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Effect of puerarin on bone formation
OsteoArthritis and Cartilage (2007) 15, 894e899 ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2007.02.009
International Cartilage Repair Society
Effect of puerarin on bone formation R. Wong Ph.D.* and B. Rabie Ph.D. University of Hong Kong, Department of Orthodontics, Prince Philip Dental Hospital, Hong Kong Summary Objective: Puerarin is one of the major phytoestrogens isolated from Pueraria lobata, a Chinese medicine known as Gegen. Our laboratory compared the amount of new bone produced by puerarin in collagen matrix (carrier) to that produced by the collagen matrix alone. Method: Eighteen bone defects, 5 mm by 10 mm were created in the parietal bone of nine New Zealand White rabbits. In the experimental group, six defects were grafted with puerarin solution mixed with collagen matrix. In the control groups, six defects were grafted with collagen matrix alone (active control) and six were left empty (passive control). Animals were killed on day 14 and the defects were dissected and prepared for histological assessment. Serial sections were cut across each defect. No new bone was formed in the passive control group. Quantitative analysis of new bone formation was made on 100 sections (10 sections in each defect, in five defects randomly selected in each of the experimental group and active control group) using image analysis. Results: A total of 554% more new bone was present in defects grafted with puerarin in collagen matrix than those grafted with the collagen matrix alone. Conclusion: Puerarin in collagen matrix has the effect of increasing new bone formation locally and can be used for bone grafting or for bone induction often required in surgery. ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Bone repair, Bone graft, Phytoestrogen, Puerarin, Pueraria lobata.
three classes: isoflavones, coumestans and lignans. In addition, some flavonoids, such as naringenin, are also classed as phytoestrogens10. They have attracted much attention among public and medical communities because of their potential beneficial role in prevention and treatment of cardiovascular diseases, osteoporosis, diabetes and obesity, menopausal symptoms, renal diseases and various cancers11,12. Kanno et al.13 report the effects of phytoestrogens and environmental estrogens on osteoblast differentiation using MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line. They increased alkaline phosphatase activity and enhanced bone mineralization in these cells. Puerarin (Fig. 1), 4H-1-benzopyran-4-one,8-b-D-glueopyranosyl-7-hydroxy-3-(4-hydroxy-phenyl), C12H20C9, is one of the major phytoestrogens isolated from the root of a wild leguminous creeper, Pueraria lobata (Willd.) Ohwi14. This is a commonly used traditional Chinese medicine known as Gegen ( ) and has been demonstrated to have effects on decreasing loss in bone density in ovariectomized mice15. It also has other important uses on treatment of fever14, liver diseases16 and cardiovascular diseases17. In China, P. lobata is also used as a health supplement for reducing risk factors of cardiovascular diseases. There are wide application of P. lobata in clinical prescriptions and dietary supplements18. It is possible that puerarin may also increase the bone formation. The significance is, puerarin is commonly taken as a health supplement in dishes and soups in the Asian meals. Therefore, if puerarin can be shown to increase bone in an animal model of bone defect healing, it may be the long-sought-after safe and ideal agent for bone induction and bone defect repair.
Introduction Bone induction is needed in treating bone trauma and filling osseous defects often required for successful surgery. Over the years autogenous cancellous bone grafting has been considered the gold standard in replacing bone1. The major limitations of using autogenous grafting are the inadequacy of supply and surgical morbidity; including donor site pain, paresthesia, and infection, which can approach 8e10%2. Moreover, graft resorption posed a severe problem. In an experimental study, endochondral bone grafts showed 65% volume loss3. Allografts, an alternative to autogenous grafting, seem to be biologically inferior and are associated with infection and inflammation4. The decrease in serum estrogen after menopause is associated with bone loss and osteoporosis, and estrogen replacement therapy is considered to be effective in preventing bone loss5. It has been shown that estrogen enhances osteoblast differentiation and bone formation6e8 and the conditioned medium of estrogen-treated osteoblast cultures inhibits osteoclast development9. Thus, estrogen is one of the most important sex steroids for the maintenance of bone balance. Phytoestrogens are plant-derived non-steroidal compounds that bind to estrogen receptors (ERs) and have estrogen-like activity10. Phytoestrogens are divided into *Address correspondence and reprint requests to: R. Wong, Ph.D., University of Hong Kong, Department of Orthodontics, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong. Tel: þ852-28590554; fax: þ852-25593803; E-mail: fyoung@ hkucc.hku.hk Received 3 July 2006; revision accepted 7 February 2007.
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Fig. 1. Puerarin.
In the present study our laboratory decided to examine its bone forming ability in vivo for the repair of bone defects by using a carrier to enable its use in the clinical setting. To achieve this, our laboratory measured the amount of new bone produced by puerarin with collagen matrix carrier grafted into bony defects and compared with that of the collagen carrier alone.
