Animal Cells and Systems, 2015 Vol. 19, No. 5, 312–320, http://dx.doi.org/10.1080/19768354.2015.1087429 Heparin increases the osteogenic effect of recombinant human bone morphogenetic protein-2 in the rabbit bone defect model a,b c d Ju Yeon Ban *, Sang Wook Kang and Gyeong-Ju Park a b Department of Dental Pharmacology, School of Dentistry, Dankook University, Cheonan 330-714, Republic of Korea; Department of Medical Laser, Graduate School, Dankook University, Cheonan 330-714, Republic of Korea; Kohwang Medical Research Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea; Department of Oral Histology, School of Dentistry, Dankook University, Cheonan 330-714, Republic of Korea (Received 20 June 2015; received in revised form 18 August 2015; accepted 23 August 2015) In order to perform successful dental implantations and solve other problems such as bone deficiencies, it is important to find suitable biomaterials to enhance osteoinduction. Heparin has been known to enhance bone morphogenetic protein 2 (BMP-2) induced-bone formation. We histomorphometrically investigated the enhancing effects and roles of heparin to recombinant human bone morphogenetic proteins-2 (rhBMP-2). For morphologic study, stem cells obtained from rabbit adipose tissue were divided into four groups based on heparin concentrations, with a constant rhBMP-2 concentration. They were cultured for 2, 4, and 8 days. Alkaline phosphatase and calcium content were measured as time schedules. In addition, a mixture of rhBMP-2 and heparin was blotted into multiporous anorganic bovine bone and was inserted into calvarial defects in rabbits. The harvested tissues were stained using the Hematoxylin & Eosin (H&E) and Masson’s trichrome (MT) stain. And the areas of newly formed bone in the grafted material were analyzed. In morphologic results, the degree of osteoblastic differentiation was significantly increased with increasing heparin concentrations, but the cellular degeneration was accelerated at a higher concentration of heparin as time passed. In histological results, the more higher the concentration of heparin, larger newly formed bone in grafted materials was also observed in the initial period. However, the increased amount of the newly formed bone in grafted materials was progressively decreased at higher concentrations of heparin as time passed. In conclusion, heparin might have an influence on the osteoinductive effects of rhBMP-2 during the initial stage of bone formation. Keywords: recombinant human bone morphogenetic proteins; heparin; anorganic bovine bone; osteoinduction; multiporous 1. Introduction and to play a key role in blood vessel formation in skeletal and tumoral development in mammals (Oppenheimer et al. Dental implants are widely used to rehabilitate the mastica- 2008; Siegel et al. 2008; Oppenheimer et al. 2012). BMPs tory function but their use could fail by delayed or failed were generally identified as inducers of ectopic bone for- osteogenesis due to poor bone quality or deficiency of mations when implanted into muscle tissues (Urist 1965). the jaw bone. Although various types of grafting materials Except for BMP-1, all BMPs are members of the trans- have been widely evaluated to augment bone deficiencies, forming growth factor-β (TGF-β) superfamily (Hazama their use can result in unpredictable complications, such as et al. 1995; Kubler et al. 1998). Among BMPs, BMP-2 foreign body reactions and bacterial infection on various has shown an excellent ability to enhance bone formation grafting materials (Fiorellini & Nevins 2003; Rocchietta (Nakashima et al. 2002). Although some complications et al. 2008). such as inflammatory reactions, edema, and heterotropic Many studies have been focused on the development of bone formation have been reported (Benglis et al. 2008), new surfaces, which increased the osteogenesis on titanium many researchers insisted on the safety and efficacy of implant surfaces (Wall et al. 2009; Hsu et al. 2011). BMP-2 in spinal and maxillofacial applications (Dicker- However, surface improvements alone are not sufficient man et al. 2007;Fu etal. 2013; Simmonds et al. 2013; to assure the success of dental implantation and solve Freitas et al. 2015). other problems such as bone deficiencies in the implanted The activity of BMPs is suppressed or enhanced by sites and the slow integration of grafted material to the tita- various molecules (Balemans & Van Hul 2002;Wan nium surface. Therefore, considerable investigations have et al. 2005). Noggin, chordin, and follistatin are examples been made to find suitable biomaterials to enhance osteoin- of proteins which suppress BMP activity in the