Bone mesenchymal stem cells co‑expressing VEGF and BMP‑6 genes to combat avascular necrosis of the femoral head

Liao,Hongxing; Zhong,Zhixiong; Liu,Zhanliang; Li,Liangping; Ling,Zemin; Zou,Xuenong

doi:10.3892/etm.2017.5455

Bone mesenchymal stem cells co‑expressing VEGF and BMP‑6 genes to combat avascular necrosis of the femoral head

Authors:
- Hongxing Liao
- Zhixiong Zhong
- Zhanliang Liu
- Liangping Li
- Zemin Ling
- Xuenong Zou
View Affiliations

Affiliations: Department of Orthopedics, Meizhou People's Hospital, Meizhou, Guangdong 514000, P.R. China, Department of Cardiovascular Medicine, Meizhou People's Hospital, Meizhou, Guangdong 514000, P.R. China, Guangdong Provincial Key Laboratory of Orthopedics and Traumatology, The First Affiliated Hospital of Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China
Published online on: November 7, 2017 https://doi.org/10.3892/etm.2017.5455
Pages: 954-962
Copyright: © Liao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The aim of the present study was to investigate the potential of bone mesenchymal stem cells (BMSCs) treated with a combination of vascular endothelial growth factor (VEGF) and bone morphogenetic protein‑6 (BMP‑6) genes for the treatment of avascular necrosis of the femoral head (ANFH). Rat BMSCs were isolated and purified using a density gradient centrifugation method. The purity and characteristics of the BMSCs were detected by cell surface antigens identification using flow cytometry. The experimental groups were administered with one of the following adeno‑associated virus (AAV) vector constructs: AAV‑green fluorescent protein (AAV‑GFP), AAV‑BMP‑6, AAV‑VEGF or AAV‑VEGF‑BMP‑6. The expression of VEGF and BMP‑6 was detected by reverse transcription‑quantitative polymerase chain reaction, western blotting and ELISA assays. The effects of VEGF and BMP‑6 on BMSCs were evaluated by angiogenic and osteogenic assays. The transfected BMSCs were combined with a biomimetic synthetic scaffold poly lactide‑co‑glycolide (PLAGA) and they were then subcutaneously implanted into nude mice. After four weeks, the implants were analyzed with histology and subsequent immunostaining to evaluate the effects of BMSCs on blood vessel and bone formation in vivo. In the AAV‑VEGF‑BMP‑6 group, the expression levels of VEGF and BMP‑6 were significantly increased and human umbilical vein endothelial cells tube formation was significantly enhanced compared with other groups. Capillaries and bone formation in the AAV‑VEGF‑BMP‑6 group was significantly higher compared with the other groups. The results of the present study suggest that BMSCs expressing both VEGF and BMP‑6 induce an increase in blood vessels and bone formation, which provides theoretical support for ANFH gene therapy.