Roche). In order to maintain the level of neuroleptanalgesia, increments of Hypnorm (0.1 ml/kg) were given at 30-min intervals during the operation. The surgical procedure consisted of the creation of one or two 10 5 mm full-thickness (approximately 2 mm) cranial defects, devoid of periosteum, using templates, in the parietal bones (Fig. 2). The defects were produced using round stainless steel burs (1 mm in diameter) on a low speed dental drill. Outlines of the defects were made initially by making holes of full thickness the parietal bone using a stainless steel wire template bent to the required size of the defect. The holes were joined to complete the process. During the cutting of bone, copious amount of sterile saline was used for irrigation and to minimize thermal damage to the tissues. Depending on which groups in which the rabbit belonged, the defects were grafted with different materials. In the experimental group, the defects were filled with puerarin in collagen matrix which consisted of 0.2 ml puerarin solution (Sigma-Aldrich, MO, USA, dissolved in water for injection to the concentration of 100 mg/ml) mixed with 0.02 g of collagen matrix (purified absorbable fibrillar collagen, Collagen Matrix, Inc., NJ, USA). The grafts were prepared 15 min before grafting. In the control groups, the defects were either left alone (negative control) or grafted with 0.02 g of collagen matrix (purified fibrillar collagen, Collagen Matrix, Inc., NJ, USA) mixed with 0.2 ml water for injection (positive control). POSTOPERATIVE CARE
Materials and methods EXPERIMENTAL AND CONTROL GROUPS
Eighteen 10 5 mm full-thickness bone defects were created in the parietal bones of nine New Zealand White rabbits from an inbred colony. The rabbits were 5 months old (adult stage) and weighed 3.5e4.0 kg. The handling of the animals and the experimental protocol were approved by the Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong. In the experimental groups, six defects were grafted with puerarin solution mixed with collagen matrix (as carrier for puerarin). In the control groups, six defects were grafted with collagen matrix alone (positive control) and six were left empty (negative control). The sample size was based on previous research using this model. Our laboratory uses a small sample size because there was a large difference in bone formation between different groups so that statistical significant difference can be detected with minimal number of animals, also from the animal ethic committee that the smallest sample size that allows a significant difference to be detected should be used. For the puerarin group and the control groups, two defects were created on the parietal bone of each rabbit. In each group six defects were created and surgery performed but after sacrifice only five were randomly drawn and prepared for analyses.
All wounds were closed with interrupted 3/0 black silk sutures. No attempt was made to approximate the periosteum to prevent the barrier effect. Postoperatively, the rabbits were given oxytetracycline hydrochloride daily for 10 days and buprenorphine hydrochloride for 2 weeks. Two weeks after surgery, the animals were killed with sodium pentobarbitone. Immediately upon death, defects
SURGICAL PROCEDURES
The details of the operation and the postoperative care of the animals were previously described Wong and Rabie19. In short, the animals were premedicated 1 h before surgery with oxytetracycline hydrochloride (200 mg/ml, 30 mg/kg body weight, Tetroxyla, Bimeda, Dublin, Ireland) and buprenorphine hydrochloride (0.3 ml/kg body weight, Hypnorm, Janssen Pharmaceutical, Beerse, Belgium), supplemented with diazepam (5 mg/ml, 1 mg/kg body weight, valium 10,
Fig. 2. Diagram of the dorsal view of the skull of a rabbit, with the anterior region orientated to the right, showing the sites of two surgically created bone defects on the parietal bones and five regions within the defect from which sections were taken for quantitative analyses. From more than 10 sections cut in each region, two sections were selected randomly and measured for area new bone formed, giving a total of 10 sections from each defect. Therefore, the amount of new bone formation was assessed throughout the whole defect.
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and surrounding tissue were removed for histological preparation. HISTOLOGICAL PREPARATION AND ANALYSIS
Tissues were fixed in 10% neutral buffered formal saline solution, demineralized with K’s Decal Fluid (sodium formate/ formic acid), and finally double embedded in celloidin/paraffin wax. Each tissue sample containing the defect was embedded intact. Serial, 5-mm-thick sections of the whole defect were cut perpendicular to the long axis. Quantitative analysis was made on serial sections of defects in the experimental and the active control groups. Defects were divided into five regions spaced 1500 mm apart (Fig. 2). From the serial sections in each region, two sections were selected randomly, giving a total of 10 sections from each defect. Therefore, the amount of new bone formation was assessed throughout the whole defect. The total amount of new bone formed (N, Figs. 3 and 4) within the surgically created defect was measured on 100 sections with a technique previously described by Wong and Rabie19,20. Briefly, each section included the graft and host bone of both sides of the defect. Thus, there were two graftehost interfaces. The total amount of new bone (N, Figs. 3 and 4) formed in both graftehost interfaces within the surgically created defect was quantified by one observer (blinded e who did not know which group he was measuring), by outlining the periphery of the newly formed bone image in the computer screen (Leica Qwin Image Processing & Analysis Software, V2.3, Leica Microsystems Imaging Solutions, Ltd., UK) using a transmitted light microscope (Leica, DMLB, Germany) fitted with a video camera (Single CCD Color Camera, Tk-C1380E, JVC, Japan). Differences in staining properties and morphology between newly formed bone and mature bone made identification easy (Figs. 3 and 4). The computer image analysis system then calculated the area of the outlined new bone.
Fig. 4. Photomicrograph of defect grafted with collagen matrix (positive control) in day 14. No bone could be seen across the defect except for a little new bone (N) near the ends of the host bone (H). Collagen matrix (C) remained across the bone defect (Periodic acideSchiff stain, original magnification 40).
t test which does not assume equal variances, with P < 0.05 chosen as the critical level of statistical significance. The size of the method error in digitizing of new ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pthe P areas bone was calculated by the formula d 2 =2n, where d was the difference between the two registrations of a pair and n was the number of double registrations. The size of the method error is 0.014 mm2. Ten randomly drawn histological sections were digitized on two separate occasions at least 3 months apart by the same observer and also by an independent observer. Paired t tests were also performed to compare the intra-observer and the interobserver registrations. The two-tailed P value to compare the intra-observer registrations was 0.5652, that to compare the inter-observer registrations was 0.5911, both considered not significant.
Results CLINICAL AND PHYSICAL EXAMINATIONS
All animals remained in excellent health throughout the course of the experiment and recovered rapidly after operation. There was no evidence of side effects or infection in any of the animals.
STATISTICAL METHODS
Data were analyzed using a statistical analysis computer software (Graphpad Instat, v.2.04a, 1993, San Diego, CA, USA). The one-way analysis of variance (ANOVA) method was used to compare sections drawn from the five regions in each defect. The arithmetic mean, standard deviation (SD) and 95% confidence intervals were calculated for each experimental group. The means (puerarin group and control group) were compared by the Welch’s unpaired
Fig. 3. Photomicrograph showing healing of a bony defect grafted with puerarin in collagen matrix on day 14. New bone (N) can be seen spanning the defect. H ¼ Host bone. Some collagen matrix (C) remained at the center of the bony defect (Periodic acideSchiff stain, original magnification 40).
