Abstract
We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (PVEGF) or BMP-6 (PBMP-6) or both (PVEGF+BMP-6) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with PVEGF+BMP-6 scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the PVEGF+BMP-6 group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in PVEGF, PBMP-6 and PVEGF+BMP-6 but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with PVEGF, PBMP-6, or PVEGF+BMP-6 but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on PVEGF+BMP-6 but not on P, PVEGF, or PBMP-6. Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.
Similar content being viewed by others
References
Arzi B, Versstraete FJ, Huey DJ, Cissel DD, Athanasiou KA (2015) Regenerating mandibular bone using rhBMP-2. Part 1 Immediate reconstruction of segmental mandibulectomies. Vet Surg 44:403-409
Brown JL, Peach MS, Nair LS, Kumbar SG, Laurencin CT (2010) Composite scaffolds: briding nanofiber and microsphere architectures to improve bioactiity of mechanically competent constructs. J Biomed Mater Res A 95:1150–1158
Cheng Y, Ramos D, Lee P, Liang D, Yu X, Kumbar SG (2014) Collagen functionalized bioactive nanofibers matrices for osteogenic differentiation of mesenchymal stem cells: bone tissue engineering. J Biomed Nanotechnol 10:287–298
Costantino PD, Hiltzik D, Govindaraj S, Moche J (2002) Bone healing and bone substitutes. Facial Plast Surg 18:13–26
Cui F, Wang X, Liu X, Dighe AS, Balian G, Cui Q (2010) VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells. Growth Factors 28:306–317
Cui Q, Dighe AS, Irvine JN (2013) Combined angiogenic and osteogenic factor delivery for bone regenerative engineering. Curr Pharm Des 19:3374–3383
Delimar D, Smoljanovic T, Bojanic I (2012) Could the use of bone morphogenetic proteins in fracture healing do more harm than good to our patients? Int Orthop 36:683
Diefenderfer DL, Osyczka AM, Reilly GC, Leboy PS (2003a) BMP responsiveness in human mesenchymal stem cells. Connect Tissue Res 44:305–311
Diefenderfer DL, Osyczka AM, Garino JP, Leboy PS (2003b) Regulation of BMP induced transcription in cultured human bone marrow stromal cells. J Bone Joint Surg Am 85:19–28
Fajardo M, Liu CJ, Egol K (2009) Levels of expression for BMP-7 and several BMP antagonists may play an integral role in a fracture nonunion: a pilot study. Clin Orthop Relat Res 467:3071–3078
Fiedler J, Roderer G, Gunther KP, Brenner RE (2002) BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells. J Cell Biochem 87:305–312
Fiedler J, Leucht F, Waltenberger J, Dehio C, Brenner RE (2005) VEGF-A and PlGF-1 stimulate chemotactic migration of human mesenchymal progenitor cells. Biochem Biophys Res Commun 334:561–568
Fong KD, Nacamuli RP, Song HM, Warren SM, Lorenz HP, Longaker MT (2003) New strategies for craniofacial repair and replacement: a brief review. J Craniofacial Surg 14:333–339
Hanada K, Dennis JE, Caplan AI (1997) Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J Bone Miner Res 12:1606–1614
Hausman MR, Schaffler MB, Majeska RJ (2001) Prevention of fracture healing in rats by an inhibitor of angiogenesis. Bone 29:560–564
Hollinger JO, Kleinschmidt JC (1990) The critical size defect as an experimental model to test bone repair materials. J Craniofacial Surg 1:60–68
Hsieh SC, Tang CM, Huang WT, Hsieh LL, Lu CM, Chang CJ, Hsu SH (2011) Comparison between two different methods of immobilizing NGF in poly(DL-lactic acid-co-glycolic acid) conduit for peripheral nerve regeneration by EDC/NHS/MES and genipin. J Biomed Mater Res A 99:576–585