View Figures

View References

1	Chamberlain JR, Schwarze U, Wang PR, Hirata RK, Hankenson KD, Pace JM, Underwood RA, Song KM, Sussman M, Byers PH and Russell DW: Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science. 303:1198–1201. 2004. View Article : Google Scholar : PubMed/NCBI
2	Yang C, Yang S, Du J, Li J, Xu W and Xiong Y: Experimental study of vascular endothelial growth factor gene therapy for avascular necrosis of the femoral head. J Huazhong Univ Sci Technolog Med Sci. 23:297–299, 316. 2003. View Article : Google Scholar : PubMed/NCBI
3	Wilson CG, Martin-Saavedra FM, Vilaboa N and Franceschi RT: Advanced BMP gene therapies for temporal and spatial control of bone regeneration. J Dent Res. 92:409–417. 2013. View Article : Google Scholar : PubMed/NCBI
4	Kuh SU, Zhu Y, Li J, Tsai KJ, Fei Q, Hutton WC and Yoon ST: Can TGF-beta1 and rhBMP-2 act in synergy to transform bone marrow stem cells to discogenic-type cells? Acta Neurochir (Wien). 150:1073–1079. 2008. View Article : Google Scholar : PubMed/NCBI
5	Evans CH and Huard J: Gene therapy approaches to regenerating the musculoskeletal system. Nat Rev Rheumatol. 11:234–242. 2015. View Article : Google Scholar : PubMed/NCBI
6	Herberg S, Susin C, Pelaez M, Howie RN, Moreno de Freitas R, Lee J, Cray JJ Jr, Johnson MH, Elsalanty ME, Hamrick MW, et al: Low-dose bone morphogenetic protein-2/stromal cell-derived factor-1β cotherapy induces bone regeneration in critical-size rat calvarial defects. Tissue Eng Part A. 20:1444–1453. 2014. View Article : Google Scholar : PubMed/NCBI
7	White AP, Vaccaro AR, Hall JA, Whang PG, Friel BC and McKee MD: Clinical applications of BMP-7/OP-1 in fractures, nonunions and spinal fusion. Int Orthop. 31:735–741. 2007. View Article : Google Scholar : PubMed/NCBI
8	La WG, Jin M, Park S, Yoon HH, Jeong GJ, Bhang SH, Park H, Char K and Kim BS: Delivery of bone morphogenetic protein-2 and substance P using graphene oxide for bone regeneration. Int J Nanomed. 9 Suppl 1:S107–S116. 2014.
9	Wang YJ, Chen S, Deng C, Li F, Wang Y, Hu X, Shi F and Dong N: MicroRNA-204 Targets Runx2 to Attenuate BMP-2-induced osteoblast differentiation of human aortic valve interstitial cells. J Cardiovasc Pharmacol. 66:63–71. 2015. View Article : Google Scholar : PubMed/NCBI
10	Mundy C, Gannon M and Popoff SN: Connective tissue growth factor (CTGF/CCN2) negatively regulates BMP-2 induced osteoblast differentiation and signaling. J Cell Physiol. 229:672–681. 2014. View Article : Google Scholar : PubMed/NCBI
11	Hanada K, Solchaga LA, Caplan AI, Hering TM, Goldberg VM, Yoo JU and Johnstone B: BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis. J Cell Biochem. 81:284–294. 2001. View Article : Google Scholar : PubMed/NCBI
12	Wang L, Park P, La Marca F, Than KD and Lin CY: BMP-2 inhibits tumor-initiating ability in human renal cancer stem cells and induces bone formation. J Cancer Res Clin Oncol. 141:1013–1024. 2015. View Article : Google Scholar : PubMed/NCBI
13	Dohin B, Dahan-Oliel N, Fassier F and Hamdy R: Enhancement of difficult nonunion in children with osteogenic protein-1 (OP-1): Early experience. Clin Orthop Relat Res. 467:3230–3238. 2009. View Article : Google Scholar : PubMed/NCBI
14	Takahashi T, Hatakeyama S and Machida T: Ductal adenocarcinoma of the pancreas with psammomatous calcification: Report of a case with immunohistochemical study for bone morphogenetic protein. Pathol Int. 61:603–607. 2011. View Article : Google Scholar : PubMed/NCBI
15	Rahman MS, Akhtar N, Jamil HM, Banik RS and Asaduzzaman SM: TGF-β/BMP signaling and other molecular events: Regulation of osteoblastogenesis and bone formation. Bone Res. 3:150052015. View Article : Google Scholar : PubMed/NCBI
16	Scharpfenecker M, van Dinther M, Liu Z, van Bezooijen RL, Zhao Q, Pukac L, Löwik CW and Ten Dijke P: BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis. J Cell Sci. 120:964–972. 2007. View Article : Google Scholar : PubMed/NCBI
