J Periodontol • November 2014
Effect of Enamel Matrix Derivative on Periodontal Wound Healing and Regeneration in an Osteoporotic Model Richard J. Miron,*† Lingfei Wei,* Shuang Yang,* Oana M. Caluseru,† Anton Sculean,† and Yufeng Zhang*
Background: Despite the worldwide increased prevalence of osteoporosis, no data are available evaluating the effect of an enamel matrix derivative (EMD) on the healing of periodontal defects in patients with osteoporosis. This study aims to evaluate whether the regenerative potential of EMD may be suitable for osteoporosis-related periodontal defects. Methods: Forty female Wistar rats (mean body weight: 200 g) were used for this study. An osteoporosis animal model was carried out by bilateral ovariectomy (OVX) in 20 animals. Ten weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. Animals were randomly assigned to four groups of 10 animals per group: 1) control animals with unfilled periodontal defects; 2) control animals with EMD-treated defects; 3) OVX animals with unfilled defects; and 4) OVX animals with EMD-treated defects. The animals were euthanized 28 days later, and the percentage of defect fill and thickness of newly formed bone and cementum were assessed by histomorphometry and microcomputed tomography (micro-CT) analysis. The number of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP), and angiogenesis was assessed by analyzing formation of blood vessels. Results: OVX animals demonstrated significantly reduced bone volume in unfilled defects compared with control defects (18.9% for OVX animals versus 27.2% for control animals) as assessed by microCT. The addition of EMD in both OVX and control animals resulted in significantly higher bone density (52.4% and 69.2%, respectively) and bone width (134 versus 165mm) compared with untreated defects; however, the healing in OVX animals treated with EMD was significantly lower than that in control animals treated with EMD. Animals treated with EMD also demonstrated significantly higher cementum formation in both control and OVX animals. The number of TRAP-positive osteoclasts did not vary between untreated and EMD-treated animals; however, a significant increase was observed in all OVX animals. The number of blood vessels and percentage of new vessel formation was significantly higher in EMDtreated samples. Conclusions: The results from the present study suggest that: 1) an osteoporotic phenotype may decrease periodontal regeneration; and 2) EMD may support greater periodontal regeneration in patients suffering from the disease. Additional clinical studies are necessary to fully elucidate the possible beneficial effect of EMD for periodontal regeneration in patients suffering from osteoporosis. J Periodontol 2014;85:1603-1611. KEY WORDS Cementum; cementum, bone; enamel matrix proteins; osteoporosis; osteoporosis, regeneration; periodontal guided tissue regeneration. * The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China. † Department of Periodontology, School of Dental Medicine, University of Bern, Bern, Switzerland.
doi: 10.1902/jop.2014.130745
1603
Periodontal Regeneration of Osteoporotic Defects With EMD
T
he increasing longevity resulting from epidemiologic transition has key implications relating to the elderly population. Diseases specific to this age group, such as osteoporosis and periodontal disease, have become prominent social healthcare issues.1,2 Osteoporosis is a worldwide chronic disease characterized by low bone mass, poor bone strength, and micro-architectural deterioration of bone tissue leading to increased bone fragility and fracture risk.3 It is an age-related disease caused by the imbalance between bone-forming osteoblasts and bone-resorbing osteoclasts commonly resulting from postmenopausal estrogen deficiency.4-7 An estimated 200 million people are affected worldwide, 80% being women.8 For decades, studies have demonstrated that bone healing in postmenopausal osteoporotic women is delayed, mainly because of estrogen deficiencies that lead to an imbalance between bone-forming osteoblasts and osteoclast number.5-7,9-11 In relation to periodontal treatment, osteoporosis is believed to affect the condition of the periodontal tissues after therapy, given that it may prevent proper healing and ultimately cause increased severity of the preexisting periodontal disease.12-14 Diminished bone density increases susceptibility for periodontal