extracellu- duction (Werner et al. 2009). lar space by inhibiting the binding of BMPs to cell-surface Among these, BMPs are known to improve the osteoin- receptors (Balemans & Van Hul 2002). In contrast, heparin ductive effect by differentiating mesenchymal cells to enhances the action of BMPs, but heparin alone cannot osteoblasts (Katagiri et al. 1994; Nakashima et al. 2002) *Corresponding author. Email: email@example.com © 2015 Korean Society for Integrative Biology DEVELOPMENTAL BIOLOGY Animal Cells and Systems 313 differentiate mesenchymal progenitor cells from osteo- detached from the flasks by trypsin (BioWhittaker), dis- blasts. The capacity of sulfated polysaccharides such as persed into another flask at a density of 2×10 cells/ml, heparin to enhance the actions of BMPs is likely to and cultured (secondary culture). depend on the size and number of sulfated residues Verification of stem cell lineage was performed by (Takada et al. 2003). A previous study demonstrated that using monoclonal antibodies for STRO-1 and CD 90 heparin potentiated ectopic bone formation induced by (Santa Cruz Biotechnology, Dallas, TX, USA; Figure 1 BMP-2 in vivo (Zhao et al. 2006), but morphologic and his- (B) and Figure 1(C)), which are expressed in mesenchymal tometric studies to determine the effects of heparin on bony stem cells. defects of critical size have still not been carried out. In con- trast, many studies have been conducted regarding the 2.3. Determining of osteoblastic differentiation effects of heparin on BMP-2 in peripheral new bone for- mation around dental implants or grafted materials (Ishibe The verified stem cells were divided into four groups: a et al. 2009). Their studies are not specific to osteoinduction control group and three groups with increasing concen- because the newly formed peripheral bone may contain both trations of heparin (0.25, 2.5, and 25 μg/ml). In all four osteoinductive and osteoconductive bones. groups, rhBMP-2 concentration remained constant (150 In this study, we evaluated morphologic changes in cul- ng/ml). The cells were cultured for 2, 4, and 8 days. Alka- tured adipose stem cells obtained from rabbits by adding line phosphatase activity was used as a marker of osteoblast various concentrations of heparin (0, 0.25, 2.5, and 25 differentiation and degree of osteoblastic activity. Cells μg/ml) along with a constant concentration of rhBMP-2 treated with an acetone/ethanol mixture (50:50, v/v) were (150 ng/ml) to culture media. In addition, we measured incubated with a substrate solution composed of 0.1 M the amount of internally formed bone in grafted material diethanolamine, 0.1 mM MgCl , and 10 mg/ml p-nitrophe- using a mixture of different concentrations of heparin and nyl phosphate. The reaction was terminated by adding constant concentration of rhBMP-2 (5 μg/ml), and then NaOH, and values were measured at 405 nm light. We placed the grafts into critically sized calvarial defects in used naphthol AS phosphate–fast blue BB to determine rabbits. Finally, we also investigated the action-pattern of alkaline phosphatase content and alizarin red staining to heparin based on time elapsed and different concentrations determine calcium content at specific times, and the cul- of heparin. tured adipose stem cells were inspected for morphology and osteoblastic activity. 2. Materials and methods 2.4. Preparation of grafting material 2.1. Preparation of rhBMP-2, heparin, and anorganic bovine bone The four groups of mixture – containing rhBMP-2 (5 μg/ ml) alone (the control group) or rhBMP-2 (5 μg/ml) Purified rhBMP-2 was purchased from Cowellmedi Co, mixed with 0.25, 2.5, or 25 μg/ml of heparin (Groups 1, Ltd (Seoul, Korea) and has been produced by the Escher- 2, and 3, respectively) – were blotted into 0.5 g of anor- ichia coli expression system. The rhBMP-2 was dissolved ganic bovine bone, freeze-dried, and were stored at −60° in sterile distilled water. Purified heparin for injection was C until they were grafted into rabbit calvarial defects 8 obtained from JW Pharmaceutical Corp. (Seoul, Korea). mm in diameter. Both the rhBMP-2 and heparin were diluted with sterile distilled water. Anorganic bovine bone (OCS-B ) was sup- plied by Nano Intelligent Biomedical Engineering Corp. 2.5. Surgical procedure (Seoul, Korea). The animal experiments were reviewed, and approved by the animal ethics committee at Dankook University 2.2. Preparation of progenitor cells for morphologic (Cheonan, Korea). All procedures were carried out under study sterile conditions. Forty-eight rabbits (8-month old; male; Sterile adipose tissue was harvested from the foreleg New Zealand White) were housed and acclimated in axillae of a rabbit (4-month old; New Zealand White), cages with access to food and water for 1 week ad and was very finely minced and chopped with sterile eye libitum. The rabbits were divided into four groups accord- scissors. This prepared adipose tissue was dispersed in ing to heparin concentration (0, 0.25, 2.5, and 25 μg/ml). 