Effect of rhbmp-2 produced by E-coli expression system on bone formation in rat calvarial defects Suk-Hoon Kwon The Graduate School Yonsei University Department of Dental Science
Effect of rhbmp-2 produced by E-coli expression system on bone formation in rat calvarial defects 비교 연구 A Dissertation Thesis Submitted to the Department of Dental Science, the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy of Dental Science Suk-Hoon Kwon
June 2008 This certifies that the dissertation thesis of Suk-Hoon Kwon is approved. Thesis Supervisor:Kyoo-Sung Cho Jung-Kiu Chai Seong-Ho Choi Keun-Woo Lee Hyung-Jun Kim The Graduate School Yonsei University
June 2008 감사의글 본논문이완성되기까지부족한저를항상격려해주시고사랑과관심으로논문지도를해주신조규성교수님께깊은감사를드립니다. 그리고, 많은조언과따뜻한관심으로지켜봐주신김종관교수님, 채중규교수님, 최성호교수님께도진심으로감사드립니다. 논문심사를맡아주신이근우교수님, 김형준교수님께도감사드립니다. 실험및연구를도와준채경준선생님, 이지현선생님그리고김민수선생님을비롯한의국원후배님들에게도감사드립니다. 무엇보다도늘아낌없는사랑과헌신적인도움으로든든하고따뜻한버팀목이되어주고부족한나를믿고따라준사랑하는나의아내와사랑하는딸수린이에게도진정으로사랑과고마움의마음을전합니다. 항상격려를해주신양가부모님과친지들에게도감사드리며앞으로더욱좋은모습으로보답하겠습니다. 2008 년 7 월 저자씀
Table of Contents Abstract (English) iii I. Introduction 1 II. Materials and Methods 5 1. Animals 5 2. rhbmp-2 Implants 5 3. Surgical Procedures 6 4. Histologic and Histometric Procedures 7 5. Statistical Analysis 9 III. Results 10 1. Clinical Observations 10 2. Histologic Observations 10 3. Histometric Analysis 11 IV. Discussion 14 V. Conclusion 18 References 19 Figures 25 Abstract(Korean) 31 i
List of Figures Figure 1. Schematic diagram of the calvarial osteotomy defect showing 8 the histometric analysis Figure 2. Sham surgery control group(2 week, x20 magnification) 26 Figure 3. Sham surgery control group(8 week, x20 magnification) 26 Figure 4. Positive control group(2 week, x20 magnification) 26 Figure 5. Positive control group(8 week, x20 magnification) 26 Figure 6. Experimental group(2 week, x20 magnification) 27 Figure 7 Experimental group(8 week, x20 magnification) 27 Figure 8. Experimental group(2 week, x100 magnification) 28 Figure 9. Experimental group(2 week, x100 magnification) 28 Figure10. Experimental group(8 week, x100 magnification) 29 Figure 11. Experimental group(8 week, x100 magnification) 29 Figure 12. Experimental group(8 week, x100 magnification) 30 List of Tables Table 1. Study design 6 Table 2. Defect closure 12 Table 3. New bone area 13 Table 4. Augmented area 13 ii
Abstract Effect of rhbmp-2 produced by E-coli expression system on Bone Formation in Rat Calvarial Defects Bone morphogenetic proteins (BMPs) are being evaluated as potential candidates for periodontal and bone regenerative therapy. In spite of good prospects in BMP applications, there is economically inviable for clinical use in dental area. The purpose of this study was to evaluate the osteogenic effect of rhbmp-2 produced by E-coli expression system. Eight-mm critical-size calvarial defects were created in 48 male Sprague-Dawley rats. The animals were divided into 6 groups of 8 animals each. Each group received one of the following: sham-surgery control(no material applied), positive control(acs alone), experimental(acs loaded with BMP). Defects were evaluated by histologic and histometric parameters following 2- and 8-week healing intervals. The experimental group showed significantly defect closure at 2 and 8weeks than the sham surgery and positive control groups. Moreover, the experimental group showed significantly greater new bone and augmented area than the other groups at both 2 and 8weeks. rhbmp-2 produced by E-coli expression system may be effective for bone regeneration. Key Words: Osteogenic effect; bone morphogenetic protein-2; E-coli expression system; ACS; rat calvarial defect model iii
Effect of rhbmp-2 produced by E-coli expression system on bone formation in rat calvarial defects. Suk-Hoon Kwon, D.D.S., M.S.D. Department of Dental Science Graduate School, Yonsei University (Directed by Prof. Kyoo-Sung Cho, D.D.S., M.S.D., PhD.) I. Introduction The principal objective of periodontal treatments is to regenerate the original periodontal tissue. Several studies have been carried out to achieve this aim. Among the various procedures available to reconstruct bone, the application of bone morphogenetic proteins (BMPs) is believed to be a promising advancement. Urist reported that demineralized bone matrix implanted in the extraskeletal sites created osseous tissue in rats (Urist, 1965). In 1971, Urist et al. identified the factors within the matrix responsible for the effects of bone morphogenetic proteins ((BMPs) Urist 1