HISTOLOGICAL FINDINGS
Experimental group: In the group grafted with puerarin in collagen matrix, new bone was formed at the host bonee graft interface and tended to grow across the defect (Fig. 3). Integration of the puerarin and collagen with the recipient bed was characterized by the presence of new bone. No cartilage was found. At higher magnification (Fig. 5), new bone could be seen growing around a collagen matrix fragment and tended to grow toward and amalgamate with the collagen matrix, bone cells were present showing that the collagen was not just calcified, rather, new bone was formed. Control groups: In the active control group little new bone was formed at the host boneegraft interface. Some collagen fibers were present at the center of the defects (Fig. 4). In the passive control group, the defect was healed, with fibrous tissue bridging across the defect. Very little new bone had formed at the ends of the host bone, so no quantitative analysis was performed. The newly formed bone was woven bone and histologically looked similar in different groups, bone cells could be seen so they were not just calcifications, it was more abundance near the ends of the bone defect. In the puerarin group it tended to grow toward the collagen matrix where puerarin was added.
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Table II Comparison of amounts of new bone (mm2) in five defects grafted with collagen matrix (positive control). Mean and SD of the 10 sections of each defects, and results of ANOVA (degree of freedom [df], F, P) comparing five regions within each defect
Fig. 5. High power photomicrograph showing the formation of new bone in a bony defect grafted with puerarin in collagen matrix. New bone (N) and osteocytes (O) could be seen growing around the collagen matrix (C). Capillaries (cap) were present. No cartilage was found (Periodic acideSchiff stain, original magnification 200).
Region
Defect 1
A
0.68 0.74
0.64 0.82
0.18 0.18
0.12 0.11
0.41 0.42
B
0.57 0.51
0.68 0.71
0.15 0.16
0.31 0.38
0.29 0.34
C
0.70 0.70
0.85 0.96
0.35 0.32
0.23 0.18
0.31 0.35
D
0.58 0.74
0.86 0.95
0.20 0.21
0.22 0.24
0.07 0.08
E
0.60 0.80
0.51 0.62
0.06 0.06
0.15 0.13
0.11 0.09
Mean SD df F P
Defect 2
0.662 0.76 0.09223 0.1503 4,5 4,5 1.379 6.494 0.3603 0.0324
Defect 3
Defect 4
0.187 0.145 0.09393 0.07412 4,5 4,5 179.23 34.071 <0.0001 0.0008
Defect 5
0.247 0.1431 4,5 96.734 <0.0001
Discussion QUANTITATIVE ANALYSIS
A total of 100 sections of the experimental group and the active control group were digitized and analyzed. The amount of newly formed bone was significantly greater in the defects grafted with puerarin in collagen matrix than in those grafted with collagen matrix only (Tables I and II), (Fig. 6). In the experimental group, 50 sections were measured; the mean area of newly formed bone in each defect was 2.68 mm2, with a SD of 0.6460 mm2. In the active control group, 50 sections were measured; the mean area of newly formed bone in each defect was 0.41 mm2, with a SD of 0.2728 mm2. Welch’s unpaired t test which does not assume equal variances was used to test the difference between the two groups, the two-tailed P value is <0.0001, considered significant.
Our laboratory demonstrated that puerarin in collagen matrix significantly increased new bone formation locally when grafted into skull defects. It produced 554% more new bone than the absorbable collagen sponge alone (active control) (Figs. 3e6). The difference was significant (P < 0.0001, unpaired t test). This comparison showed that puerarin was osteogenic when used with collagen matrix. It is important to understand the underlying mechanisms of this effect. As mentioned, puerarin is an isoflavone phytoestrogen. Isoflavones stimulate osteogenesis at low concentrations and inhibit osteogenesis at high concentrations in osteoblasts and osteoprogenitor cells21,22. Isoflavones also influence the production of cytokines derived from osteoblasts and osteoprogenitor cells in a similar biphasic manner23.
Table I Comparison of amounts of new bone (mm2) in five defects grafted with puerarin in collagen matrix. Mean and SD of the 10 sections of each defects, and results of ANOVA (degree of freedom [df], F, P) comparing five regions within each defect Region
Defect 1
Defect 2
Defect 3
Defect 4
Defect 5
A
4.21 3.61
2.94 2.76
2.29 2.15
2.75 3.08
2.01 2.03
B
2.92 2.85
3.04 2.93
2.37 2.31
2.89 3.07
2.08 1.97
C
3.89 2.65
3.15 3.32
2.79 2.72
2.68 2.73
2.26 2.37
D
3.23 2.74
3.46 3.56
2.02 1.88
3.11 2.99
1.76 1.68
E
3.44 3.26
3.11 3.42
1.47 1.71
2.40 2.51
1.58 1.46
Mean SD df F P
3.28 0.5140 4,5 1.483 0.3335
3.169 0.2626 4,5 7.395 0.025
2.171 0.4176 4,5 36.012 0.001
2.821 0.2479 4,5 6.869 0.029
Mean and Standard Error
1.92 0.2938 4,5 41.525 0.001
2
1
0 A
B
Column Fig. 6. Comparison of areas (mm2) of newly formed bone between defects grafted with collagen matrix (A, 0.41, SD ¼ 0.2728), areas of defects grafted with puerarin in collagen matrix (B, 2.68, SD ¼ 0.6460).
