Histomorphometric Analysis of Heparin Effects on the rhBMP-2

다국어 초록 (Multilingual Abstract)

Although recombinant human Bone Morphogenetic Proteins-2 (rhBMP-2) is clinically useful for bone regeneration, induction of new bone formation requires a large amount of rhBMP-2 in humans. Many investigators have been concentrated efforts on searching materials which enhance the effect of rhBMP-2, and then heparin was found as a potentiating material to rhBMP-2. The purpose of this study were histomorphometrically to analyze the enhancing effect of heparin to rhBMP-2 and to study the mode of actions of heparin to rhBMP-2. Stem cells obtained from rabbit adipose tissue were divided into 4 groups according to heparin concentrations(0, 0.25, 2.5, and 25 μg/ml) with a constant rhBMP-2 concentration(150 ng/ml) and cultured for 2, 4, and 8 days. Naphtol AS phosphate-fast blue BB staining for alkaline phosphatase content and Alizarin red staining for calcium content were performed as time schedules and the morphology and osteoblastic activity were observed closely. 5 μg/ml rhBMP-2 was mixed with the following doses of heparin: 0, 0.25, 2.5, and 25 μg/ml. Each mixture was blotted into 0.5 g of multiporous anorganic bovine bone and was inserted into the critical sized calvarial defects(diameter of 0.8-mm) of rabbits. After 1, 3, and 6 weeks, the harvested tissues were processed and stained using H&E and Masson’s trichrome methods. And the areas of newly formed bone in the grafted material were measured and statistically analyzed.
During culture experiment of adipose stem cells with rhBMP-2 and heparin, the degree of osteoblastic differentiation was increased with increasing heparin concentration, but the cellular degeneration was accelerated at higher concentration of heparin as time passed.
Consequently the osteoblastic differentiation of progenitor cells were accelerated as the concentration of heparin increased. In addition, the progenitor cells exhibited full differentiations early showing fast degeneration. The higher the concentration of heparin, larger newly formed bone in grafted materials was obtained in initial period. However, the increased amount of the newly formed bone in grafted materials was progressively decreased at the higher concentration of heparin as time passed. In conclusion, the heparin has influence on the osteoinductive effect of rhBMP-2 in the initial stage of bone formation. The use of heparin with rhBMP-2 could offer cheaper, safer, and improve clinical results in grafting procedures.

참고문헌 (Reference)

1 박경주, "탈회동결건조골과 수종의 생물학적 물질들을 티타늄 매식체와 함께 이식 후 골형성" 대한구강악안면병리학회 32 (32): 241-252, 2008

2 Dickerman RD, "rh-BMP-2 can be used safely in the cervical spine : dose and containment are the keys!" 7 : 508-509, 2007

3 Bieniasz M, "The positive correlation between gene expression of the two angiogenic factors : VEGF and BMP-2 in lung cancer patients" 66 : 319-326, 2009

4 Siegel HJ, "The outcome of composite bone graft substitute used to treat cavitary bone defects" 31 : 754-, 2008

5 Nakashima K, "The novel zinc fingercontaining transcription factor osterix is required for osteoblast differentiation and bone formation" 108 : 17-29, 2002

6 Estes JW, "The kinetics of heparin" 179 : 187-204, 1971

7 Werner S, "The effect of microstructured surfaces and laminin-derived peptide coatings on soft tissue interactions with titanium dental implants" 30 : 2291-2301, 2009

8 Takada T, "Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins" 278 : 43229-43235, 2003

9 Takada T, "Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins" 278 : 43229-, 2003

10 Kumar V, "Robbins basic pathology" Saunders 2003

11 Carreon LY, "RhBMP-2 versus iliac crest bone graft for lumbar spine fusion in patients over 60 years of age: a cost-utility study" 34 : 238-243, 2009

12 Glassman SD, "RhBMP-2 versus iliac crest bone graft for lumbar spine fusion : a randomized, controlled trial in patients over sixty years of age" 33 : 2843-, 2008

13 Albrektsson T, "Osteoinduction, osteoconduction and osseointegration" 10 (10): S96-S101, 2001

14 Kusumoto K, "Osteoinduction by recombinant human bone morphogenetic protein-2 in muscles of non-human primates" 30 : 251-259, 2002