et al., 1971). BMPs are considered members of the transforming growth factor-β superfamily through their characteristic amino acid sequences (Reddi et al., 1976; Wozney et al., 1988). BMPs contain potent growth and differentiation factors that have an effect on mesenchymal cells causing them to differentiate into mature osteoblasts, resulting in new bone formation (Reddi et al., 1976). Many studies have reported the biological activities of BMPs in a variety of cells in vitro, including the induction of osteoblastic phenotype expression during the course of osteoblastic differentiation. BMPs include alkaline phosphatase(alp), typeⅠ collagen, osteocalcin, osteopontin and bone sialoprotein (Hiraki et al., 1991; Katagiri et al., 1994; Knutsen et al., 1993; Kobayashi et al.,1999; Takuwa et al., 1991; Thies et al., 1992; Wang et al., 1993; Yamagichi et al., 1996). When BMPs are implanted in vivo, the osteoinductive effect of BMPs initiates a series of cellular events, culminating in bone formation (Sampath et al., 1992; Urist, 1965; Wozney et al., 1988). Therefore, BMPs need accessible and bioactive delivery systems. Carrier systems for delivering BMP should be both biocompatible and biodegradable in order to minimize the local tissue response and be replaced by newly formed bone(aldinger et al., 1991). An absorbable collagen sponge ((ACS) Sampath et al., 1981; King et al., 1998; Barboza et al., 2000), and β-tricalcium phosphate (β-tcp) have been studied and used extensively as carriers for BMPs (Urist et al., 1984; Urist et al., 1987; Gao et al., 1996; Alam et al., 2001). A collagen carrier acts only as a temporary template for the 2
osteoinduction of BMP at the early stage of bone formation, and is unsuitable for maintaining bone defects because it is quite absorbable. β-tcp has a multiporous structure, which is believed to entrap BMP and protect it from diffusion(alam et al., 2001; Laffargue et al., 1999; Gao et al., 1996). In addition, the effects of Fibrin- Fibronectin Sealing System as a carrier for rhbmp-4 have been studied in rat calvarial defects(han et al.,2005; Hong et al.,2005). Therefore, BMPs are considered to be the most suitable osteoinductive material and are expected to be used clinically for bone regeneration and reconstruction. In 2007, the FDA approved the use of rhbmp-2(infuse) in the dental area. Although there are good effects after applying BMPs, there is still a gap between research and the realistic clinical use of BMP, due to the difficulty of a large quantity production. This difficulty results in the high cost of BMP production, which makes uneconomically viable for use in the dental area. BMP-2 is produced from mammalian cell cultures, albeit in low yield (Wang. et al.,1990). Biologically active BMP-2 has been produced from E-coli through the in vitro refolding of inclusion bodies. However, the refolding procedure is complicated and the overall yield is either low or the refolding buffer contains expensive regents. One method involves several time consuming purification and concentration steps and produces a low yield of 0.2mg active BMP-2 per gram cell wet weight(r.ruppert et al.,1996). Although another procedure improved the yield of the active BMP-2 3
dimer to 10mg/g cell wet weight, a high concentration of expensive reagent was required (Vallejo et al., 1996). Therefore, new, simple and inexpensive methods are needed to make them economically viable. rhbmp-2 produced by an E-coli expression system was reported to solve these productivity and economic viability issues (Choi et al., 2008). The aim of this study was to evaluate the osteogenic effect of rhbmp-2 produced by an E-coli expression system in a rat calvarial defect model. 4