4×5 cm culture flasks with 0.5 ml of α-MEM and 10% They were anesthetized with isoflurane (initiated at 5% fetal bovine serum (BioWhittaker, Walkersville, MD, and maintained at 2%) in 25% O /75% N , and then 2 2 USA) and incubated at 37°C with 5% CO for 4 days. local anesthesia was performed in the calvarial region by After verifying cell migration from prepared adipose lidocaine HCl with epinephrine (1:80,000). An incision tissue (Figure 1(A), the culture media were changed at 2- was made on the scalp and four 8-mm craniotomy holes day intervals for 8 days (primary culture). Cells were were prepared on the exposed calvaria using a trephine 314 J.Y. Ban et al. Figure 1. Cell migration from the minced and chopped adipose tissue of rabbits (A). Positive reactions are shown to antibodies for STRO-1 (B) and CD90 (C). bur. When the dura mater was exposed, the trephination The results were similar on histologic examination was stopped and the bone chips were removed. Sub- using naphthol AS phosphate–fast blue BB staining and ali- sequently, the prepared grafting materials were inserted zarin red staining. At 2 days, when 150 ng/ml of rhBMP-2 into the holes (Figure 2). According to the prespecified pro- alone was added to the culture (control group), cells did not tocol, four rabbits from each group were sacrificed at 1, 3, exhibit the morphologic characteristics of osteoblasts. Few and 6 weeks after the procedure. cells reacted to naphthol AS phosphate–fast blue BB stain- ing and alizarin red staining, respectively (Figure 4(A) and Figure 5(A)). The cells in Group 1 that were treated with 2.6. Histologic study 0.25 μg/ml of heparin and 150 ng/ml of rhBMP-2 only The harvested bony tissues were fixed in 10% buffered slightly displayed an early form of osteoblasts and mildly neutral formalin and demineralized with 10% formic acid. reacted to the two types of staining (Figure 4(D) and The prepared sections were stained with H&E and MT Figure 5(D), respectively). The cells in Group 2 that were stains. Photographs of each stained section (two sections treated with 2.5 μg/ml of heparin and 150 ng/ml of per rabbit; a total of eight specimens per group) were taken rhBMP-2 exhibited morphologic characteristics of osteo- at ×50 magnification, and the images were analyzed using blasts to a moderate degree and reacted somewhat positively IPTK version 5.0 software (Reindeer Graphics, Asheville, on staining (Figures 4(G) and 5(G)). The cells in Group 3 NC, USA) to measure the total area of internally formed that were treated with 25 μg/ml of heparin and 150 ng/ml new bone in the grafted material (1–2 mm) and the total of rhBMP-2 exhibited morphologic characteristics of osteo- area of grafted material. Subsequently, the total area of intern- blasts to a greater extent and showed highly positive reac- ally formed new bone was divided by the total area of grafted tions on staining (Figures 4(J) and 5(J)). At 4 days, the material, and the results were expressed as percent ratios. cells in the control group began to show morphologic simi- larities to osteoblasts and positive reactions on staining (Figures 4(B) and 5(B)). Group 1 cells produced features 2.7. Statistical analysis more similar to osteoblasts and reacted more strongly at Statistical analysis was performed using SPSS version 17.0 day 4 than at day 2 (Figures 4(E) and 5(E)). Group 2 (SPSS, Chicago, IL, USA). The differences in the percent showed more advanced forms of osteoblasts and features values were evaluated by one-way analysis of variance similar to the morphology seen in Group 3 at 2 days (ANOVA). Post hoc tests of ANOVA tests (Scheffe’s (Figures 4(H) and 5(H)). At 8 days, the cell shapes of method) were then performed to verify the significance Group 1, 2, and 3 exhibited morphologically degenerating of the results. A p-value of less than .05 was considered forms, except for the control group, and the degree of mor- statistically significant. phological degeneration increased with increasing concen- trations of heparin. Cellular reactivity to the naphthol AS phosphate–fast blue BB and alizarin red staining also 3. Results decreased. Especially at the heparin concentration of 25 3.1. Morphologic effects of heparin with rhBMP-2 on μg/ml, the cells were least active in terms of morphology adipose stem cells in culture