breakdown, ultimately causing a more complicated regenerative periodontal procedure. One well-established method for enhancing periodontal regeneration is the use of enamel matrix derivative (EMD) harvested from developing porcine teeth.15-18 EMD was introduced to regenerate root cementum, periodontal ligament, and alveolar bone and has since been used successfully for a variety of clinical applications including intrabony, recession, and furcation defects.15 In a systematic review on wound healing properties following application of EMD, EMD was found to significantly decrease interleukin-1 and receptor activator of nuclear factorkB ligand expression, increase prostaglandin E2 and osteoprotegerin expression, increase proliferation and migration of T lymphocytes, induce monocyte differentiation, increase bacterial and tissue debris clearance, and increase angiogenesis by inducing endothelial cell proliferation, migration, and capillarylike sprout formation (R.J. Miron Michel Dard, and Miron Weinreb, unpublished observations). Despite its widespread use for different periodontal indications, to date no single in vitro, in vivo, or clinical study has tested the effects of EMD on periodontal regeneration in osteoporosis-related defects, a scenario with compromised wound healing properties. Therefore, the aims of this study are: 1) to establish a postmenopausal osteoporotic model by bilateral ovariectomy (OVX) in animals and 2) to determine the effects of EMD on periodontal wound healing and
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Volume 85 • Number 11
regeneration in a rat periodontal defect. To evaluate the specific effects of EMD on periodontal regeneration, a periodontal model not exposed to a bacterial biofilm was used. MATERIALS AND METHODS Animals and Surgical Procedures Forty mature female Wistar rats (10 weeks old; mean body weight: 230 g) were purchased and used for this study with all handling and surgical procedures in accordance with the policies of the Ethics Committee for Animal Research, Wuhan University, China. Animals had food and water ad libitum with constant temperature at 22C. After 1 week for acclimatization to the new laboratory surroundings, osteoporosis animal models were carried out by OVX on half the animals under sterile conditions with a minimally invasive surgical technique as previously described.19,20 Briefly, general anesthesia was achieved by intraperitoneal injection of chloral hydrate (10%, 4 mL/kg body weight), and the rats were given 10-mm linear bilateral lumbar lateral skin incisions. The enterocele was exposed by blunt dissection of muscle and peritoneum. The bilateral ovaries were removed gently following ligation of the ovarian artery and vein. Then the small incisions were sutured in layers. Postoperatively, penicillin (40,000 IU/mL, 1 mL/kg) was injected for 3 days, and there was no sign of inflammation or other notable anomaly. Control animals were subject to sham operations as previously described.20 Periodontal fenestration defects (standardized with 2.8 mm in length, 1.4 mm in height, and 0.5 mm in depth) were created 2 months later when the osteoporotic model was established. At that time, defects were also created in control animals. Rats were subjected to bilateral extraoral incisions at the base of the mandible. The buccal mandibular bone overlying the first molar roots was drilled to create a defect by use of a size 4 round bur as depicted in Fig. 1. The procedure was performed under an operating microscope to avoid perforation of intraoral mucosa. The buccal bone covering the first mandibular molar was carefully denuded of its periodontal ligament, overlying cementum, and superficial dentin. The height was standardized to the width of the round bur (diameter 1.4 mm) and extended longitudinally to either side. Animals were divided into four groups of 10 animals per group as follows: 1) control unfilled periodontal defects; 2) control EMD-treated defects; 3) OVX unfilled defects; and 4) OVX EMD-treated defects, with the split-mouth design. EMD,‡ consisting of enamel matrix proteins and a polyglycolic acid carrier, was directly applied to fill the defects ‡ Emdogain, Institute Straumann, Basel, Switzerland.
Miron, Wei, Yang, Caluseru, Sculean, Zhang
J Periodontol • November 2014
and 3D images were generated by the built-in software of the micro-CT.
Figure 1. Schematic diagram of horizontally sectioned rat mandible.