II. Materials & methods 1. Animals Forty-eight male Sprague-Dawley rats (weight 250-300g) were used in this study. The rats were maintained in plastic cages in a room with a 12 h-day/night cycle and at 21 C. The rats were given access to water and standard laboratory pellets ad libitum. Animal selection and management, surgical protocol, and preparation were carried out in accordance with the routines approved by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea. 2. rhbmp-2 implants The rhbmp-2 produced by the E-coli expression system * was reconstituted and diluted in a buffer to produce a concentration of 0.1 mg/ml. A sterile 8-mm diameter ACS was then loaded with 0.1 ml of the rhbmp-2 solutions. The buffer was used alone for the control experiments. No material was applied in sham surgery. The rhbmp-2 implants were fitted to a calvarial defect after a 5-minute binding period. 5
3. Surgical Procedures The animals were anaesthetized by an intramuscular injection (5 mg/kg body wt.) of a 4:1 solution of ketamine hydrochloride ** :Xylazine. Routine infiltration anaesthesia was used at the surgical site. An incision was made in the sagittal plane across the cranium and a full thickness flap was reflected, exposing the calvarial bone. A standardized, circular, transosseous defect, 8 mm in diameter, was created on the cranium using a saline-cooled trephine drill #. The animals were divided into 6 groups containing 48 animals each. The wounds were allowed to heal for 2 (8 rats) or 8 (8 rats) weeks. Each animal received 1 of 3 experimental treatments. The periosteum and skin were closed and sutured with 4-0 coated Monosyn sutures for primary intention healing. Table 1 gives a summary of the study design Table 1. Study design Group(n) Sham surgery 2,8week(8,8) Positive control 2,8week(8,8) Experimental 2,8week(8,8) Treatment no material apply absorbable collagen sponge alone absorbable collagen sponge loaded with rhbmp-2 6
4. Histologic and Histometric Procedures The animals were sacrificed by CO 2 asphyxiation at 2 and 8 weeks post-surgery. Block sections, including the experimental sites, were removed and fixed in a 10% neutral buffered formalin solution for a 10 day period. The samples were decalcified in a 5% formic acid solution over a 14 day period and embedded in paraffin. Serial sections, 5µm in thickness, were prepared at 80 µm intervals, stained with hematoxylin/eosin (H-E), and examined by optical microscopy. The most central sections from each block were selected for the histological evaluation. Computer-assisted histometric measurements were obtained using an automated image analysis system coupled with a video camera on the optical microscope. The sections were examined at x 20 and x 100 magnifications. The histometric parameters are defined as follows (Figure 1). : 7
a b Original bone New bone Biomaterial Fibrovascular tissue Defect closure (%) = (a - b) / a x 100 New bone area = n Biomaterial,Fibrovascular tissue,bone Marrow= m Augmented area = n + m Figure 1. Schematic diagram of the calvarial osteotomy defect showing the histometric analysis The level of defect closure was determined by measuring the distance between the defect margin and new bone margin, and is expressed in mm and as a percentage of the total defect width. The augmented bone area (mm 2 ) was measured including all the tissues within the boundaries of the newly formed bone, i.e., mineralized bone and fatty marrow and fibrovascular tissue/marrow and residual biomaterial. 8
5. Statistical Analysis Histometric recordings from the samples were used to calculate the mean and standard deviations (m±sd). Two-way analysis of variance was used (two-way ANOVA) to detect the interactions between the healing interval and treatment conditions. ANOVA and post hoc t-tests were used to analyze the differences between the treatment groups at each healing interval. A paired t-test was used for the comparisons between the 2- and 8-week healing interval within the same group. A p- value <0.05 was considered significant. * Cowellmedi co.ltd. Busan,Korea Collatape, Calcitek, Carlsbad, CA, USA ** Ketalar, Yuhan Co., Seoul, Korea Rompun, Bayer Korea, Seoul, Korea 2% lidocaine, 1:100,000 epinephrine, Kwangmyung Pharm., Seoul, Korea # 3i, Palm Beach Gardens, FL, USA Glyconate monofilament, absorbable. VIOLET, BRAUN Int. Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA Olympus BX50, Olympus Optical Co., Tokyo, Japan 9