898 In addition, isoflavones inhibit osteoclast formation and activity24. Pettersson et al.25 and An et al.26, proposed the molecular mechanisms of the osteogenic effects of phytoestrogens based on their ERs-mediated and/or enzyme-inhibiting effects. ER-mediated action has focused on the preferential binding of phytoestrogens to ERb, which acts as a dominant-negative regulator of estrogen signaling. In addition, some phytoestrogens inhibit tyrosine kinases, and mitogenactivated protein kinases, which are crucial for cellular functions27. However, the biological effects of phytoestrogens cannot be fully explained by these mechanisms28. Dang et al.21 identified peroxisome proliferator-activated receptors (PPARs) as additional molecular targets of phytoestrogens22. Regardless of their enzyme inhibitory actions, phytoestrogens can dose dependently activate PPARs and induce divergent effects on osteogenesis and adipogenesis. As a result, the balance between concurrently activated ERs and PPARs determines the dosedependent biological effects of phytoestrogens. As shown in their studies, dominant ER-mediated effects (an increase in osteogenesis and a decrease in adipogenesis) could only be seen at low concentrations of phytoestrogens, whereas dominant PPARg-mediated effects (a decrease in osteogenesis and an increase in adipogenesis) were only evident at high concentrations. Phytoestrogens can target Runx2 transcription factor and PPARg, via binding and/or phosphorylation, resulting in the commitment of osteoprogenitor cells to differentiate into either osteoblasts or adipocytes21,29. Bone morphogenetic protein-2 (BMP-2), a potent inducer of osteogenic differentiation, is a target for estrogen and isoflavones30,31. Similar to estrogen, isoflavones increased the expression of messenger RNA encoding BMP-2 in mouse bone marrow mesenchymal stem cells, in C3H10T1/2 cells and in primary rat osteoblastic cells30,31. These effects are probably mediated by ERb, and not ERa31. Further research on various gene expressions in osteoblasts, mesenchymal cells and endothelial cells during the early healing of puerarin in collagen matrix is needed to confirm the mechanism of its osteogenic effect. The rabbit model used in this study was relevant because non-grafted control bone defects have been found not to heal with new bone formation within 14 days after their creation. In addition, there was minimal bone healing across the defect with collagen matrix as shown by the results of the control group (Fig. 4). There was minimal morbidity due to this procedure as all the rabbits were in good health and condition after the surgery. Two weeks was chosen to examine the bone formation during the early healing of the bone defect was based on another study32. It gave better indication of the ability of new bone to grow across the bone defect. It was also the time span chosen for other studies on bone formation using the same animal model19,20,33,34 so that comparisons can be made. The results of the ANOVA of the different regions within each defect showed the necessity to analyze multiple regions within each defect (Tables I and II). This was indicated by the P value, the difference in the amount of new bone formed between the different regions within each defect was statistically significant in many defects. There was variability because the two sections in each region to be measured were randomly selected among the serial sections. Despite the variations in the amount of bone formation in different regions and in different defects, the amount of bone formation of the puerarin group was greater than that in the control groups.
R. Wong et al.: Effect of puerarin on bone formation Collagen matrix (purified absorbable fibrillar collagen) was used in this study because it had been used successfully as a carrier for growth factors like BMP-2 to induce bone formation in animals and in humans35e37. It was derived from bovine tendon, in the fibrillar form and was suggested from the Manufacturer (Collagen Matrix, Inc., NJ, USA) to be useful for delivering cells and growth factors and for gene therapy. Recently, it was successfully used with rhBMP-2 in the repair of alveolar clefts in humans38. Bouxsein et al.39 assessed the retention time of 125IrhBMP-2 in absorbable collagen sponge using gamma scintigraphy and showed that about 37 (10)% of the initial dose remained at the site 1 week after surgery, and 8 (7)% remained after 2 weeks. It is possible that puerarin could have been retained similarly by the collagen matrix carrier and released over time to exert their effect that led to an increase in bone formation. The dose for puerarin in this study was estimated from a clinical study using puerarin injection40, by multiplying the dose for human with the body mass ratio between rabbit and human. Using pilot study this estimated dose did caused an increase in bone formation. In this study we have tried to demonstrate the osteogenic effect of puerarin using one commonly used carrier for bone repair. Further research is needed to determine the optimum dose, best choice of carrier and the release kinetics of puerarin in different carriers for bone grafting. This study has considerable significance as puerarin is a commonly used health supplement in Asia. It gained interests in research by its multiple health maintenance effects. This is the first study that demonstrated its local osteogenic effect. Puerarin has tremendous potential to be utilized in any application that requires increase in bone formation. Further studies are needed to determine the systemic effect of puerarin on bone metabolism, for example, on the prevention of osteoporosis.
Conclusion Puerarin in collagen matrix has the effect of increasing new bone formation locally and can be used for bone grafting or for bone induction. Further research is needed to optimize its use and to gain further understanding on its bone forming mechanism.
Acknowledgements This work was supported by the University Research Grant no.: 10205789/36170/08003/323/01, the University of Hong Kong.