17	Simic P, Culej JB, Orlic I, Grgurevic L, Draca N, Spaventi R and Vukicevic S: Systemically administered bone morphogenetic protein-6 restores bone in aged ovariectomized rats by increasing bone formation and suppressing bone resorption. J Biol Chem. 281:25509–25521. 2006. View Article : Google Scholar : PubMed/NCBI
18	Mizrahi O, Sheyn D, Tawackoli W, Kallai I, Oh A, Su S, Da X, Zarrini P, Cook-Wiens G, Gazit D and Gazit Z: BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells. Gene Ther. 20:370–377. 2013. View Article : Google Scholar : PubMed/NCBI
19	Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, et al: Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA. 99:pp. 9656–9661. 2002; View Article : Google Scholar : PubMed/NCBI
20	Moens S, Goveia J, Stapor PC, Cantelmo AR and Carmeliet P: The multifaceted activity of VEGF in angiogenesis-Implications for therapy responses. Cytokine Growth Factor Rev. 25:473–482. 2014. View Article : Google Scholar : PubMed/NCBI
21	Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z and Ferrara N: VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med. 5:623–628. 1999. View Article : Google Scholar : PubMed/NCBI
22	Ferrara N: VEGF-A: A critical regulator of blood vessel growth. Eur Cytokine Netw. 20:158–163. 2009.PubMed/NCBI
23	Joensuu K, Uusitalo L, Alm JJ, Aro HT, Hentunen TA and Heino TJ: Enhanced osteoblastic differentiation and bone formation in co-culture of human bone marrow mesenchymal stromal cells and peripheral blood mononuclear cells with exogenous VEGF. Orthop Traumatol Surg Res. 101:381–386. 2015. View Article : Google Scholar : PubMed/NCBI
24	Ramazanoglu M, Lutz R, Rusche P, Trabzon L, Kose GT, Prechtl C and Schlegel KA: Bone response to biomimetic implants delivering BMP-2 and VEGF: An immunohistochemical study. J Craniomaxillofac Surg. 41:826–835. 2013. View Article : Google Scholar : PubMed/NCBI
25	Zhang W, Zhu C, Wu Y, Ye D, Wang S, Zou D, Zhang X, Kaplan DL and Jiang X: VEGF and BMP-2 promote bone regeneration by facilitating bone marrow stem cell homing and differentiation. Eur Cells Mater. 27:1–12. 2014. View Article : Google Scholar
26	Kempen DH, Lu L, Heijink A, Hefferan TE, Creemers LB, Maran A, Yaszemski MJ and Dhert WJ: Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration. Biomaterials. 30:2816–2825. 2009. View Article : Google Scholar : PubMed/NCBI
27	Peng H, Usas A, Olshanski A, Ho AM, Gearhart B, Cooper GM and Huard J: VEGF improves, whereas sFlt1 inhibits, BMP2-induced bone formation and bone healing through modulation of angiogenesis. J Bone Miner Res. 20:2017–2027. 2005. View Article : Google Scholar : PubMed/NCBI
28	Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 284:143–147. 1999. View Article : Google Scholar : PubMed/NCBI
29	Büning H, Perabo L, Coutelle O, Quadt-Humme S and Hallek M: Recent developments in adeno-associated virus vector technology. J Gene Med. 10:717–733. 2008. View Article : Google Scholar : PubMed/NCBI
30	Shi ZB and Wang KZ: Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: A preliminary study in vitro. Tissue Cell. 42:314–321. 2010. View Article : Google Scholar : PubMed/NCBI
31	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2012. View Article : Google Scholar
32	Xin CW, Zheng LW, Lei LC, Ma L, Ehrbar M, Weber FE and Zwahlen RA: Effect of different rhBMP-2 and TG-VEGF ratios on the formation of heterotopic bone and neovessels. Biomed Res Int. 2014:5715102014.PubMed/NCBI
33	Ali RR, Reichel MB, Thrasher AJ, Levinsky RJ, Kinnon C, Kanuga N, Hunt DM and Bhattacharya SS: Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum Mol Genet. 5:591–594. 1996. View Article : Google Scholar : PubMed/NCBI
34	Merten OW, Gény-Fiamma C and Douar AM: Current issues in adeno-associated viral vector production. Gene Ther. 12 Suppl 1:S51–S61. 2005. View Article : Google Scholar : PubMed/NCBI
35	Grieger JC, Choi VW and Samulski RJ: Production and characterization of adeno-associated viral vectors. Nat Protoc. 1:1412–1428. 2006. View Article : Google Scholar : PubMed/NCBI