III. Results 1. Clinical Observations The level of wound healing was similar in all groups. There was no material exposure or other complications observed at the surgical sites. 2. Histologic Observations Sham-surgery control group: At 2 and 8 weeks after surgery, the defects were filled with thin fibrous connective tissue. There was a minimal amount of new bone formation originating from the defect margins, and the defect center appeared to have collapsed. Moreover, there was minimal inflammatory cell infiltration at the defect site.(figure 2,3) Positive control group: At 2 weeks after surgery, there was dense, fibrous connective tissue at the defect site and the ACS had partially degraded but still present. There was a small amount of new bone formation adjacent to the defect margins, and obvious host bone-to-new bone interface. No inflammatory cell infiltration was 10
observed at the defect site. At 8 weeks, a similar pattern to that observed at 2 weeks was noted. However, the ACS was completely degraded.(figure 4,5,6) Experimental group: At 2 weeks after surgery, there was not only a consolidation of woven bone along the dural aspect but also marked bone regeneration. The degradation of the ACS had advanced considerably without significant adverse reactions, and some degraded ACS fragments were embedded within the new bone, without connective tissue intervention. At 8 weeks, the quantity of the new bone was greater than that observed at 2 weeks, and the appearance of the new bone was more lamellar than that observed at 2 weeks. No remnants of the ACS could be detected. There was no clear border between the preexisting bone and new bone.(figure 7-13) 3. Histometric Analysis Table 2-4 show the results of the histomorphometric analysis are shown in. At 2 weeks after surgery, the mean defect closure (±SD) for the sham surgery control, positive control, experimental groups was 13.53±3.58%, 17.98±.7.66%, 85.9±16.25%, respectively, with a significant difference being observed between the groups (P<0.01). At 8 weeks after surgery, the corresponding values were 18.3±8.65%, 21.88±8.34%, 100%, respectively, with significant difference observed between the 11
groups (P<0.01). The experimental group had a significantly longer closure length at both 2 and 8 weeks than the other groups (P<0.05). The defects were completely closed in the experimental group at 8 weeks. (Table 2.) The results of the new bone and augmented bone areas were similar to the defect closure result. The experimental group had significantly greater new bone and augmented areas at both 2 and 8weeks than the other groups.(table 3,4.) Two-way ANOVA revealed interaction between the healing interval and treatment conditions in the defect closure and new bone area (p <0.01). The treatment had a strong influence on defect closure, new bone area and augmented area (p <0.01), whereas the healing interval had an influence on defect closure and the new bone area (p <0.01). Table 2. Defect closure (group means ± SD; n=8, %) 2 weeks 8 weeks Sham-surgery control 13.53 ± 3.58 18.30 ± 8.65 Positive control 17.98 ± 7.66 21.88 ± 8.34 Experimental 85.90 ± 16.25 * 100 * *: Statistically significant difference compared with the sham-surgery control group (P<0.01) : Statistically significant difference compared with the positive control group (P<0.01) : Statistically significant difference compared with that observed at 2 weeks (P<0.05) 12