(0.1 mL EMD gel into each defect) without any previous root surface conditioning. Control defects were not filled with the PGA carrier to determine the rate of wound healing in these defects without any additive material. The muscle and skin were repositioned and sutured separately. Postoperatively, penicillin (40,000 IU/mL, 1 mL/kg) was injected intramuscularly for 3 days. Four weeks after surgery, the animals were sacrificed by an overdose of chloral hydrate, and samples were removed and prepared for analysis. Microcomputed Tomography Analyses The samples were fixed in 4% formaldehyde for 12 hours at 4C. A microcomputed tomography (microCT) imaging system§ was used to reveal new bone formation within the defect region. Scanning was performed at 70 kV and 114 mA with a thickness of 0.048 mm per slice in medium-resolution mode. For three-dimensional (3D) reconstruction, the mineralized bone tissue was differentially segmented with a fixed low threshold (value = 212). Representative sections were cut from buccal and mesio-distal views. The mean distance in a bucco-lingual direction between the new bone outer surface and inner surface within the defect was measured as the bone width. For conformation of the established osteoporosis model, a series of slices starting at a distance of 1 mm proximal from the end of the growth plate with a length of 2 mm were chosen for evaluation. For analysis of the bone regeneration process within the defect, the selected region of the defect was defined by drawing a circular contour as an area of measurement per slice, thus obtaining a consistent volume of interest (VOI) and avoiding the native bone margins. After 3D reconstruction, the bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were automatically determined for identification of the osteoporotic model, whereas BV/TV in defect regions was used to evaluate new bone formation, using a protocol provided by the manufacturer of the micro-CT scanner.21 All digitalized data
Histologic Analyses The mandibular samples were decalcified in 10% EDTA for 4 weeks, gradient dehydrated for embedding in paraffin, and sectioned perpendicular to the long axis of the molar roots. Serial sections of 5 mm were cut and mounted on polylysine-coated slides and then given hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) stainingi in accordance with the manufacturer’s protocol. For histomorphometry, three individual sections were selected from three different locations situated in the middle, coronal, and apical (400 mm apart from the central) levels. The histometric measurements were determined by processing the images, which were captured with an upright microscope,¶ in imaging software.# Newly formed bone was identified by different staining of eosin and morphologic structure. The following histometric parameters of new bone formation were measured according to the methods in the literature: 1) bone width, the mean distance in a bucco-lingual direction between the new bone outer surface and inner surface within defect; 2) percentage of new cementum, the ratio of the extent of new cementum extension and the total extent of the exposed root surface; 3) percentage of angiogenesis, the ratio between the area of regenerated vessels and newly formed bone; and 4) number of TRAP-positive cells, the number of TRAP-positive cells in a 500-mm square of the newly formed bone. Statistical Analyses All data analysis was performed using software,** and statistically significant values were adopted as P
Effect of Enamel Matrix Derivative on Periodontal Wound Healing and Regeneration in an Osteoporotic Model Richard J. Miron,*† Lingfei Wei,* Shuang Yang,* Oana M. Caluseru,† Anton Sculean,† and Yufeng Zhang*
Background: Despite the worldwide increased prevalence of osteoporosis, no data are available evaluating the effect of an enamel matrix derivative (EMD) on the healing of periodontal defects in patients with osteoporosis. This study aims to evaluate whether the regenerative potential of EMD may be suitable for osteoporosis-related periodontal defects. Methods: Forty female Wistar rats (mean body weight: 200 g) were used for this study. An osteoporosis animal model was carried out by bilateral ovariectomy (OVX) in 20 animals. Ten weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. Animals were randomly assigned to four groups of 10 animals per group: 1) control animals with unfilled periodontal defects; 2) control animals with EMD-treated defects; 3) OVX animals with unfilled defects; and 4) OVX animals with EMD-treated defects. The animals were euthanized 28 days later, and the percentage of defect fill and thickness of newly formed bone and cementum were assessed by histomorphometry and microcomputed tomography (micro-CT) analysis. The number of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP), and angiogenesis was assessed by analyzing formation of blood vessels. Results: OVX animals demonstrated significantly reduced bone volume in unfilled defects compared with control defects (18.9% for OVX animals versus 27.2% for control animals) as assessed by microCT. The addition of EMD in both OVX and control animals resulted in significantly higher bone density (52.4% and 69.2%, respectively) and bone width (134 versus 165mm) compared with untreated defects; however, the healing in OVX animals treated with EMD was significantly lower than that in control animals treated with EMD. Animals treated with EMD also demonstrated significantly higher cementum formation in both control and OVX animals. The number of TRAP-positive osteoclasts did not vary between untreated and EMD-treated animals; however, a significant increase was observed in all OVX animals. The number of blood vessels and percentage of new vessel formation was significantly higher in EMDtreated samples. Conclusions: The results from the present study suggest that: 1) an osteoporotic phenotype may decrease periodontal regeneration; and 2) EMD may support greater periodontal regeneration in patients suffering from the disease. Additional clinical studies are necessary to fully elucidate the possible beneficial effect of EMD for periodontal regeneration in patients suffering from osteoporosis. J Periodontol 2014;85:1603-1611. KEY WORDS Cementum; cementum, bone; enamel matrix proteins; osteoporosis; osteoporosis, regeneration; periodontal guided tissue regeneration. * The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China. † Department of Periodontology, School of Dental Medicine, University of Bern, Bern, Switzerland.