and reactivity (Figures 4(C) and 5(C); Figures 4(F) and 5 The effect of heparin with rhBMP-2 on osteoblastic activity (F); Figures 4(I) and 5(I); and Figures 4(L) and 5(L)). is shown in Figure 3. Osteoblastic activity increased in pro- portion with heparin concentration at days 2 and 4, whereas 3.2. Histologic effects of heparin with rhBMP-2 on in the control group (150 ng/ml of rhBMP-2 alone), osteo- rabbit calvarial defects blastic activity increased slightly over time but was lowest at days 2 and 4. However, osteoblastic activity decreased in Control group. After 1 week, newly formed collagen fibers proportion to heparin concentration at day 8. were observed around the multiporous grafted material Animal Cells and Systems 315 Figure 2. Operative procedure (insertion of prepared grafting materials in rabbit calvarial defects). Holes, 8 mm in diameter, were formed by trephine bur craniotomy (A). Dura mater can be seen at the bases of the four holes (B). Each hole was filled with anorganic bovine bone, which was blotted with 5 μg/ml of rhBMP-2 alone (control group) and with 5 μg/ml of rhBMP-2 with 0.25, 2.5, or 25 μg/ml of heparin (Groups 1, 2, and 3, respectively) (C). After the holes were filled with the prepared grafting materials, the periosteum was sutured (D) and then the scalp flap was sutured (E). (Figure 6(A), blue color), as were a few lymphocytes changes were detected within the graft material itself (Figure 6(D), thick arrow), some macrophages, and many (Figure 6(C), thin arrow), but these internal changes did capillaries. Cuboidal cells with prominent nuclei that not meet the accepted histologic features of new bone for- resembled osteoblasts were seen in the area where the mation because they lacked a clear boundary, lacunae, and grafted material met the connective tissue (Figure 6(D), the specific colors that indicate bony material (blue for thin arrow), but no changes were observed in the grafted newly formed bone and brown to red for formed bone on material itself (Figure 6(D)). At 3 weeks, the amount of Masson’s trichrome staining). In contrast, the peripheral newly formed collagen fibers increased in the connective bone around the grafted material did exhibit the histologic tissue around the grafted material, but there were no features of newly formed bone (Figure 6(E)). changes in the grafted material (Figure 6(B)). At 6 Group 1. After 1 week, the amount of newly formed weeks, collagen fibers and peripheral bone had formed bone around the grafted material was very small, but around the grafted material (Figure 6(C)), and some clear internal changes were observed in the grafted material (Figure 6(F)). These new internal structures were linear or ring-shaped, and higher magnification revealed newly formed bone containing osteocytes and lacunae (Figure 6 (I)). In the center of the newly formed bone was a space containing what appeared to be red blood cells, white blood cells, and mesenchymal cells. At 3 weeks, the amount of newly formed peripheral bone and internal bone had increased from what was observed at 1 week (Figure 6(G)). At 6 weeks, the amount of peripheral new bone and internal new bone had increased from 3 weeks (Figure 6(H)). Group 2. In a week, significantly increased formation of new internal bone was observed in grafted material (Figure 6(J)). At 3 weeks, the amount of peripheral and internal new bone was greater than that observed at 1 week. In addition, internally formed bone was partially interconnected with peripheral bone (Figure 6(K), arrow). At 6 weeks, the amount of newly formed peripheral bone Figure 3. Osteoblastic activity of differentiated adipose stem cells and internal bone was greater than that observed at 3 exposed to rhBMP-2 (150 ng/ml) alone with different concen- weeks (Figure 6(L)). Higher magnification indicated that trations of heparin (0, 0.25, 2.5, and 25 μg/ml). 316 J.Y. Ban et al. Figure 4. Photographs showing the effects of naphthol AS phosphate–fast blue BB staining in the four groups of cells (magnification ×50). (Control, 150 ng/ml of rhBMP-2 alone; Group 1, 150 ng/ml of rhBMP-2+0.25 μg/ml of heparin; Group 2, 150 ng/ml of rhBMP-2+2.5 μg/ ml of heparin; Group 3, 150 ng/ml of rhBMP-2+25 μg/ml of heparin). the internal new bone had complete bony structures (i.e. (0.25, 2.5, and 25 μg/ml) up to a maximum of fivefold osteocytes, osteoblasts, and lacunae) (Figure 6(M)). The (Figure 7). central area contained blood vessels that presumably con- tained red and white blood cells and progenitor cells 4. Discussion (Figure 6(M)). Group 3. After 1 week, significant amounts of new In this study we evaluated the osteoinductive effects of internal bone