Effects of Enamel Matrix Derivative on Bioactive Glass in Rat Calvarium Defects

Replacement of local bone loss is a significant clinical challenge. There are a variety of techniques available to surgeons to manage this problem, each technique with its own advantages and disadvantages. Several treatment procedures including bone grafts, guided tissue regeneration, combined approaches, and growth factors have been suggested for bone regeneration.

The treatment of bony defects with various grafting materials has provided a baseline for what can be achieved in reference to regenerative efforts to create bone fill. While the use of autograft material is the preferred technique, it has limitations, such as donor site morbidity. Allograft has the disadvantage of eliciting an immunologic response due to genetic differences and the risk of inducing transmissible diseases.1 Bone grafting procedures are undergoing a major shift from autologous and allogeneic bone grafts to synthetic bone graft substitutes. Bioactive glasses are groups of synthetic silica-based bioactive materials with bone bonding properties first discovered by Larry Hench in the early 1970s.2 Bone regeneration techniques increasingly rely on the use of exogenous molecules that are able to enhance tissue formation in pathologic and traumatic defects.3 The bioactive glass is considered to be an attractive vehicle for delivering osteogenic agents to the regenerative sites. It has the property to promote adsorption and concentration of proteins utilized by osteoblasts to form a mineralized extracellular matrix4 and thus, promote osteogenesis by allowing rapid formation of bone.

Bioactive glasses are hard, solid (nonporous), and can be regarded as a 3-dimensional silica (SiO₂) network. Silicon dioxide (also known as silicate) forms the main component. By varying the proportions of sodium, calcium, phosphates, and silicon dioxide, various forms of the bioactive glass can be produced, ranging from those are soluble in vivo (solubility being proportional to the sodium oxide content) to those that are essentially nonresorbable.2 

The basis of the bonding property of bioactive glasses is their chemical reactivity in body or tissue fluid, which result in the formation of a hydroxyapatite (HA) layer to which bone can bond. Three general processes are leaching, dissolution, and precipitation. The end result is a bonded interface consisting of a series of layers: glass-silica, gel-hydroxycarbonate, and apatite-bone. A mechanically strong bond between bioactive glass and bone is formed as a result of a silica-rich gel layer that formed on the surface of the bioactive glass when exposed to physiologic aqueous solutions. Within this gel, Ca²⁺ and PO₄²⁻ ions combine to form crystals of HA similar to that of bone.1,5 It has been reported that the leaching reaction of bioactive glass particles in vivo results in the formation of niches in which bone can form.6 When used as a preformed implant, bioactive glass particles have significantly greater mechanical strength when compared with calcium phosphate preparations such as ceramic HA.2 In an animal model, the speed of bone growth around bioactive glass particles was much faster than bone formed around HA particles.7 In addition, the bone formed around bioactive glass was much denser and more mature compared with the bone formed around HA particles.

Biograns (Orthovita, Implant Innovations, Palm Beach Gardens, Fla) is a resorbable amorphous bioactive glass supplied in granules that are approximately 300–355 µm in diameter. They consist of 45% SiO₂, 24.5% CaO, 24.5% Na₂O, and 6% P₂O₅. It is conceivable that this material appears to be resorbed by dissolution rather than by osteoclastic activity.

Enamel matrix derivatives (EMDs) are harvested from around developing pig teeth, following special processing procedures. EMDs are the major component of commercially available Emdogain (Biora AB, Malmö, Sweden). Most of the initial work with this material has been aimed at regenerating periodontal attachment apparatus lost due to periodontitis, but other applications are being explored. The main biologic effects of EMD have been attributed to their predominant protein, amelogenin. Amelogenin is not a classic growth factor, but rather a cell-adhesion matrix-bound protein. Specific amelogenin gene products are thought to have activity as epithelial–mesenchymal signaling molecules.8 In addition, there is evidence that the alternate splice variant of the amelogenin gene, leucine-rich amelogenin peptide, may also have direct signaling activities on cementoblasts and osteoblasts.3 