doi: 10.1902/jop.2014.130745
1603
Periodontal Regeneration of Osteoporotic Defects With EMD
T
he increasing longevity resulting from epidemiologic transition has key implications relating to the elderly population. Diseases specific to this age group, such as osteoporosis and periodontal disease, have become prominent social healthcare issues.1,2 Osteoporosis is a worldwide chronic disease characterized by low bone mass, poor bone strength, and micro-architectural deterioration of bone tissue leading to increased bone fragility and fracture risk.3 It is an age-related disease caused by the imbalance between bone-forming osteoblasts and bone-resorbing osteoclasts commonly resulting from postmenopausal estrogen deficiency.4-7 An estimated 200 million people are affected worldwide, 80% being women.8 For decades, studies have demonstrated that bone healing in postmenopausal osteoporotic women is delayed, mainly because of estrogen deficiencies that lead to an imbalance between bone-forming osteoblasts and osteoclast number.5-7,9-11 In relation to periodontal treatment, osteoporosis is believed to affect the condition of the periodontal tissues after therapy, given that it may prevent proper healing and ultimately cause increased severity of the preexisting periodontal disease.12-14 Diminished bone density increases susceptibility for periodontal breakdown, ultimately causing a more complicated regenerative periodontal procedure. One well-established method for enhancing periodontal regeneration is the use of enamel matrix derivative (EMD) harvested from developing porcine teeth.15-18 EMD was introduced to regenerate root cementum, periodontal ligament, and alveolar bone and has since been used successfully for a variety of clinical applications including intrabony, recession, and furcation defects.15 In a systematic review on wound healing properties following application of EMD, EMD was found to significantly decrease interleukin-1 and receptor activator of nuclear factorkB ligand expression, increase prostaglandin E2 and osteoprotegerin expression, increase proliferation and migration of T lymphocytes, induce monocyte differentiation, increase bacterial and tissue debris clearance, and increase angiogenesis by inducing endothelial cell proliferation, migration, and capillarylike sprout formation (R.J. Miron Michel Dard, and Miron Weinreb, unpublished observations). Despite its widespread use for different periodontal indications, to date no single in vitro, in vivo, or clinical study has tested the effects of EMD on periodontal regeneration in osteoporosis-related defects, a scenario with compromised wound healing properties. Therefore, the aims of this study are: 1) to establish a postmenopausal osteoporotic model by bilateral ovariectomy (OVX) in animals and 2) to determine the effects of EMD on periodontal wound healing and
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Volume 85 • Number 11
regeneration in a rat periodontal defect. To evaluate the specific effects of EMD on periodontal regeneration, a periodontal model not exposed to a bacterial biofilm was used. MATERIALS AND METHODS Animals and Surgical Procedures Forty mature female Wistar rats (10 weeks old; mean body weight: 230 g) were purchased and used for this study with all handling and surgical procedures in accordance with the policies of the Ethics Committee for Animal Research, Wuhan University, China. Animals had food and water ad libitum with constant temperature at 22C. After 1 week for acclimatization to the new laboratory surroundings, osteoporosis animal models were carried out by OVX on half the animals under sterile conditions with a minimally invasive surgical technique as previously described.19,20 Briefly, general anesthesia was achieved by intraperitoneal injection of chloral hydrate (10%, 4 mL/kg body weight), and the rats were given 10-mm linear bilateral lumbar lateral skin incisions. The enterocele was exposed by blunt dissection of muscle and peritoneum. The bilateral ovaries were removed gently following ligation of the ovarian artery and vein. Then the small incisions were sutured in layers. Postoperatively, penicillin (40,000 IU/mL, 1 mL/kg) was injected for 3 days, and there was no sign of inflammation or other notable anomaly. Control animals were subject to sham operations as previously described.20 Periodontal fenestration defects (standardized with 2.8 mm in length, 1.4 mm in height, and 0.5 mm in depth) were created 2 months later when the osteoporotic model was established. At that time, defects were also created in control animals. Rats were subjected to bilateral extraoral incisions at the base of the mandible. The buccal mandibular bone overlying the first molar roots was drilled to create a defect by use of a size 4 round bur as depicted in Fig. 1. The procedure was performed under an operating microscope to avoid perforation of intraoral mucosa. The buccal bone covering the first mandibular molar was carefully denuded of its periodontal ligament, overlying cementum, and superficial dentin. The height was standardized to the width of the round bur (diameter 1.4 mm) and extended longitudinally to either side. Animals were divided into four groups of 10 animals per group as follows: 1) control unfilled periodontal defects; 2) control EMD-treated defects; 3) OVX unfilled defects; and 4) OVX EMD-treated defects, with the split-mouth design. EMD,‡ consisting of enamel matrix proteins and a polyglycolic acid carrier, was directly applied to fill the defects ‡ Emdogain, Institute Straumann, Basel, Switzerland.