Table 3. New bone area (group means ± SD; n=8, mm 2 ) 2 weeks 8 weeks Sham-surgery control 0.18 ± 0.04 0.81 ± 0.27 Positive control 0.23 ± 0.13 0.46± 0.07 Experimental 1.73 ± 0.64 * 7.4±1.56 * *: Statistically significant difference compared with the sham surgery control group (P<0.01) : Statistically significant difference compared with the positive control group (P<0.01) : Statistically significant difference compared with that observed at 2 weeks (P<0.05) Table 4. Augmented area (group means ± SD; n=8, mm 2 ) 2 weeks 8 weeks Sham-surgery control 0.22 ± 0.07 0.89 ± 0.32 Positive control 2.00 ± 1.01 * 3.5± 0.30 * Experimental 5.09± 1.13 * 9.8± 1.56 * *: Statistically significant difference compared with the sham surgery control group (P<0.01) : Statistically significant difference compared with the positive control group (P<0.01) : Statistically significant difference compared with that observed at 2 weeks (P<0.05) 13
Ⅳ. Discussion The aim of this study was to evaluate the bone regenerative effect of rhbmp-2 produced by the E-coli expression system and delivered with an absorbable collagen sponge (ACS). Previous studies reported the osteoinductive effects of rhbmp-2 both in vivo and in vitro. In 2005, Hong et al. examined effect of a fibrin-fibronectin sealing system in combination with ß-Tricalcium Phosphate as a carrier for recombinant human bone morphogenetic protein-2 on the bone formation effects in rat calvarial defects(hong et al.,2005). In this study, 6 groups containing 48 animals each received one of the following: sham-surgery control(no material applied), positive control(acs alone), experimental(acs loaded with BMP). These groups were evaluated using the histologic and histometric parameters after a 2- and 8-week healing period. The BMP produced by the E.coli expression system at a concentration of 0.1 mg/ml was used. Preexisting BMP studies mainly used a BMP concentration of 0.025~0.05 mg/ml. However, 0.1 mg/ml of BMP was used in this study because the biological activity concentrations of the rhbmp-2 obtained from CHO cell and E-coli are different. The experimental model used in this study was based on the model reported by Takagi and Urist (Takagi et al., 1982). A critical-size rat calvarial defect was used 14
because of its relative accessibility, simplicity and reproducibility. This model is convenient for evaluating the bone regenerative effects of biomaterials. There was no spontaneous healing in the control specimens (Frame, 1980; Schmitz et al., 1986). BMPs were first identified by Urist(Urist.,1965) in partially purified form from a rabbit and bovine demineralized bone matrix, and were isolated by Urist and coworkers in the 1980s(Mizutani H.et al, 1982; Urist et al, 1982). BMPs form a set of growth and differentiation factors that act on the early osteoprogenitor cells so they can differentiate into mature osteoblasts, resulting in the formation of new bone and cartilage when implanted in animals(gerhart T.N.et al., 1993; Ferguson D.et al, 1987) In order to observe the osteoinductive effect of BMP, an appropriate carrier system is essential for the delivery, retention, and release of BMPs at the implantation site (Wikesjö et al., 2001). Ahn.et al.(2002) reported that an ACS was a superior carrier to β-tcp for the bone regenerative effect of rhbmp-4(ahn. et al., 2002). Pang et al.(2004) reported that rhbmp-4 using ACS or ß-TCP carrier technologies have significant potential to induce bone formation in the rat calvarial critical size defect model, and both ACS and ß-TCP might be effective carriers for rhbmp-4 (Pang. et al., 2004). In this study, ACS was used as the BMP carrier. BMP is produced from mammalian cells. BMP-2 was also found to be expressed from Chinese hamster ovary (CHO) cells. However, the BMPs used thus far have a 15
limitation in mass production, which results in the high cost of BMP production giving them no economic value when used in dentistry. More economical and less difficult methods are being examined in an attempt to solve this problem. rhbmp-2 produced by E-coli expression system solves these productivity and economic viability issues (Choi et al., 2008). rhbmp-2 produced from E-coli is different from the rhbmp-2 produced from CHO cells without glycosylation but there is no difference in biological activity between both rhbmp-2 (Vallejo et al.,2002). In this study, the experimental group showed significantly greater defect closure at 2 and 8weeks than the sham surgery and positive control groups. In particular, the defects in the experimental group had closed completely at 8 weeks, which is unlike that observed in the sham surgery and positive control groups (18.30±8.65% and 21.88±8.34%, respectively). The new bone and augmented bone areas were similar to the defect closure results. The experimental group had significantly larger new bone and augmented area than the other groups at both 2 and 8weeks. The new bone area in the experimental group at 8weeks was significantly greater than at 2 weeks (p<0.05). These new bone inductive effects of rhbmp-2 produced by the e-coli expression system are similar to the preexisting rhbmp-2 produced by CHO cells (Hong et al.,2005). 16