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stimulates the production of osteoprotegerin by human trabecular osteoblasts. J Cell Biochem 2002;84: 725e35. Setchell KD, Lydeking-Olsen E. Dietary phytoestrogens and their effect on bone: evidence from in vitro and in vivo, human observational, and dietary intervention studies. Am J Clin Nutr 2003;78:593Se609S. Pettersson K, Delaunay F, Gustafsson JA. Estrogen receptor b acts as a dominant regulator of estrogen signaling. Oncogene 2000;19:4970e8. An J, Tzagarakis-Foster C, Scharschmidt TC, Lomri N, Leitman DC. Estrogen receptor b-selective transcriptional activity and recruitment of coregulators by phytoestrogens. J Biol Chem 2001;276:17808e14. Agarwal R. Cell signaling and regulators of cell cycle as molecular targets for prostate cancer prevention by dietary agents. Biochem Pharmacol 2000;60:1051e9. Barnes S. Soy isoflavones-phytoestrogens and what else? J Nutr 2004;134:1225Se8S. Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung UI, Kubota N, et al. PPAR insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J Clin Invest 2004; 113:846e55. Jia TL, Wang HZ, Xie LP, Wang XY, Zhang RQ. Daidzein enhances osteoblast growth that may be mediated by increased bone morphogenetic protein (BMP) production. Biochem Pharmacol 2003;65: 709e15. Zhou S, Turgeman G, Harris SE, Leitman DC, Komm BS, Bodine PVN, et al. Estrogens activate bone morphogenetic protein-2 gene transcription in mouse mesenchymal stem cells. Mol Endocrinol 2003;17:56e66. Rabie ABM, Lie KJ. Integration of endochondral bone grafts in the presence of demineralized bone matrix. Int J Oral Maxillofac Surg 1996;25:311e8. Rabie ABM, Wong RWK, Ha¨gg U. Bone induction using autogenous bone mixed with demineralised bone matrices. Aust Orthod J 1999;15:269e75. Rabie ABM, Wong RWK, Ha¨gg U. Composite autogenous bone and demineralised bone matrices used to repair defects in the parietal bone of rabbits. Br J Oral Maxillofac Surg 2000;38:565e70. Nevins M, Kirker-Head C, Nevins M. Bone formation in the goat maxillary sinus induced by absorbable collagen sponge implants impregnated with recombinant human bone morphogenetic protein e 2. Int J Periodont Restorative Dent 1996;16:9e19. Li ST. Collagen as a delivery vehicle for bone morphogenetic protein (BMP). Trans Orthop Res Soc 1996; 21:647. Boyne PJ, Marx RE, Nevins M. A feasibility study evaluating rhBMP e 2/absorbable collagen sponge for maxillary sinus floor augmentation. Int J Periodont Restorative Dent 1997;17:11e25. Chin M, Ng T, Torn WK, Carstens M. Repair of alveolar clefts with recombinant human bone morphogenetic protein (rhBMP-2) in patients with clefts. J Craniofac Surg 2005;5:778e89. Bouxsein ML, Turek TJ, Blake CA. Recombinant human bone morphogenetic protein e 2 accelerates healing in a rabbit ulnar osteotomy model. J Bone Joint Surg Am 2001;83A:1219e30. Chen X. The clinical observation of puerarin injection on unstable angina pectoris. Zhong Yao Cai 2004;27: 77e8.
International Cartilage Repair Society
Effect of puerarin on bone formation R. Wong Ph.D.* and B. Rabie Ph.D. University of Hong Kong, Department of Orthodontics, Prince Philip Dental Hospital, Hong Kong Summary Objective: Puerarin is one of the major phytoestrogens isolated from Pueraria lobata, a Chinese medicine known as Gegen. Our laboratory compared the amount of new bone produced by puerarin in collagen matrix (carrier) to that produced by the collagen matrix alone. Method: Eighteen bone defects, 5 mm by 10 mm were created in the parietal bone of nine New Zealand White rabbits. In the experimental group, six defects were grafted with puerarin solution mixed with collagen matrix. In the control groups, six defects were grafted with collagen matrix alone (active control) and six were left empty (passive control). Animals were killed on day 14 and the defects were dissected and prepared for histological assessment. Serial sections were cut across each defect. No new bone was formed in the passive control group. Quantitative analysis of new bone formation was made on 100 sections (10 sections in each defect, in five defects randomly selected in each of the experimental group and active control group) using image analysis. Results: A total of 554% more new bone was present in defects grafted with puerarin in collagen matrix than those grafted with the collagen matrix alone. Conclusion: Puerarin in collagen matrix has the effect of increasing new bone formation locally and can be used for bone grafting or for bone induction often required in surgery. ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Bone repair, Bone graft, Phytoestrogen, Puerarin, Pueraria lobata.
three classes: isoflavones, coumestans and lignans. In addition, some flavonoids, such as naringenin, are also classed as phytoestrogens10. They have attracted much attention among public and medical communities because of their potential beneficial role in prevention and treatment of cardiovascular diseases, osteoporosis, diabetes and obesity, menopausal symptoms, renal diseases and various cancers11,12. Kanno et al.13 report the effects of phytoestrogens and environmental estrogens on osteoblast differentiation using MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line. They increased alkaline phosphatase activity and enhanced bone mineralization in these cells. Puerarin (Fig. 1), 4H-1-benzopyran-4-one,8-b-D-glueopyranosyl-7-hydroxy-3-(4-hydroxy-phenyl), C12H20C9, is one of the major phytoestrogens isolated from the root of a wild leguminous creeper, Pueraria lobata (Willd.) Ohwi14. This is a commonly used traditional Chinese medicine known as Gegen ( ) and has been demonstrated to have effects on decreasing loss in bone density in ovariectomized mice15. It also has other important uses on treatment of fever14, liver diseases16 and cardiovascular diseases17. In China, P. lobata is also used as a health supplement for reducing risk factors of cardiovascular diseases. There are wide application of P. lobata in clinical prescriptions and dietary supplements18. It is possible that puerarin may also increase the bone formation. The significance is, puerarin is commonly taken as a health supplement in dishes and soups in the Asian meals. Therefore, if puerarin can be shown to increase bone in an animal model of bone defect healing, it may be the long-sought-after safe and ideal agent for bone induction and bone defect repair.
Introduction Bone induction is needed in treating bone trauma and filling osseous defects often required for successful surgery. Over the years autogenous cancellous bone grafting has been considered the gold standard in replacing bone1. The major limitations of using autogenous grafting are the inadequacy of supply and surgical morbidity; including donor site pain, paresthesia, and infection, which can approach 8e10%2. Moreover, graft resorption posed a severe problem. In an experimental study, endochondral bone grafts showed 65% volume loss3. Allografts, an alternative to autogenous grafting, seem to be biologically inferior and are associated with infection and inflammation4. The decrease in serum estrogen after menopause is associated with bone loss and osteoporosis, and estrogen replacement therapy is considered to be effective in preventing bone loss5. It has been shown that estrogen enhances osteoblast differentiation and bone formation6e8 and the conditioned medium of estrogen-treated osteoblast cultures inhibits osteoclast development9. Thus, estrogen is one of the most important sex steroids for the maintenance of bone balance. Phytoestrogens are plant-derived non-steroidal compounds that bind to estrogen receptors (ERs) and have estrogen-like activity10. Phytoestrogens are divided into *Address correspondence and reprint requests to: R. Wong, Ph.D., University of Hong Kong, Department of Orthodontics, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong. Tel: þ852-28590554; fax: þ852-25593803; E-mail: fyoung@ hkucc.hku.hk Received 3 July 2006; revision accepted 7 February 2007.
