Cell sheets of co‑cultured BMP‑2‑modified bone marrow stromal cells and endothelial progenitor cells accelerate bone regeneration in vitro

He,Jia; Han,Xuesong; Wang,Songmei; Zhang,Ying; Dai,Xiaoming; Liu,Boyan; Liu,Liu; Zhao,Xian

doi:10.3892/etm.2019.7982

Cell sheets of co‑cultured BMP‑2‑modified bone marrow stromal cells and endothelial progenitor cells accelerate bone regeneration in vitro

Authors:
- Jia He
- Xuesong Han
- Songmei Wang
- Ying Zhang
- Xiaoming Dai
- Boyan Liu
- Liu Liu
- Xian Zhao
View Affiliations

Affiliations: Department of Plastic Surgery, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China, Department of Obstetrics and Gynecology, Kunming Medical University, Kunming, Yunnan 650031, P.R. China, School of Public Health, Kunming Medical University, Kunming, Yunnan 650031, P.R. China, Department of Cardiology, The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650101, P.R. China
Published online on: September 6, 2019 https://doi.org/10.3892/etm.2019.7982
Pages: 3333-3340
Copyright: © He 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

Bone tissue engineering provides a substitute for bone transplantation to address various bone defects. However, bone regeneration involves a large number of cellular events. In addition, obtaining sufficient source material for autogenous bone or alloplastic bone substitutes remains an unsolved issue. In previous studies, it was confirmed that bone marrow stromal cells (BMSCs) and endothelial progenitor cells (EPCs) had the capacity to promote bone regeneration. Additionally, bone morphogenetic protein‑2 (BMP‑2) has been demonstrated to be an active inducer of osteoblast differentiation. Therefore, the aim of the present study was to produce an effective integration system, including a scaffold, reparative cells and growth factors, that may enhance bone regeneration. Firstly, bone marrow‑derived BMSCs and EPCs were isolated and identified by flow cytometry. Cell proliferation ability, secreted BMP‑2 levels and alkaline phosphatase (ALP) activity were highest in the cell sheets containing BMP‑2‑modified BMSCs and EPCs. In addition, the expression levels of osteogenesis‑associated genes, including runt related transcription factor 2 (Runx2), distal‑less homeobox 5 (Dlx5), ALP and integrin‑binding sialoprotein (Ibsp), and osteogenesis‑associated proteins, including Runx2, Dlx, ALP, Ibsp, vascular endothelial growth factor, osteonectin, osteopontin and type I collagen, gradually increased during the co‑culture of ad‑BMP‑2‑BMSCs/EPCs. The levels of these genes and proteins were increased compared with those observed in the BMSC, EPC and BMP‑2‑modified BMSC groups. Finally, scanning electron microscopy observation also demonstrated that the BMP2‑modified BMSCs were able to combine well with EPCs to construct a cell sheet for bone formation. Collectively, these results describe an adenovirus (ad)‑BMP2‑BMSCs/EPCs co‑culture system that may significantly accelerate bone regeneration compared with a BMSCs/EPCs co‑culture system or ad‑BMP2‑BMSCs alone.