Miron, Wei, Yang, Caluseru, Sculean, Zhang
J Periodontol • November 2014
and 3D images were generated by the built-in software of the micro-CT.
Figure 1. Schematic diagram of horizontally sectioned rat mandible.
(0.1 mL EMD gel into each defect) without any previous root surface conditioning. Control defects were not filled with the PGA carrier to determine the rate of wound healing in these defects without any additive material. The muscle and skin were repositioned and sutured separately. Postoperatively, penicillin (40,000 IU/mL, 1 mL/kg) was injected intramuscularly for 3 days. Four weeks after surgery, the animals were sacrificed by an overdose of chloral hydrate, and samples were removed and prepared for analysis. Microcomputed Tomography Analyses The samples were fixed in 4% formaldehyde for 12 hours at 4C. A microcomputed tomography (microCT) imaging system§ was used to reveal new bone formation within the defect region. Scanning was performed at 70 kV and 114 mA with a thickness of 0.048 mm per slice in medium-resolution mode. For three-dimensional (3D) reconstruction, the mineralized bone tissue was differentially segmented with a fixed low threshold (value = 212). Representative sections were cut from buccal and mesio-distal views. The mean distance in a bucco-lingual direction between the new bone outer surface and inner surface within the defect was measured as the bone width. For conformation of the established osteoporosis model, a series of slices starting at a distance of 1 mm proximal from the end of the growth plate with a length of 2 mm were chosen for evaluation. For analysis of the bone regeneration process within the defect, the selected region of the defect was defined by drawing a circular contour as an area of measurement per slice, thus obtaining a consistent volume of interest (VOI) and avoiding the native bone margins. After 3D reconstruction, the bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were automatically determined for identification of the osteoporotic model, whereas BV/TV in defect regions was used to evaluate new bone formation, using a protocol provided by the manufacturer of the micro-CT scanner.21 All digitalized data
Histologic Analyses The mandibular samples were decalcified in 10% EDTA for 4 weeks, gradient dehydrated for embedding in paraffin, and sectioned perpendicular to the long axis of the molar roots. Serial sections of 5 mm were cut and mounted on polylysine-coated slides and then given hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) stainingi in accordance with the manufacturer’s protocol. For histomorphometry, three individual sections were selected from three different locations situated in the middle, coronal, and apical (400 mm apart from the central) levels. The histometric measurements were determined by processing the images, which were captured with an upright microscope,¶ in imaging software.# Newly formed bone was identified by different staining of eosin and morphologic structure. The following histometric parameters of new bone formation were measured according to the methods in the literature: 1) bone width, the mean distance in a bucco-lingual direction between the new bone outer surface and inner surface within defect; 2) percentage of new cementum, the ratio of the extent of new cementum extension and the total extent of the exposed root surface; 3) percentage of angiogenesis, the ratio between the area of regenerated vessels and newly formed bone; and 4) number of TRAP-positive cells, the number of TRAP-positive cells in a 500-mm square of the newly formed bone. Statistical Analyses All data analysis was performed using software,** and statistically significant values were adopted as P