Jørgensen NR, Henriksen Z, Sørensen OH, Civitelli R (2004) Dexamethasone, BMP-2, and 1, 25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids 69:219–226
Kaban LB, Glowacki J, Murray JE (1979) Repair of experimental mandibular bony defects in rats. Surg Forum 30:519–521
Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, Deyrup AT, Jiang W, Luu HH, Luo J, Szatkowski JP, Vanichakarn P, Park JY, Li Y, Haydon RC, He TC (2004) Characterization of the distinct orthotopic boneforming activity of 14 B.P. using recombinant adenovirus-mediated gene delivery. Gene Ther 11:1312–1320
Kempen DH, Lu L, Heijink A, Hefferan TE, Creemers LB, Maran A, Yaszemski MJ, Dhert WJ (2009) Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration. Biomaterials 30:2816–2825
Kimelman N, Pelled G, Helm GA, Huard J, Schwarz EM, Gazit D (2007) Review: gene- and stem cell-based therapeutics for bone regeneration and repair. Tissue Eng 13:1135–1150
Kofron MD, Cooper JA, Kumbar SG, Laurencin CT (2007) Novel tubular composite matrix for bone repair. J Biomed Mater Res A 82:415–425
Kofron MD, Griswold A, Kumbar SG, Martin K, Wen X, Laurencin CT (2009) The implications of polymer selection in regenerative medicine: a comparison of amorphous and semi-crystalline polymer for tissue regeneration. Adv Funct Mater 19:1351–1359
Lee DH, Park BJ, Lee MS, Lee JW, Kim JK, Yang HC, Park JC (2006) Chemotactic migration of human mesenchymal stem cells and MC3T3-E1 osteoblast-like cells induced by COS-7 cell line expressing rhBMP-7. Tissue Eng 12:1577–1586
Lee MH, Kwon TG, Park HS, Wozney JM, Ryoo HM (2003) BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 309:689–694
Li JZ, Li H, Sasaki T, Holman D, Beres B, Dumont RJ, Pittman DD, Hankins GR, Helm GA (2003) Osteogenic potential of five different recombinant human bone morphogenetic protein adenoviral vectors in the rat. Gene Ther 10:1735–1743
Lissenberg-Thunnissen SN, Gorter DJ de, Sier CF, Schipper IB (2011) Use and efficacy of bone morphogenetic proteins in fracture healing. Int Orthop 35:1271–1280
Madhu V, Li CJ, Dighe AS, Balian G, Cui Q (2014) BMP-non-responsive Sca1+ CD73+ CD44+ mouse bone marrow derived osteoprogenitor cells respond to combination of VEGF and BMP-6 to display enhanced osteoblastic differentiation and ectopic bone formation. PLOS One 9:e103060
Mathew A, Fukuda T, Nagaoka Y, Hasumura T, Morimoto H, Yoshida Y, Maekawa T, Venugopal K, Kumar DS (2012) Curcumin loaded-PLGA nanoparticles conjugated with Tet-1 peptide for potential use in Alzheimer’s disease. PLoS One 7: e32616
Mathieu M, Rigutto S, Ingels A, Spruyt D, Stricwant N, Kharroubi I, Albarani V, Jayankura M, Rasschaert J, Bastianelli E, Gangji V (2013) Decreased pool of mesenchymal stem cells is associated with altered chemokines serum levels in atrophic nonunion fractures. Bone 53:391–398
Matsubara T, Kida K, Yamaguchi A, Hata K, Ichida F, Meguro H, Aburatani H, Nishimura R, Yoneda T (2008) BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem 283:29119–29125
Mayr-Wohlfart U, Waltenberger J, Hausser H, Kessler S, Gunther KP, Dehio C, Puhl W, Brenner RE (2002) Vascular endothelial growth factor stimulates chemotactic migration of primary human osteoblasts. Bone 30:472–477
Mizuno D, Agata H, Furue H, Kimura A, Narita Y, Watanabe N, Ishii Y, Ueda M, Tojo A, Kagami H (2010) Limited but heterogeneous osteogenic response of human bone marrow mesenchymal stem cells to bone morphogenetic protein-2 and serum. Growth Factors 28:34–43