View Figures

View References

1	Younger EM and Chapman MW: Morbidity at bone graft donor sites. J Orthop Trauma. 3:192–195. 1989. View Article : Google Scholar : PubMed/NCBI
2	Banwart JC, Asher MA and Hassanein RS: Iliac crest bone graft harvest donor site morbidity. A statistical evaluation. Spine (Phila Pa 1976). 20:1055–1060. 1995. View Article : Google Scholar : PubMed/NCBI
3	Wang C, Meng G, Zhang L, Xiong Z and Liu J: Physical properties and biocompatibility of a core-sheath structure composite scaffold for bone tissue engineering in vitro. J Biomed Biotechnol. 2012:5791412012. View Article : Google Scholar : PubMed/NCBI
4	Xie H, Wang Z, Zhang L, Lei Q, Zhao A, Wang H, Li Q, Chen Z and Zhang W: Development of an angiogenesis-promoting microvesicle-alginate-polycaprolactone composite graft for bone tissue engineering applications. PeerJ. 4:e20402016. View Article : Google Scholar : PubMed/NCBI
5	Wu W, Le AV, Mendez JJ, Chang J, Niklason LE and Steinbacher DM: Osteogenic performance of donor-matched human adipose and bone marrow mesenchymal cells under dynamic culture. Tissue engineering. Part A. 21:1621–1632. 2015.
6	Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, et al: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 418:41–49. 2002. View Article : Google Scholar : PubMed/NCBI
7	de Girolamo L, Lucarelli E, Alessandri G, Avanzini MA, Bernardo ME, Biagi E, Brini AT, D'Amico G, Fagioli F, Ferrero I, et al: Mesenchymal stem/stromal cells: A new ‘cells as drugs’ paradigm. Efficacy and critical aspects in cell therapy. Curr Pharm Des. 19:2459–2473. 2013. View Article : Google Scholar : PubMed/NCBI
8	Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 276:71–74. 1997. View Article : Google Scholar : PubMed/NCBI
9	Egashira K, Sumita Y, Zhong W, Ohba S, Nagai K and Asahina I: Bone marrow concentrate promotes bone regeneration with a suboptimal-dose of rhBMP-2. PLoS One. 13:e01910992018. View Article : Google Scholar : PubMed/NCBI
10	Hokugo A, Saito T, Li A, Sato K, Tabata Y and Jarrahy R: Stimulation of bone regeneration following the controlled release of water-insoluble oxysterol from biodegradable hydrogel. Biomaterials. 35:5565–5571. 2014. View Article : Google Scholar : PubMed/NCBI
11	Atesok K, Li R, Stewart DJ and Schemitsch EH: Endothelial progenitor cells promote fracture healing in a segmental bone defect model. J Orthop Res. 28:1007–1014. 2010.PubMed/NCBI
12	Shirota T, Yasui H, Shimokawa H and Matsuda T: Fabrication of endothelial progenitor cell (EPC)-seeded intravascular stent devices and in vitro endothelialization on hybrid vascular tissue. Biomaterials. 24:2295–2302. 2003. View Article : Google Scholar : PubMed/NCBI
13	Li R, Atesok K, Nauth A, Wright D, Qamirani E, Whyne CM and Schemitsch EH: Endothelial progenitor cells for fracture healing: A microcomputed tomography and biomechanical analysis. J Orthop Trauma. 25:467–471. 2011. View Article : Google Scholar : PubMed/NCBI
14	Seebach C, Henrich D, Kähling C, Wilhelm K, Tami AE, Alini M and Marzi I: Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance early vascularization and bone healing in a critical-sized bone defect in rats. Tissue Eng Part A. 16:1961–1970. 2010. View Article : Google Scholar : PubMed/NCBI
15	Carreira AC, Lojudice FH, Halcsik E, Navarro RD, Sogayar MC and Granjeiro JM: Bone morphogenetic proteins: Facts, challenges, and future perspectives. J Dent Res. 93:335–345. 2014. View Article : Google Scholar : PubMed/NCBI
16	Ho SS, Vollmer NL, Refaat MI, Jeon O, Alsberg E, Lee MA and Leach JK: Bone morphogenetic protein-2 promotes human mesenchymal stem cell survival and resultant bone formation when entrapped in photocrosslinked alginate hydrogels. Adv Healthc Mater. 5:2501–2509. 2016. View Article : Google Scholar : PubMed/NCBI