15 Chen X, "Osteogenic protein-1 induced bone formation in an infected segmental defect in the rat femur" 20 : 142-150, 2002

16 Fiorellini JP, "Localized ridge augmentation/preservation. A systematic review" 8 : 321-327, 2003

17 Hiremath GK, "Is it safe to use recombinant human bone morphogenetic protein in posterior cervical fusion?" 34 : 885-, 2009

18 Kubler NR, "Inductive properties of recombinant human BMP-2 produced in a bacterial expression system" 27 : 305-309, 1998

19 Khosla S, "Increasing options for the treatment of osteoporosis" 361 : 818-820, 2009

20 Kierszenbaum AL, "Histology and cell biology: an introduction to pathology" Mosby Inc 2002

21 Zhao B, "Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2" 281 : 23246-23253, 2006

22 Zhao B, "Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2" 281 : 23246-, 2006

23 Mak KK, "Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression" 14 : 674-688, 2008

24 Gjesdal K, "Half-life of platelet factor 4(PF-4)in plasma and platelets from macaca mulatta" 37 : 73-80, 1977

25 Balemans W, "Extracellular regulation of BMP signaling in vertebrates : a cocktail of modulators" 250 : 231-250, 2002

26 Balemans W, "Extracellular regulation of BMP signaling in vertebrates : a cocktail of modulators" 250 : 231-250, 2002

27 Hazama M, "Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system" 209 : 859-866, 1995

28 Hentunen TA, "Effects of recombinant human osteogenic protein-1 on the differentiation of osteoclast-like cells and bone resorption" 209 : 433-443, 1995

29 Miller CP, "Effects of recombinant human bone morphogenetic protein 2 on surgical infections in a rabbit posterolateral lumbar fusion model" 38 : 578-584, 2009

30 Hsu SK, "Effects of near-field ultrasound stimulation on new bone formation and osseointegration of dental titanium implants in vitro and in vivo" 37 : 403-416, 2011

31 Oppenheimer AJ, "Current and Emerging Basic Science Concepts in Bone Biology : Implications in Craniofacial Surgery" 23 : 30-36, 2012

32 Oppenheimer AJ, "Craniofacial Bone Grafting : Woff’s Law Revisited" 1 : 49-61, 2008

33 Rocchietta I, "Clinical outcomes of vertical bone augmentation to enable dental implant placement : a systematic review" 35 : 203-215, 2008

34 Emodi G, "Circulating interferon in man after administration of exogenous human leukocyte interferon" 54 : 1045-1049, 1975

35 Langenfeld EM, "Bone morphogenetic protein-2stimulates angiogenesis in developing tumors" 2 : 141-149, 2004

36 Katagiri T, "Bone morphogenetic protein-2 converts the differentiation pathway of C2C12myoblasts into the osteoblast lineage" 127 : 1755-1766, 1994

37 Ishibe T, "Bone formation on apatite-coated titanium with incorporated BMP-2/heparin in vivo" 108 : 867-875, 2009

38 Hall J, "Bone formation at rhBMP 2 coated titanium implants in the rat ectopic model" 34 : 444-451, 2007

39 Urist MR, "Bone : formation by autoinduction" 150 : 893-899, 1965

40 Wan M, "BMP signaling in skeletal development" 328 : 651-657, 2005

41 Wan M, "BMP signaling in skeletal development" 328 : 651-657, 2005

42 Boakye M, "Anterior cervical discectomy and fusion involving a polyetheretherketone spacer and bone morphogenetic protein" 2 : 521-525, 2005

43 Palmieri A, "Anorganic bovine bone(Bio-Oss)regulates miRNA of osteoblast-like cells" 30 : 83-87, 2010

44 Shah MM, "Adverse facial edema associated with off-label use of recombinant human bone morphogenetic protein-2 in cranial reconstruction for craniosynostosis. Case report" 1 : 255-257, 2008

45 Latzman JM, "Administration of human recombinant bone morphogenetic protein-2 for spine fusion may be associated with transient postoperative renal insufficiency" 35 : E231-E237, 2010

46 Benglis D, "A comprehensive review of the safety profile of bone morphogenetic protein in spine surgery" 62 : ONS423-, 2008