Moghadam HG, Urist MR, Sandor GK, Clokie CM (2001) Successful mandibular reconstruction using a BMP bioimplant. J Craniofac Surg 12:119–128
Nauth A, Ristiniemi J, McKee MD, Schemitsch EH (2009) Bone morphogenetic proteins in open fractures: past, present, and future. Injury 40:27–31
Osyczka AM, Diefenderfer DL, Bhargave G, Leboy PS (2004) Different effects of BMP-2 on marrow stromal cells from human and rat bone. Cells Tissues Organs 176:109–119
Patel ZS, Young S, Tabata Y, Jansen JA, Wong ME, Mikos AG (2008) Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model. Bone 43:931–940
Puleo DA (1997) Dependence of mesenchymal cell responses on duration of exposure to bone morphogenetic protein-2 in vitro. J Cell Physiol 173:93–101
Seamon J, Wang X, Cui F, Keller T, Dighe AS, Balian G, Cui Q (2013) Adenoviral delivery of the VEGF and BMP-6 genes to rat mesenchymal stem cells potentiates osteogenesis. Bone Marrow Res 2013:737580
Street J, Lenehan B (2009) Vascular endothelial growth factor regulates osteoblast survival—evidence for an autocrine feedback mechanism. J Orthop Surg Res 4:1
Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, van Bruggen N, Redmond HP, Carano RA, Filvaroff EH (2002) Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A 99:9656–9661
Wang XX, Allen RJ Jr, Tutela JP, Sailon A, Allori AC, Davidson EH, Paek GK, Saadeh PB, McCarthy JG, Warren SM (2011a) Progenitor cell mobilization enhances bone healing by means of improved neovascularization and osteogenesis. Plast Reconstr Surg 128:395–405
Wang X, Cui F, Madhu V, Dighe AS, Balian G, Cui Q (2011b) Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation. Growth Factors 29:36–48
Wolff KD, Ervens J, Herzog K, Hoffmeister B (1996) Experience with the osteocutaneous fibula flap: an analysis of 24 consecutive reconstructions of composite mandibular defects. J Craniomaxillofac Surg 24:330–338
Young S, Patel ZS, Kretlow JD, Murphy MB, Mountziaris PM, Baggett LS, Ueda H, Tabata Y, Jansen JA, Wong M, Mikos AG (2009) Dose effect of dual delivery of vascular endothelial growth factor and bone morphogenetic protein-2 on bone regeneration in a rat critical-size defect model. Tissue Eng Part A 15:2347–2362
Zaidi N, Nixon AJ (2007) Stem cell therapy in bone repair and regeneration. Ann N Y Acad Sci 1117:62–72
Zhang Y, Madhu V, Dighe AS, Irvine JN Jr, Cui Q (2012) Osteogenic response of human adipose-derived stem cells to BMP-6, VEGF, and combined VEGF plus BMP-6 in vitro. Growth Factors 30:333–343
Acknowledgments
We thank Dr. Vedavathi Madhu, Orthopaedic Surgery Research Center, University of Virginia, Charlottesville, USA and Mr. Weitao Wang, School of Medicine, University of Virginia, Charlottesville, USA for their help in the standardization of the EDC-NHS-MES cross-linking method in our group. The research is supported by Orthopaedic Research and Education Foundation/Zachary B. Friedenberg Clinician Scientist Award and by Orthopaedic Research Fund, University of Virginia.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Das, A., Fishero, B.A., Christophel, J.J. et al. Poly(lactic-co-glycolide) polymer constructs cross-linked with human BMP-6 and VEGF protein significantly enhance rat mandible defect repair. Cell Tissue Res 364, 125–135 (2016). https://doi.org/10.1007/s00441-015-2301-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-015-2301-x