were observed in the grafted material different concentrations of heparin (0, 0.25, 2.5, and 25 (Figure 6(N)). At 3 weeks, the amounts of new peripheral μg/ml) with a fixed concentration of rhBMP-2 (5 μg/ml) and internal bone had increased to a greater extent than on cultured rabbit adipose stem cells. In addition, we those observed at 1 week (Figure 6(O)). At 6 weeks, the blotted multiporous anorganic bovine bone with these amounts of new peripheral bone and internal bone were same concentrations of heparin and a fixed concentration greater than those observed at 3 weeks (Figure 6(P)). The of rhBMP-2 (150 ng/ml) to fill critically sized rabbit cal- formed spaces within the internal bone exhibited both varial defects. These concentrations were chosen based hematopoietic and fatty marrow (Figure 6(P)). on previous studies (Zhao et al. 2006; Ishibe et al. 2009). An anorganic bovine bone substitute (i.e. Bio-Oss) was used as a bone substitute because of its osteoconducting 3.3. Histometric analysis capacity (Palmieri et al. 2010). Throughout the experimental period, no critical changes Currently, the most serious and obvious clinical com- indicating bone formation were seen in the grafted material plications of using rhBMP-2 in humans are swelling or in the control group. In general, the percent ratios of edema (Shah et al. 2008), transient renal insufficiency, internal new bone to grafted material increased over time and ectopic bone formation with increasing doses of in three groups of heparin-treated cells (Figure 7). The rhBMP-2 (Boakye et al. 2005; Latzman et al. 2010). overall percent ratio of internal new bone to graft material Dose and containment of rhBMP-2 are key to avoiding also increased with increasing heparin concentrations such complications (Dickerman et al. 2007). Therefore, Animal Cells and Systems 317 Figure 5. Alizarin red staining (magnification ×50) of adipose stem cells in culture in the four study groups. (Control, 150 ng/ml of rhBMP-2 alone; Group 1, 150 ng/ml of rhBMP-2+0.25 μg/ml of heparin; Group 2, 150 ng/ml of rhBMP-2+2.5 μg/ml of heparin; and Group 3, 150 ng/ml of rhBMP-2+25 μg/ml of heparin). enhancing the activity of rhBMP-2 as a way to reduce the responses can be explained in three ways. First, like most dose may prove more economical and decrease the associ- cytokines, the half-lives of rhBMP-2 and heparin are ated side effects and morbidity as compared with the use of very short. Hence, cytokine activity might be transient autogenous bone grafts (Carreon et al. 2009). There are and limited to a local area (Estes 1971; Emodi et al. many molecules that enhance or inhibit the activity of 1975; Gjesdal & Pepper 1977). Second, the release of BMPs (Balemans & Van Hul, 2002; Wan et al. 2005). rhBMP-2 and heparin could be delayed. In this study, Noggin, chordin, follistatin, and the DNA family proteins rhBMP-2 and heparin were blotted into multiporous anor- bind to BMPs in the extracellular space and inhibit the ganic bovine bone. The multiporous structure could result binding of ligands to cell-surface receptors (Balemans & in the slow release of rhBMP-2 into the adjacent environ- Van Hul, 2002). In contrast, heparin enhances the activity ment and prevent rapid increases in rhBMP-2 and of BMPs and consequently increases BMP-induced osteo- heparin concentrations at the periphery of the grafted blast differentiation (Takada et al. 2003). Because heparin material. Third, rhBMP-2 might potentiate the immune itself does not induce osteoblast differentiation from system response and prevents infections in surgical mesenchymal progenitor cells (Takada et al. 2003), more wounds. Osteoblasts can produce RANKL (Khosla definite confirmation of the enhancing effect of heparin 2009), and the destruction of RANKL can retard the on rhBMP-2 is needed. immune system (Khosla 2009). The application of Some previous studies have reported that rhBMP-2 rhBMP-2 can increase RANKL by osteoblast differen- causes inflammation and edema, but in our study only a tiation and potentiate the immune system in human or few lymphocytes and some macrophages were seen to animals (Chen et al. 2002; Khosla 2009; Miller et al. have infiltrated the connective tissue around the grafted 2009). In this study, none of the rabbits grafted with material, and we observed little edema in the connective rhBMP-2/heparin-blotted anorganic bovine bone showed tissue (Figure 6(D)). These reduced inflammatory any signs of infection. Based on the results of this and 318 J.Y. Ban et al. Figure 6. Histologic findings for heparin with rhBMP-2 in rabbit calvarial defects. (MT stain, magnification ×50; bar=150 μm). 