Enamel matrix proteins have been suggested to exert influence locally by stimulating cellular activation in cell culture that resembles a process critical for healing. In vitro studies show that EMDs were able to increase proliferation and differentiation of human and murine osteoblast cell lines, with the stimulation of phenotypic bone markers in some osteoblast cell lines.9–12 Narukawa et al13 demonstrated that EMDs stimulated osteoblastic differentiation via the induction of mRNA of osteogenesis-related transcription factors. EMDs could create a favorable osteogenic microenvironment by reducing the receptor activator of nuclear factor-kappa B ligand (RANKL) release and enhancing osteoblastic osteoprotegerin (OPG) production.12 In 2006, Reseland et al9 found that EMD had a positive effect on factors involved in mineralization in vitro, causing an increased alkaline phosphatase activity in the medium as well as increased expression of osteocalcin and collagen type I of the primary osteoblastic cells.

It has been demonstrated that bioactive glass coated with Emdogain had the ability to support the growth of osteoblast-like cells in vitro and to promote osteoblast differentiation by stimulating the expression of major phenotypic markers including bone sialoprotein and osteocalcin.14 Boyan et al tested the ability of EMD to induce new bone formation in nude mouse calf muscle or to enhance bone induction ability of a demineralized freeze-dried bone allograft.15 It was concluded that EMD is not osteoinductive, but it could be osteopromotive.11,15 

For clinical application, EMD had a controversial effect on new bone formation in an animal model.16–19 In rats, EMDs had been demonstrated to have osteopromotive effects on new bone formation in the femur16,17 and skull bone defects.20 EMD was also an effective biologic matrix for enhancing new bone formation around titanium implantation in the rat femur.17 However, EMD did not increase the new bone formation using the rabbit tibia model18 or around titanium implants.19 

The aim of this study was to evaluate the effect of EMD on bioactive glass in enhancing bone formation in rat calvarium defects.

Twenty male, 5-month-old albino rats of the Wistar strain were used in the study. All animals were kept separately in cages at the animal house with 24°C and 55% relative humidity and with at least 12 hours of light per day. This study has been approved by the animal experiment ethical committee of Prince of Songkla University.

The animals were anesthetized preoperatively with an intramuscular injection of tiletamine and zolazepam, 20 mg/kg about 5 minutes prior to surgery. Hair over the calvarium was shaved and disinfected with Betadine solution. A midline incision was cut through the skin of the calvaria, and the cranial vertex was exposed. Two bone defects were created in the left and right parietal bone using a trephine bur (5.0 mm diameter) in a slow-speed micromotor under copious saline irrigation with depth equal to the full thickness of the calvarial bone. The calvarial bone was removed carefully to avoid injury to the dura mater. Two reference markers were made with a round bur at 2 mm anterior and posterior to the margin of each defect. They were filled with gutta-percha and served as radiographic landmarks (Figure 1).

The rats were randomly allocated into group A and group B, 15 and 5 rats, respectively. In group A, bioactive glass (Biograns, Biomet 3i, Palm Beach Garden, Fla) mixed with EMD (Emdogain gel, Biora AB) was filled randomly in one side of the defect, and the contralateral side was filled with bioactive glass alone. The healing period in group A was divided into 3 subgroups (2, 4, and 8 weeks), 5 rats per subgroup. In group B (control), EMD was randomly filled in one side, and the contralateral side was empty (CSD). The healing period for group B was 8 weeks. Soft tissue and skin were closed with 4-0 coated Vicryl resorbable sutures (Ethicon, Johnson & Johnson, Somerville, NJ). At the end of the surgical procedures, all animals received a single intramuscular injection of antibiotic (ampicillin, 100 mg/kg) and were fed with rat chow and water until the date of sacrifice.

The rats were sacrificed by an overdose intraperitoneal injection of pentobarbitone (Nembutal). The area of the original surgical calvarial defect and the surrounding tissues were visually examined for signs of inflammation before removal en bloc. Osteogenesis was assessed both radiologically and histomorphologically.