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Fig. 1. Puerarin.
In the present study our laboratory decided to examine its bone forming ability in vivo for the repair of bone defects by using a carrier to enable its use in the clinical setting. To achieve this, our laboratory measured the amount of new bone produced by puerarin with collagen matrix carrier grafted into bony defects and compared with that of the collagen carrier alone.
Roche). In order to maintain the level of neuroleptanalgesia, increments of Hypnorm (0.1 ml/kg) were given at 30-min intervals during the operation. The surgical procedure consisted of the creation of one or two 10 5 mm full-thickness (approximately 2 mm) cranial defects, devoid of periosteum, using templates, in the parietal bones (Fig. 2). The defects were produced using round stainless steel burs (1 mm in diameter) on a low speed dental drill. Outlines of the defects were made initially by making holes of full thickness the parietal bone using a stainless steel wire template bent to the required size of the defect. The holes were joined to complete the process. During the cutting of bone, copious amount of sterile saline was used for irrigation and to minimize thermal damage to the tissues. Depending on which groups in which the rabbit belonged, the defects were grafted with different materials. In the experimental group, the defects were filled with puerarin in collagen matrix which consisted of 0.2 ml puerarin solution (Sigma-Aldrich, MO, USA, dissolved in water for injection to the concentration of 100 mg/ml) mixed with 0.02 g of collagen matrix (purified absorbable fibrillar collagen, Collagen Matrix, Inc., NJ, USA). The grafts were prepared 15 min before grafting. In the control groups, the defects were either left alone (negative control) or grafted with 0.02 g of collagen matrix (purified fibrillar collagen, Collagen Matrix, Inc., NJ, USA) mixed with 0.2 ml water for injection (positive control). POSTOPERATIVE CARE
Materials and methods EXPERIMENTAL AND CONTROL GROUPS
Eighteen 10 5 mm full-thickness bone defects were created in the parietal bones of nine New Zealand White rabbits from an inbred colony. The rabbits were 5 months old (adult stage) and weighed 3.5e4.0 kg. The handling of the animals and the experimental protocol were approved by the Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong. In the experimental groups, six defects were grafted with puerarin solution mixed with collagen matrix (as carrier for puerarin). In the control groups, six defects were grafted with collagen matrix alone (positive control) and six were left empty (negative control). The sample size was based on previous research using this model. Our laboratory uses a small sample size because there was a large difference in bone formation between different groups so that statistical significant difference can be detected with minimal number of animals, also from the animal ethic committee that the smallest sample size that allows a significant difference to be detected should be used. For the puerarin group and the control groups, two defects were created on the parietal bone of each rabbit. In each group six defects were created and surgery performed but after sacrifice only five were randomly drawn and prepared for analyses.
All wounds were closed with interrupted 3/0 black silk sutures. No attempt was made to approximate the periosteum to prevent the barrier effect. Postoperatively, the rabbits were given oxytetracycline hydrochloride daily for 10 days and buprenorphine hydrochloride for 2 weeks. Two weeks after surgery, the animals were killed with sodium pentobarbitone. Immediately upon death, defects
SURGICAL PROCEDURES
The details of the operation and the postoperative care of the animals were previously described Wong and Rabie19. In short, the animals were premedicated 1 h before surgery with oxytetracycline hydrochloride (200 mg/ml, 30 mg/kg body weight, Tetroxyla, Bimeda, Dublin, Ireland) and buprenorphine hydrochloride (0.3 ml/kg body weight, Hypnorm, Janssen Pharmaceutical, Beerse, Belgium), supplemented with diazepam (5 mg/ml, 1 mg/kg body weight, valium 10,
Fig. 2. Diagram of the dorsal view of the skull of a rabbit, with the anterior region orientated to the right, showing the sites of two surgically created bone defects on the parietal bones and five regions within the defect from which sections were taken for quantitative analyses. From more than 10 sections cut in each region, two sections were selected randomly and measured for area new bone formed, giving a total of 10 sections from each defect. Therefore, the amount of new bone formation was assessed throughout the whole defect.
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and surrounding tissue were removed for histological preparation. HISTOLOGICAL PREPARATION AND ANALYSIS
Tissues were fixed in 10% neutral buffered formal saline solution, demineralized with K’s Decal Fluid (sodium formate/ formic acid), and finally double embedded in celloidin/paraffin wax. Each tissue sample containing the defect was embedded intact. Serial, 5-mm-thick sections of the whole defect were cut perpendicular to the long axis. Quantitative analysis was made on serial sections of defects in the experimental and the active control groups. Defects were divided into five regions spaced 1500 mm apart (Fig. 2). From the serial sections in each region, two sections were selected randomly, giving a total of 10 sections from each defect. Therefore, the amount of new bone formation was assessed throughout the whole defect. The total amount of new bone formed (N, Figs. 3 and 4) within the surgically created defect was measured on 100 sections with a technique previously described by Wong and Rabie19,20. Briefly, each section included the graft and host bone of both sides of the defect. Thus, there were two graftehost interfaces. The total amount of new bone (N, Figs. 3 and 4) formed in both graftehost interfaces within the surgically created defect was quantified by one observer (blinded e who did not know which group he was measuring), by outlining the periphery of the newly formed bone image in the computer screen (Leica Qwin Image Processing & Analysis Software, V2.3, Leica Microsystems Imaging Solutions, Ltd., UK) using a transmitted light microscope (Leica, DMLB, Germany) fitted with a video camera (Single CCD Color Camera, Tk-C1380E, JVC, Japan). Differences in staining properties and morphology between newly formed bone and mature bone made identification easy (Figs. 3 and 4). The computer image analysis system then calculated the area of the outlined new bone.