36	Roedersheimer M, West J, Huffer W, Harral J and Benedict J: A bone-derived mixture of TGF beta-superfamily members forms a more mature vascular network than bFGF or TGF-beta 2 in vivo. Angiogenesis. 8:327–338. 2005. View Article : Google Scholar : PubMed/NCBI
37	Samee M, Kasugai S, Kondo H, Ohya K, Shimokawa H and Kuroda S: Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation. J Pharmacol Sci. 108:18–31. 2008. View Article : Google Scholar : PubMed/NCBI
38	Sukul M, Nguyen TB, Min YK, Lee SY and Lee BT: Effect of local sustainable release of BMP2-VEGF from Nano-cellulose loaded in sponge biphasic calcium phosphate on bone regeneration. Tissue Eng Part A. 21:1822–1836. 2015. View Article : Google Scholar : PubMed/NCBI
39	Cao QS, Zhang T and Zhang J: Correlation analysis of STAT3 and VEGF expression and eosinophil infiltration in nasal polyps. Eur Arch Otorhinolaryngol. 272:1955–1960. 2015. View Article : Google Scholar : PubMed/NCBI
40	Kanczler JM, Ginty PJ, White L, Clarke NM, Howdle SM, Shakesheff KM and Oreffo RO: The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation. Biomaterials. 31:1242–1250. 2010. View Article : Google Scholar : PubMed/NCBI
41	Jiang J, Wang SH, Chai F, Ai CC and Chen SY: The study on vascularisation and osteogenesis of BMP/VEGF co-modified tissue engineering bone in vivo. RSC Adv. 6:41800–41808. 2016. View Article : Google Scholar
42	Zhang C, Wang KZ, Qiang H, Tang YL, Li Q, Li M and Dang XQ: Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo. Acta Pharmacol Sin. 31:821–830. 2010. View Article : Google Scholar : PubMed/NCBI
43	Yung LM, Sánchez-Duffhues G, Ten Dijke P and Yu PB: Bone morphogenetic protein 6 and oxidized Low-density lipoprotein synergistically recruit osteogenic differentiation in endothelial cells. Cardiovasc Res. 108:278–287. 2015. View Article : Google Scholar : PubMed/NCBI
44	Wei Z, Salmon RM, Upton PD, Morrell NW and Li W: Regulation of bone morphogenetic protein 9 (BMP9) by redox-dependent proteolysis. J Biol Chem. 289:31150–31159. 2014. View Article : Google Scholar : PubMed/NCBI
45	Friedman MS, Long MW and Hankenson KD: Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6. J Cell Biochem. 98:538–554. 2006. View Article : Google Scholar : PubMed/NCBI
46	Cui F, Wang X, Liu X, Dighe AS, Balian G and Cui Q: VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells. Growth Factors. 28:306–317. 2010. View Article : Google Scholar : PubMed/NCBI
47	Derubeis AR and Cancedda R: Bone marrow stromal cells (BMSCs) in bone engineering: Limitations and recent advances. Ann Biomed Eng. 32:160–165. 2004. View Article : Google Scholar : PubMed/NCBI
48	Xiong J, Menicanin D, Zilm PS, Marino V, Bartold PM and Gronthos S: Investigation of the cell surface proteome of human periodontal ligament stem cells. Stem Cells Int. 2016:19471572016. View Article : Google Scholar : PubMed/NCBI
49	Hung SC, Chen NJ, Hsieh SL, Li H, Ma HL and Lo WH: Isolation and characterization of size-sieved stem cells from human bone marrow. Stem Cells. 20:249–258. 2002. View Article : Google Scholar : PubMed/NCBI
50	Bongio M, van den Beucken JJJP, Leeuwenburgh SCG and Jansen JA: Development of bone substitute materials: From ‘biocompatible’ to ‘instructive’. J Mater Chem. 20:8747–8759. 2010. View Article : Google Scholar
51	Nugroho RWN, Odelius K, Hoglund A and Albertsson AC: Highlighting the importance of surface grafting in combination with a Layer-by-Layer approach for fabricating advanced 3D Poly(L-lactide) microsphere scaffolds. Chem Mater. 28:3298–3307. 2016. View Article : Google Scholar
52	Madhu V, Li CJ, Dighe AS, Balian G and Cui Q: 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:e1030602014. View Article : Google Scholar : PubMed/NCBI
53	Fiedler J, Leucht F, Waltenberger J, Dehio C and Brenner RE: VEGF-A and PlGF-1 stimulate chemotactic migration of human mesenchymal progenitor cells. Biochem Biophys Res Commun. 334:561–568. 2005. View Article : Google Scholar : PubMed/NCBI