Radiographs of all specimens were taken using Gendex X-ray machine (Gendex Co, Des Plaines, Ill) with 75 kVp, 10 mA, 0.26 sec. The distance between the film and the defect was kept at 50 cm. The films were automatically processed using Dent-X 9000 processor (Dent-X, LogEtronics GmbH, Dieselstrasse, Germany). The radiographs were scanned using a Bio-Rad Model GS-700 imaging densitometer (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) to obtain digital radiographic images of the specimens, which were then analyzed with Molecular Analyst software (version 1.5, Bio Rad, Hercules, Calif). The mean value of the radiographic optical density was measured and calculated for comparing the amount of mineralized tissue produced in response to each type of graft material.

All specimens were fixed in 10% neutral buffered formalin and decalcified with 10% formic acid. Three histologic sections, representing the center of the original surgical defect, were stained with hematoxylin and eosin, and study blinded under a light microscope for histologic and histomorphometric analyses.

Computer-assisted histomorphometry was performed to measure the amount of newly formed bone within the defect. The images of the histologic sections were captured by a digital camera equipped with a light microscope (AxionCam MR, Carl Zeiss, Göttingen, Germany) with an original magnification of ×32. The digital images were saved on a computer, and histomorphometric analysis was completed using Image-Pro Plus, version 5.0 (Media Cybernetics Inc, Bethesda, Md). The criteria used in this study to standardize the histomorphometric analysis of the digital images followed the work of Furlaneto et al.21 The percentage of newly formed bone area was calculated and compared.

The data were analyzed using the SPSS program (version 14.0, Standard Software Package Inc, Chicago, Ill). A nonparametric analysis of variance (Kruskal-Wallis test) was used to detect statistically significant differences between the treatments of the different groups. Following this, the Wilcoxon signed-rank test was used for paired comparisons within each rat. The probability level P < .05 was considered as the level of statistical significance.

Two rats of group AI and one of group AII were lost because of problems encountered during the surgical procedure. All other animals remained healthy during the observation period, and all implantation sides healed uneventfully. After full recovery, they were able to eat the pellet food and drink water.

From radiographs, well-delineated round bone defects were observed together with the radiopaque areas of bone graft materials in situ. The mean optical density of all defects, experimental and control sides, seemed to appear the same as shown in Figure 2, with no statistically significant difference (P > .05).

Microscopically, there was only minimal bone growth in the defect area with no bridging of the defect in all studied.

The bone edge of the defect presented an irregular morphologic appearance, with small areas of reparative bone neoformation in both groups. A large number of residual bioactive glass particles were observed. Fibrous connective tissue was found in both defects (Figures 3 and 4).

The histologic observations were similar to the 2 weeks of observation. Bone neoformation was observed mostly limited to the borders of the surgical defect. The fibrous connective tissue was cellular and vascularized. A large amount of the bioactive glass particles were presented with cracks with complete and incomplete surrounding by fibrous connective tissue. Pink amorphous material within some bioactive glass particles was also evident (Figures 5 and 6).

At the bone edges of the defect, bone neoformation with extension toward the center in direct contact with the particles of the bioactive glass was noted. A lower number of residual bioactive glass particles within the bone defects and a greater amount of dense, organized connective tissue were observed. At this time, the particles appeared to be more fragmented. The histologic appearance showed an increase in bone formation over time; noticeably, the new bone formation found in 8 weeks was greater than in 4 and 2 weeks, respectively. However, the amount of new bone ingrowth was greater in the experimental groups than in the control groups (Figures 7 and 8).

Histologic evaluation of these specimens revealed a thin fibrous connective tissue across the defect with small amounts of new bone being occasionally seen at the bony margin of the defect (Figure 9). In the central areas of the defect, the connective tissue appeared to be thinner, with a smaller number of collagen fibers. All specimens were well vascularized and rich in fibroblasts with oriented collagen fibers (Figure 10).

Means of the percentage of newly formed bone area for each group were demonstrated in Figure 11. The data showed that in groups A and B, the percentage of new bone area was greater in the experimental side than in the control side, though the difference was not statistically significant (P > .05).

