. During the first 2 weeks after the surgery, the dogs’ mouths were washed with chlorhexidine 0.2%, and the animals were fed a soft diet rich in supplementary vitamins.
The animals were checked twice per day during the first postoperative week for signs of infection. The three animals were randomly assigned to three different time intervals: 2 weeks, 4 weeks, and 6 weeks; at the end of each designated healing period, each animal received general anesthesia and was sacrificed by vital perfusion.
Each mandible was sectioned and fixed in 10% neutral buffered formalin. Eight harvested blocks containing the specimens were obtained from each mandible (four specimens from each side, one from each group). They were decalcified in 10% ethylenediaminetetraacetic acid for 8 weeks and then embedded in acrylic resin (Meliodent, Heraeus Kulzer, Newbury, Berkshire, United Kingdom). With a microtome (ACCU-Cut SRM200, Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands), buccolingual cross-sections were obtained from the middle portion of the defects and were ground to a final thickness of 4
Slides were examined by a blinded examiner under a light microscope (Olympus CX21FS, Olympus Corporation, Tokyo, Japan) at a magnification of ×100. For each section, the percentages of total generated bone, woven bone, lamellar bone, and existing fibrous connective tissue were measured.
The intensity of OPN expression in bone matrix was examined on each slide by light microscope. Based on the observation, OPN staining intensity was ranked as no expression (−), mild expression (+), moderate expression (++), or strong expression (+++).
In this study, membrane exposures were observed in some surgical sites during healing. In these cases, the membrane was removed at 4 weeks, and the site was resutured in the canine that healed for 6 weeks.
Histologic examination revealed that new bone had formed in all experimental groups, especially in the apical portions of the defects. Acute inflammation was observed in only one specimen (from a site treated with EMD/BC with membrane at 2 weeks) (Figure
Histological views of new bone formation with hematoxylin-eosin staining (magnification ×100). (a) GBR group; (b) EMD/BC without membrane; (c) EMD/BC with membrane; (d) control group. CT: connective tissue, LB: lamellar bone, WB: woven bone, OBR: osteoblastic rim.
Table
Mean percentage (± SDs) of tissue areas for each experimental group.
Experimental group | Tissue type | ||||
Total bone formation | Lamellar bone formation | Woven bone formation | Fibrous connective tissue | ||
EMD/BC with membrane | 6 | ||||
EMD/BC without membrane | 6 | ||||
Membrane only | 6 | ||||
Control | 6 |
a,b,cDifferent superscript letters indicate statistically significant differences (
The mean percentages of lamellar bone, woven bone, and fibrous connective tissue for each experimental group and time interval. EMD/BC + M: EMD/BC with membrane; EMD/BC: EMD/BC without membrane; M: membrane only; C: control.
The results of immunohistochemical evaluation and the intensity of staining for OPN in the different groups are shown in Table
OPN staining intensity in each experimental group.
Treatment group | Staining intensity | Interval | Total | ||
2 weeks | 4 weeks | 6 weeks | |||
EMD/BC with membrane | − | 0 | 0 | 0 | 0 |
+ | 0 | 0 | 0 | 0 | |
++ | 1 | 2 | 1 | 4 | |
+++ | 1 | 0 | 1 | 2 | |
EMD/BC without membrane | − | 0 | 0 | 0 | 0 |
+ | 1 | 1 | 1 | 3 | |
++ | 0 | 1 | 0 | 1 | |
+++ | 1 | 0 | 1 | 2 | |
Membrane only | − | 0 | 0 | 1 | 1 |
+ | 1 | 1 | 0 | 2 | |
++ | 1 | 1 | 1 | 3 | |
+++ | 0 | 0 | 0 | 0 | |
Control | − | 0 | 0 | 0 | 0 |
+ | 2 | 2 | 2 | 6 | |
++ | 0 | 0 | 0 | 0 | |
+++ | 0 | 0 | 0 | 0 |
Ratings for OPN expression: −: no expression, +: mild expression, ++: moderate expression, ++++: strong expression.
Immunohistochemical views of OPN stain intensity. ((a), magnification ×100) EMD/BC with membrane; ((b), ×400) EMD/BC without membrane; ((c); ×400) GBR group; ((d), ×400) control group. LB: lamellar bone, WB: woven bone, OB: osteoblast, OC: osteoclast, OPN: osteopontin-expressing cell.
In the present study, in the defects treated with EMD/BC (with or without membrane), the mean percentage of new bone formation was greater than that seen the control and membrane-only groups during each interval. Also, the quality of the newly formed bone was improved, as evidenced by the higher percentage of lamellar bone formed in the EMD/BC groups. This improvement in bone formation might have resulted from the combined effects of EMD and BC. Emdogain is a commercially available mixture of EMDs. The composition of EMDs has been described as a hydrophobic enamel matrix protein complex derived from 6-month-old porcine tooth buds containing more than 90% amelogenin as well as enamelin, tuftelin, tuft proteins, ameloblastin [
In vitro studies have demonstrated that EMDs stimulate bone cell proliferation and differentiation [
On the other hand, the HA/TCP carrier acts as a scaffold [
The increased bone formation shown in the present study is in agreement with other in vivo investigations that have demonstrated the role of enamel matrix proteins in the regeneration of dehiscence type defects around implants [
The difference in bone formation and osteogenic potential of EMD with or without bone grafting materials can be explained either by the different EMD concentrations that were used or by the bone inductive properties of specific bone substitute materials
Application of a membrane along with EMD/BC in the present study resulted in a decrease in the existence of fibrous connective tissue and an increase in lamellar bone formation. However, it did not have any significant additional effect on total bone formation or woven bone formation, which is consistent with some previous studies [
In the present research, immunohistochemical evaluation showed that the defects treated with a combination of Emdogain and BC (with or without a membrane) had the most intense staining, indicating more extracellular OPN expression in these defects in comparison with the other treatments. OPN is a noncollagenous phosphorylated acidic glycoprotein that resides in the extracellular matrix of mineralized tissues and is produced by osteoblasts, osteoclasts, osteocytes, preosteoblasts, some bone marrow cells, and many nonbone cells [
The increased expression of OPN in defects treated by Emdogain/BC suggests that osteoblast differentiation and osteoclastic activity were enhanced, indicating more bone remodeling. Also, according to previous studies, EMDs may accelerate expression of OPN [
It can be seen as a limitation of this study that BC alone, without EMD, was not tested. However, the primary purpose of the present study was to evaluate the role of a BC in bone regeneration. Historically, bone graft materials have been evaluated with and without a membrane. Future studies will investigate the role of BC in regeneration procedures.
According to the results, Emdogain combined with bone ceramic (TCP/HA) might improve bone formation in osseous defects more than the use of membrane alone. It was also observed that the use of a membrane in combination with Emdogain and bone ceramic did not confer an advantage with regard to total bone formation.