Fig. 4. Photomicrograph of defect grafted with collagen matrix (positive control) in day 14. No bone could be seen across the defect except for a little new bone (N) near the ends of the host bone (H). Collagen matrix (C) remained across the bone defect (Periodic acideSchiff stain, original magnification 40).
t test which does not assume equal variances, with P < 0.05 chosen as the critical level of statistical significance. The size of the method error in digitizing of new ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pthe P areas bone was calculated by the formula d 2 =2n, where d was the difference between the two registrations of a pair and n was the number of double registrations. The size of the method error is 0.014 mm2. Ten randomly drawn histological sections were digitized on two separate occasions at least 3 months apart by the same observer and also by an independent observer. Paired t tests were also performed to compare the intra-observer and the interobserver registrations. The two-tailed P value to compare the intra-observer registrations was 0.5652, that to compare the inter-observer registrations was 0.5911, both considered not significant.
Results CLINICAL AND PHYSICAL EXAMINATIONS
All animals remained in excellent health throughout the course of the experiment and recovered rapidly after operation. There was no evidence of side effects or infection in any of the animals.
STATISTICAL METHODS
Data were analyzed using a statistical analysis computer software (Graphpad Instat, v.2.04a, 1993, San Diego, CA, USA). The one-way analysis of variance (ANOVA) method was used to compare sections drawn from the five regions in each defect. The arithmetic mean, standard deviation (SD) and 95% confidence intervals were calculated for each experimental group. The means (puerarin group and control group) were compared by the Welch’s unpaired
Fig. 3. Photomicrograph showing healing of a bony defect grafted with puerarin in collagen matrix on day 14. New bone (N) can be seen spanning the defect. H ¼ Host bone. Some collagen matrix (C) remained at the center of the bony defect (Periodic acideSchiff stain, original magnification 40).
HISTOLOGICAL FINDINGS
Experimental group: In the group grafted with puerarin in collagen matrix, new bone was formed at the host bonee graft interface and tended to grow across the defect (Fig. 3). Integration of the puerarin and collagen with the recipient bed was characterized by the presence of new bone. No cartilage was found. At higher magnification (Fig. 5), new bone could be seen growing around a collagen matrix fragment and tended to grow toward and amalgamate with the collagen matrix, bone cells were present showing that the collagen was not just calcified, rather, new bone was formed. Control groups: In the active control group little new bone was formed at the host boneegraft interface. Some collagen fibers were present at the center of the defects (Fig. 4). In the passive control group, the defect was healed, with fibrous tissue bridging across the defect. Very little new bone had formed at the ends of the host bone, so no quantitative analysis was performed. The newly formed bone was woven bone and histologically looked similar in different groups, bone cells could be seen so they were not just calcifications, it was more abundance near the ends of the bone defect. In the puerarin group it tended to grow toward the collagen matrix where puerarin was added.
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Table II Comparison of amounts of new bone (mm2) in five defects grafted with collagen matrix (positive control). Mean and SD of the 10 sections of each defects, and results of ANOVA (degree of freedom [df], F, P) comparing five regions within each defect
Fig. 5. High power photomicrograph showing the formation of new bone in a bony defect grafted with puerarin in collagen matrix. New bone (N) and osteocytes (O) could be seen growing around the collagen matrix (C). Capillaries (cap) were present. No cartilage was found (Periodic acideSchiff stain, original magnification 200).
Region
Defect 1
A
0.68 0.74
0.64 0.82
0.18 0.18
0.12 0.11
0.41 0.42
B
0.57 0.51
0.68 0.71
0.15 0.16
0.31 0.38
0.29 0.34
C
0.70 0.70
0.85 0.96
0.35 0.32
0.23 0.18
0.31 0.35
D
0.58 0.74
0.86 0.95
0.20 0.21
0.22 0.24
0.07 0.08
E
0.60 0.80
0.51 0.62
0.06 0.06
0.15 0.13
0.11 0.09
Mean SD df F P
Defect 2
0.662 0.76 0.09223 0.1503 4,5 4,5 1.379 6.494 0.3603 0.0324
Defect 3
Defect 4
0.187 0.145 0.09393 0.07412 4,5 4,5 179.23 34.071 <0.0001 0.0008
Defect 5
0.247 0.1431 4,5 96.734 <0.0001
Discussion QUANTITATIVE ANALYSIS
A total of 100 sections of the experimental group and the active control group were digitized and analyzed. The amount of newly formed bone was significantly greater in the defects grafted with puerarin in collagen matrix than in those grafted with collagen matrix only (Tables I and II), (Fig. 6). In the experimental group, 50 sections were measured; the mean area of newly formed bone in each defect was 2.68 mm2, with a SD of 0.6460 mm2. In the active control group, 50 sections were measured; the mean area of newly formed bone in each defect was 0.41 mm2, with a SD of 0.2728 mm2. Welch’s unpaired t test which does not assume equal variances was used to test the difference between the two groups, the two-tailed P value is <0.0001, considered significant.
Our laboratory demonstrated that puerarin in collagen matrix significantly increased new bone formation locally when grafted into skull defects. It produced 554% more new bone than the absorbable collagen sponge alone (active control) (Figs. 3e6). The difference was significant (P < 0.0001, unpaired t test). This comparison showed that puerarin was osteogenic when used with collagen matrix. It is important to understand the underlying mechanisms of this effect. As mentioned, puerarin is an isoflavone phytoestrogen. Isoflavones stimulate osteogenesis at low concentrations and inhibit osteogenesis at high concentrations in osteoblasts and osteoprogenitor cells21,22. Isoflavones also influence the production of cytokines derived from osteoblasts and osteoprogenitor cells in a similar biphasic manner23.