These results suggest that the rhbmp-2 produced from E-coli is effective in new bone formation. In addition, rhbmp-2 produced by the E-coli expression system has economic value and can be applied to dentistry. Compared with CHO cellexpressed rhbmp-2, a 0.1mg/ml concentration was used in this study because the activity of the E.coli-expressed rhbmp-2 was approximately five times lower. Future experiments will examine various concentrations of rhbmp-2 produced by the E-coli expression system. 17
V. Conclusion This study evaluated the bone regenerative effect of rhbmp-2 produced by an E- coli expression system delivered with an absorbable collagen sponge (ACS). An 8-mm critical-size calvarial defect was created in 48 male Sprague-Dawley rats. The animals were divided into 6 groups containing 8 animals each. The defects were treated with rhbmp-2, a positive control (Absorbable Collagen Sponge only) or were left untreated as a sham-surgery control. The defects were evaluated using the histologic and histometric parameters after a 2- and 8-week healing interval (8 animals/group/healing intervals). The experimental group showed significantly more defect closure at 2 and 8weeks than the sham surgery and positive control groups. Moreover, the experimental group showed a significantly greater new bone and augmented area than the others groups at both 2 and 8weeks. There was greater new bone area in the experimental group at 8weeks than at 2 weeks (p<0.05). Overall, rhbmp-2 produced by an E-coli expression system may be effective for bone regeneration. 18
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FIGURES Figure 2. Sham surgery control group Figure 3. Sham surgery control group (2 week, x20 magnification) (8 week, x20 magnification) Figure 4. Positive control group Figure 5. Positive control group (2 week, x20 magnification) (8 week, x20 magnification) 26
Figure 6. x20 magnification of 2 week group (Arrow : defect margin) Figure 7. x20 magnification of 8 week group (Arrow : defect margin) 27
Figure 8. x100 magnification of 2 week group (PB : pre-existing bone, NB : new bone, Arrow : defect margin) Figure 9. x100 magnification of 2 week group (central portion) 28
Figure10. x100 magnification of 8 week group (PB : pre-existing bone, NB : new bone, Arrow : defect margin) Figure 11. x100 magnification of 8 week group (PB : pre-existing bone, NB : new bone, Arrow : defect margin) 29
Figure 12. x100 magnification of 8 week group (central portion) 30
국문요약 백서두개골결손부에서 E-coli expression system 에의해생산된 rhbmp-2 의골재생효과 < 지도교수조규성 > 연세대학교대학원치의학과 권석훈 골형성유도단백질 (bone morphogenetic protein, BMPs) 은 치주치료와골재생치료를위한효과적인골대체물질로평가되어왔다. BMPs 는그자체로도골형성을유도할수있는충분한능력이있지만 수용부에적절히작용하기위해서는 carrier 가필요하다. BMPs 는우수한골형성능력에도불구하고대량생산의어려움및이로인한높은생산비용으로 dental area 에서사용이제한적이었다. 기존의 mammalian cell 에서생산된 BMPs 와달리 E-coli 로부터생산되는 BMPs 는대량생산이상대적으로쉬워서비교적생산단가가낮고골재생효과만입증된다면치과영역에서활발이사용될수있을것이다. 본연구의 31
목적은 rat calvarial defect model 에서 E-coli expression system 으로생산된 rhbmp-2 의골재생효과를알아보는것이다. 48 마리의웅성백서에서 8mm 지름을갖는임계크기의두개부결손을형성하였다. 8 마리씩 6 개의군으로나누고, 각군은아무것도이식하지않은 sham surgery 군, ACS(absorbable collagen sponge) 만이식한군, 0.1mg/ml rhbmp-2 를 soaking 한 ACS 를이식한군으로나누어술후 2 주와 8 주에치유결과를조직학적, 조직계측학적으로비교관찰하였다. 조직학적및조직계측학적관찰결과 rhbmp-2를이식한군에서다른군들보다 defect closure, new bone area, augmented bone area에서유의성있게증가함을보였고, rhbmp-2를이식한군내에서도 8주가 2주에비해서모든항목에서유의성있게증가함을보였다. 백서두개골결손부에서 E-coli expression system으로부터생산된 rhbmp-2를사용하였을때결손부폐쇄및신생골형성에유의한효과가있다고사료된다. 핵심되는말 : 골형성효과, 골형성유도단백질, E-coli expression system, ACS(absorbable collagen sponge), 백서두개골결손부 32