17	Carreira AC, Zambuzzi WF, Rossi MC, Astorino Filho R, Sogayar MC and Granjeiro JM: Bone morphogenetic proteins: Promising molecules for bone healing, bioengineering, and regenerative medicine. Vitam Horm. 99:293–322. 2015. View Article : Google Scholar : PubMed/NCBI
18	Garrison KR, Donell S, Ryder J, Shemilt I, Mugford M, Harvey I and Song F: Clinical effectiveness and cost-effectiveness of bone morphogenetic proteins in the non-healing of fractures and spinal fusion: A systematic review. Health Technol Assess. 11:1–150, iii-iv. 2007. View Article : Google Scholar : PubMed/NCBI
19	Bloomsmith MA, Perlman JE, Hutchinson E and Sharpless M: Behavioral management programs to promote laboratory animal welfareManagement of animal care and use programs in research, education, and testing. Weichbrod RH, Thompson GAH and Norton JN: 2nd. CRC Press/Taylor & Francis; Boca Raton, FL: 2018
20	He X, Dziak R, Yuan X, Mao K, Genco R, Swihart M, Sarkar D, Li C, Wang C, Lu L, et al: BMP2 genetically engineered MSCs and EPCs promote vascularized bone regeneration in rat critical-sized calvarial bone defects. PLoS One. 8:e604732013. View Article : Google Scholar : PubMed/NCBI
21	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. 2001. View Article : Google Scholar : PubMed/NCBI
22	Amstein CF and Hartman PA: Adaptation of plastic surfaces for tissue culture by glow discharge. J Clin Microbiol. 2:46–54. 1975.PubMed/NCBI
23	Shi S, Wang XH, Guo G, Fan M, Huang MJ and Qian ZY: Preparation and characterization of microporous poly(D,L-lactic acid) film for tissue engineering scaffold. Int J Nanomedicine. 5:1049–1055. 2010.PubMed/NCBI
24	Alghazali KM, Nima ZA, Hamzah RN, Dhar MS, Anderson DE and Biris AS: Bone-tissue engineering: Complex tunable structural and biological responses to injury, drug delivery, and cell-based therapies. Drug Metab Rev. 47:431–454. 2015. View Article : Google Scholar : PubMed/NCBI
25	An ideal cell source for bone tissue engineering? Bonekey Rep. 1:2222012.PubMed/NCBI
26	Sun J, Li J, Li C and Yu Y: Role of bone morphogenetic protein-2 in osteogenic differentiation of mesenchymal stem cells. Mol Med Rep. 12:4230–4237. 2015. View Article : Google Scholar : PubMed/NCBI
27	Smajilagić A, Aljičević M, Redžić A, Filipović S and Lagumdžija A: Rat bone marrow stem cells isolation and culture as a bone formative experimental system. Bosn J Basic Med Sci. 13:27–30. 2013. View Article : Google Scholar : PubMed/NCBI
28	Denecke B, Horsch LD, Radtke S, Fischer JC, Horn PA and Giebel B: Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering. J Tissue Eng Regen Med. 9:E84–E97. 2015. View Article : Google Scholar : PubMed/NCBI
29	Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM and Asahara T: Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA. 97:3422–3427. 2000. View Article : Google Scholar : PubMed/NCBI
30	James AW, LaChaud G, Shen J, Asatrian G, Nguyen V, Zhang X, Ting K and Soo C: A review of the clinical side effects of bone morphogenetic protein-2. Tissue Eng Part B Rev. 22:284–297. 2016. View Article : Google Scholar : PubMed/NCBI
31	Luo X, Chen J, Song WX, Tang N, Luo J, Deng ZL, Sharff KA, He G, Bi Y, He BC, et al: Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects. Lab Invest. 88:1264–1277. 2008. View Article : Google Scholar : PubMed/NCBI
32	Chen D, Zhao M and Mundy GR: Bone morphogenetic proteins. Growth Factors. 22:233–241. 2004. View Article : Google Scholar : PubMed/NCBI
33	Fan T, Chen J, Pan P, Zhang Y, Hu Y, Liu X, Shi X and Zhang Q: Bioinspired double polysaccharides-based nanohybrid scaffold for bone tissue engineering. Colloids Surf B Biointerfaces. 147:217–223. 2016. View Article : Google Scholar : PubMed/NCBI
34	Li R, Liu L, Mo Z, Wang X, Xia J, Liang Z, Zhang Y, Li Y, Mao Q, Wang J, et al: An inactivated enterovirus 71 vaccine in healthy children. N Engl J Med. 370:829–837. 2014. View Article : Google Scholar : PubMed/NCBI