Table I Comparison of amounts of new bone (mm2) in five defects grafted with puerarin in collagen matrix. Mean and SD of the 10 sections of each defects, and results of ANOVA (degree of freedom [df], F, P) comparing five regions within each defect Region
Defect 1
Defect 2
Defect 3
Defect 4
Defect 5
A
4.21 3.61
2.94 2.76
2.29 2.15
2.75 3.08
2.01 2.03
B
2.92 2.85
3.04 2.93
2.37 2.31
2.89 3.07
2.08 1.97
C
3.89 2.65
3.15 3.32
2.79 2.72
2.68 2.73
2.26 2.37
D
3.23 2.74
3.46 3.56
2.02 1.88
3.11 2.99
1.76 1.68
E
3.44 3.26
3.11 3.42
1.47 1.71
2.40 2.51
1.58 1.46
Mean SD df F P
3.28 0.5140 4,5 1.483 0.3335
3.169 0.2626 4,5 7.395 0.025
2.171 0.4176 4,5 36.012 0.001
2.821 0.2479 4,5 6.869 0.029
Mean and Standard Error
1.92 0.2938 4,5 41.525 0.001
2
1
0 A
B
Column Fig. 6. Comparison of areas (mm2) of newly formed bone between defects grafted with collagen matrix (A, 0.41, SD ¼ 0.2728), areas of defects grafted with puerarin in collagen matrix (B, 2.68, SD ¼ 0.6460).
898 In addition, isoflavones inhibit osteoclast formation and activity24. Pettersson et al.25 and An et al.26, proposed the molecular mechanisms of the osteogenic effects of phytoestrogens based on their ERs-mediated and/or enzyme-inhibiting effects. ER-mediated action has focused on the preferential binding of phytoestrogens to ERb, which acts as a dominant-negative regulator of estrogen signaling. In addition, some phytoestrogens inhibit tyrosine kinases, and mitogenactivated protein kinases, which are crucial for cellular functions27. However, the biological effects of phytoestrogens cannot be fully explained by these mechanisms28. Dang et al.21 identified peroxisome proliferator-activated receptors (PPARs) as additional molecular targets of phytoestrogens22. Regardless of their enzyme inhibitory actions, phytoestrogens can dose dependently activate PPARs and induce divergent effects on osteogenesis and adipogenesis. As a result, the balance between concurrently activated ERs and PPARs determines the dosedependent biological effects of phytoestrogens. As shown in their studies, dominant ER-mediated effects (an increase in osteogenesis and a decrease in adipogenesis) could only be seen at low concentrations of phytoestrogens, whereas dominant PPARg-mediated effects (a decrease in osteogenesis and an increase in adipogenesis) were only evident at high concentrations. Phytoestrogens can target Runx2 transcription factor and PPARg, via binding and/or phosphorylation, resulting in the commitment of osteoprogenitor cells to differentiate into either osteoblasts or adipocytes21,29. Bone morphogenetic protein-2 (BMP-2), a potent inducer of osteogenic differentiation, is a target for estrogen and isoflavones30,31. Similar to estrogen, isoflavones increased the expression of messenger RNA encoding BMP-2 in mouse bone marrow mesenchymal stem cells, in C3H10T1/2 cells and in primary rat osteoblastic cells30,31. These effects are probably mediated by ERb, and not ERa31. Further research on various gene expressions in osteoblasts, mesenchymal cells and endothelial cells during the early healing of puerarin in collagen matrix is needed to confirm the mechanism of its osteogenic effect. The rabbit model used in this study was relevant because non-grafted control bone defects have been found not to heal with new bone formation within 14 days after their creation. In addition, there was minimal bone healing across the defect with collagen matrix as shown by the results of the control group (Fig. 4). There was minimal morbidity due to this procedure as all the rabbits were in good health and condition after the surgery. Two weeks was chosen to examine the bone formation during the early healing of the bone defect was based on another study32. It gave better indication of the ability of new bone to grow across the bone defect. It was also the time span chosen for other studies on bone formation using the same animal model19,20,33,34 so that comparisons can be made. The results of the ANOVA of the different regions within each defect showed the necessity to analyze multiple regions within each defect (Tables I and II). This was indicated by the P value, the difference in the amount of new bone formed between the different regions within each defect was statistically significant in many defects. There was variability because the two sections in each region to be measured were randomly selected among the serial sections. Despite the variations in the amount of bone formation in different regions and in different defects, the amount of bone formation of the puerarin group was greater than that in the control groups.
R. Wong et al.: Effect of puerarin on bone formation Collagen matrix (purified absorbable fibrillar collagen) was used in this study because it had been used successfully as a carrier for growth factors like BMP-2 to induce bone formation in animals and in humans35e37. It was derived from bovine tendon, in the fibrillar form and was suggested from the Manufacturer (Collagen Matrix, Inc., NJ, USA) to be useful for delivering cells and growth factors and for gene therapy. Recently, it was successfully used with rhBMP-2 in the repair of alveolar clefts in humans38. Bouxsein et al.39 assessed the retention time of 125IrhBMP-2 in absorbable collagen sponge using gamma scintigraphy and showed that about 37 (10)% of the initial dose remained at the site 1 week after surgery, and 8 (7)% remained after 2 weeks. It is possible that puerarin could have been retained similarly by the collagen matrix carrier and released over time to exert their effect that led to an increase in bone formation. The dose for puerarin in this study was estimated from a clinical study using puerarin injection40, by multiplying the dose for human with the body mass ratio between rabbit and human. Using pilot study this estimated dose did caused an increase in bone formation. In this study we have tried to demonstrate the osteogenic effect of puerarin using one commonly used carrier for bone repair. Further research is needed to determine the optimum dose, best choice of carrier and the release kinetics of puerarin in different carriers for bone grafting. This study has considerable significance as puerarin is a commonly used health supplement in Asia. It gained interests in research by its multiple health maintenance effects. This is the first study that demonstrated its local osteogenic effect. Puerarin has tremendous potential to be utilized in any application that requires increase in bone formation. Further studies are needed to determine the systemic effect of puerarin on bone metabolism, for example, on the prevention of osteoporosis.
Conclusion Puerarin in collagen matrix has the effect of increasing new bone formation locally and can be used for bone grafting or for bone induction. Further research is needed to optimize its use and to gain further understanding on its bone forming mechanism.
Acknowledgements This work was supported by the University Research Grant no.: 10205789/36170/08003/323/01, the University of Hong Kong.
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