5 μg/ml rhBMP-2 alone (A to C), 5 μg/ml of rhBMP-2+0.25 μg/ml of heparin (F to H), 5 μg/ml of rhBMP-2+2.5 μg/ml of heparin (J to L), and 5 μg/ml of rhBMP-2+25 μg/ml of heparin (N to P). After 1 week: 5 μg/ml of rhBMP-2 alone (H&E stain [D] and MT stain [E], magnification ×200; bar=50 μm); 5 μg/ml of rhBMP-2+0.25 μg/ml of heparin (MT stain [I]). After 6 weeks: 5 μg/ml of rhBMP-2+2.5 μg/ml of heparin (MT stain [M]). NC, newly formed collagen; GM, grafted materials; BV, blood vessels; PB, peripheral newly formed bone; IB, internal newly formed bone; MФ, macrophage; OB, osteoblast; OC, osteocyte; HM, hematopoietic marrow; FM, fatty marrow. other studies, we hypothesize that rhBMP-2 enhances the In the culture experiment, morphologic and histometric immune response in local areas, but this postulation must evaluations showed that rabbit adipose stem cells differen- be considered in future studies. tiated into osteoblasts in proportion to the heparin concen- trations. However, over time, the differentiated osteoblasts quickly showed degenerative changes in proportion to the heparin concentrations. To put it another way, heparin enhanced the effect of rhBMP-2 on osteoblastic differen- tiation and simultaneously stimulated degenerative changes in the completely differentiated osteoblasts (Figures 3–5). Previous studies have shown that with long-term administration, heparin is associated with an increasing risk for the development of osteoporosis (Wolinsky-Friedland 1995) and that heparin can suppress BMP-2 osteogenic activity by binding in high concen- trations (Kanzaki et al. 2008). These reported results are consistent with those in our study. On the histologic analysis of the control group, some cells in the control group underwent osteoblastic morpho- logical changes only in the vicinity of the grafted material Figure 7. Graph showing the percent ratio of internal newly (Figure 6(D)). This means that rhBMP-2 alone cannot formed bones to grafted materials according to heparin concen- induce mesenchymal cell migration or other changes tration at specific times. Heparin concentrations were 0.25 μg/ (vessel infiltration) in the grafted material. A previous ml in Group 1, 2.5 μg/ml in Group 2, and 25 μg/ml in Group study suggested that the heparin protected rhBMP-2 from 3. *p < .05, **p < .01, ***p < .005. Animal Cells and Systems 319 the noggin or the proteases and sustained the rhBMP-2 However, this enhancing effect was not in proportion to levels (Ishibe et al. 2009). In our study, all the experimental the heparin concentrations, and heparin influenced osteoin- groups grafted with rhBMP-2/heparin-blotted anorganic ductive effect of rhBMP-2 mainly in the initial stage of bovine bone showed internal bone formation, which bone formation. Because the most important factor in graft- seems to indicate that sustained level of rhBMP-2 in the ing procedures is the stabilization achieved during the grafted material with heparin might allow progenitor cell initial response of bone, our results suggest that the use migration and/or vessel infiltration into the grafted of heparin with rhBMP-2 could offer safer, better, and material. It seemed that heparin might assist rhBMP-2- more economical clinical results in grafting procedures. mediated osteoinduction in grafted material. The osteoinductive process, which involves recruiting progenitor cells and stimulating them to develop into pre- Disclosure osteoblasts (Albrektsson & Johansson 2001), can be eval- No potential conflict of interest was reported by the uated by undifferentiated cell migration phenomena. The authors. osteoinductive effect of a certain material must be evalu- ated by the internally formed bone in the grafted material because the peripherally formed bone around the grafted Funding material can be produced by both osteoconductive and The present research was conducted by the research fund of osteoinductive effects. Accordingly, we measured the Dankook University in 2013. area of internally formed bone in grafted material, and the total area of the newly formed internal bone was divided by the total area of the grafted material and References expressed as a percent ratio (Figure 7). Albrektsson T, Johansson C. 2001. Osteoinduction, osteoconduc- Generally speaking, the three essential components of tion and osseointegration. Eur Spine J. 10:S96–S101. osteoinduction are growth factors for osteoinduction, pro- Balemans W, Van Hul W. 2002. 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Animal Cells and Systems
– Taylor & Francis
Published: Sep 3, 2015
Keywords: recombinant human bone morphogenetic proteins; heparin; anorganic bovine bone; osteoinduction; multiporous