In the past decade, tissue engineering-based bone grafting has emerged as a viable alternative to biologic and synthetic grafts. The biomaterial component is a critical determinant of the ultimate success of the tissue-engineered graft because no single existing material possesses all the necessary properties required in an ideal bone graft. EMD is a mixture of proteins with amelogenin as a major component that has been demonstrated to promote periodontal regeneration. However, its influence may not be limited to the cementum. Using an in vitro wound model, it has been reported that EMD can enhance wound-filling for all cells exposed to it in culture medium when compared with untreated conditions.22 EMD has influenced bone metabolism through the activation of growth factor.23 Takayama et al24 reported that EMD promoted the osteogenic differentiation of pluripotent mesenchymal cells. This effect was mediated by bone morphogenetic protein-like molecules present in the EMD. Schwartz et al11 showed that EMD can affect early stages of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD also enhances differentiation. Moreover, EMD has been reported to stimulate the biosynthesis and regeneration of trabecular bone, and the volume fraction of mineralized tissue appeared to be higher in EMD-applied bone than in controls.16 EMD has also been found to be an effective matrix for enhancing new trabecular bone induction and resulting attachment of orthopedic prostheses to the recipient bone.17 The present study was designed to evaluate histometrically the effect of EMD and bioactive glass on bone healing of critical-size defects in the calvaria of rats. The size of the bone defects was in agreement with previous studies of critical-size defects in various animal models.25 Bioactive glass has documented osteoconductive and osteostimulatory properties. Hence, its use has been recommended in order to provide scaffold for the regeneration of various bone defects. In the present model, the narrow bioactive glass particles (300–355 µm) are like other bioactive glass particles of similar composition but different size—not only do the particles form a Ca-P rich layer on their outer surface, which is responsible for the extensive osteoconductive properties, but they themselves are also eroded internally by phagocytosing cells entering via small cracks.26 Most of the bioactive glass underwent resorption over the time, which reduced their diameter and widened the entrance to the internal lumen. The present study demonstrated that bioactive glass plus EMD resulted in formation of new bone area similar to those seen with bioactive glass alone after healing periods of 2, 4, and 8 weeks. The mean value of the radiographic optical density was also not significantly different. After a healing period of 8 weeks, no statistically significant differences were observed among the groups of bioactive glass plus EMD, bioactive glass alone, EMD alone, and the CSD group. Comparing the bioactive glass alone group and the CSD group, the amount of new bone formation was nearly the same in 8 weeks. According to MacNeill et al,27 graft materials that require extended periods for complete resorption will reduce the total amount of newly formed bone due to their continued presence. When EMD was applied alone, the magnitude of new bone formation was higher than in the CSD group. This observation is the same as the recent observation in which positive influence on bone formation was seen following the application of EMD on calvarial defects of rats.20 

The bioactive glass combined with EMD group showed the highest percentage of new bone formation at 8 weeks when compared with any other group, though the difference was not statistically significant. This result may be applied to tissue engineering by coating the bioactive glass with EMD in order to provide more effective scaffold in bone neoformation. Cornelini et al18 found the enamel matrix derivative implanted in rat tibia was fully resorbed after 4 weeks and did not increase the formation of the new bone. But in this study, there was more bone formation in 8 weeks, and this might be the effect of the EMD. Though bioactive glass was not directly compared with other carrier systems for EMD-driven bone formation, it would be a promising carrier material with the potential to adsorb EMD, and the magnitude of the values indicated some advantage to the combination.

This study showed that the association of bioactive glass plus EMD may positively influence bone healing since a greater percentage of new bone area was observed when compared with the other groups. More studies are necessary to evaluate the benefits of EMD associated with other regenerative procedures such as bone grafts and guided bone regeneration and also to clarify the specific components and mechanisms of action behind the observed effects.

The finding of the present study should be considered with caution due to the small sample size. In addition, direct extrapolation of data obtained from animal studies to humans should be interpreted cautiously.

From the result of this study, it may be concluded that Biograns with enamel matrix derivative did not make a significant difference in the new bone formation in the rat calvarial bone defects when compared with Biograns alone, though there was greater new bone formation in the groups of Biograns with enamel matrix derivative. Enamel matrix derivative alone used in the rat calvarial bone defects also did not make a significant difference in the new bone formation compared with unused defects (CSD).

This study was financially supported by the grants from Faculty of Dentistry and Graduate School, Prince of Songkla University.

CSD: contralateral defect

EMD: enamel matrix derivative

HA: hydroxyapatite

OPG: osteoprotegerin