Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation 
in rabbit bone marrow-derived mesenchymal stem cells

Liu,Ping; Sun,Liang; Chen,Hui; Sun,Shui; Zhou,Dongsheng; Pang,Bo; Wang,Jian

doi:10.3892/or.2015.4241

Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells

Authors:
- Ping Liu
- Liang Sun
- Hui Chen
- Shui Sun
- Dongsheng Zhou
- Bo Pang
- Jian Wang
View Affiliations

Affiliations: Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China, Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China, Department of Orthopaedics, Chinese People's Liberation Army General Hospital, Beijing 100853, P.R. China, Department of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250012, P.R. China, Department of Neurosurgery, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China
Published online on: September 1, 2015 https://doi.org/10.3892/or.2015.4241
Pages: 2618-2626

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 provide a theoretical and experimental foundation on the differentiation of stem cells through the induction of multiple genes. The lentiviral vector carrying TGF-β1 and IL-10 genes was transfected to bone marrow-derived mesenchymal stem cells (BMSCs) which differentiated into chondrogenesis. Healthy New Zealand white rabbits, 2-3 months of age were used in the present study. A 6-8 ml of bone marrow was isolated from the iliac and tibial shaft of each rabbit. The BMSCs suspension was aspired following centrifugation of the bone marrow by percoll separating medium. The BMSCs were primarily cultured and subcultured in vitro, then divided into four groups according to the difference of lentivirus vectors: group A, receiving transforming growth factor β1 (TGF‑β1); group B, receiving TGF-β1 and Interleukin-10 (IL-10); group C, empty vector transfection; and group D, receiving no cell growth factor. Fluorescence expression was detected 12 h after transfecting the lentiviral vector carrying the TGF-β1 and IL-10 gene to BMSCs. The transfection efficiency was approximately 70% with a MOI=100 after 96 h. Expression of SOX-9 aggrecan and Type Ⅱ collagen in groups A-E on day 7 and 14 was detected by RT-PCR and western blot analysis. The expression level of three genes expressed in groups A and C were higher compared to the expression in groups B, D and E. The expression level of the three genes expressed in group B was higher compared to the expression in group D. The expression level of three genes expressed in group A and C showed no statistical difference. Cytokines therefore play an important role in cell proliferation and chondrogenic differentiation. TGF-β1 has a synergistic effect in the differentiation. In addition, IL-10 may have a protective role in the restoration of cartilaginous tissue.

View Figures

View References

1	Centers for Disease Control and Prevention (CDC): Prevalence of doctor - diagnosed arthritis and arthritis - attributable activity limitation - United States, 2007–2009. MMWR Morb Mortal Wkly Rep. 59:1261–1265. 2010.
2	Buckwalter JA and Mankin HJ: Articular cartilage repair and transplantation. Arthritis Rheum. 41:1331–1342. 1998. View Article : Google Scholar : PubMed/NCBI
3	van den Berg WB, van der Kraan PM, Scharstuhl A and van Beuningen HM: Growth factors and cartilage repair. Clin Orthop Relat Res. 391(Suppl): S244–S250. 2001. View Article : Google Scholar : PubMed/NCBI
4	Fiorentino DF, Bond MW and Mosmann TR: Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med. 170:2081–2095. 1989. View Article : Google Scholar : PubMed/NCBI
5	Moore KW, de Waal Malefyt R, Coffman RL and O'Garra A: Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 19:683–765. 2001. View Article : Google Scholar : PubMed/NCBI
6	Moroguchi A, Ishimura K, Okano K, Wakabayashi H, Maeba T and Maeta H: Interleukin-10 suppresses proliferation and remodeling of extracellular matrix of cultured human skin fibroblasts. Eur Surg Res. 36:39–44. 2004. View Article : Google Scholar : PubMed/NCBI
7	Reitamo S, Remitz A, Tamai K and Uitto J: Interleukin-10 modulates type I collagen and matrix metalloprotease gene expression in cultured human skin fibroblasts. J Clin Invest. 94:2489–2492. 1994. View Article : Google Scholar : PubMed/NCBI
8	Yamamoto T, Eckes B and Krieg T: Effect of interleukin-10 on the gene expression of type I collagen, fibronectin, and decorin in human skin fibroblasts: Differential regulation by transforming growth factor-beta and monocyte chemoattractant protein-1. Biochem Biophys Res Commun. 281:200–205. 2001. View Article : Google Scholar : PubMed/NCBI
9	Virk MS, Conduah A, Park SH, Liu N, Sugiyama O, Cuomo A, Kang C and Lieberman JR: Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone. 42:921–931. 2008. View Article : Google Scholar : PubMed/NCBI
10	Naldini L, Blömer U, Gage FH, Trono D and Verma IM: Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA. 93:11382–11388. 1996. View Article : Google Scholar : PubMed/NCBI
11	Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM and Trono D: In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 272:263–267. 1996. View Article : Google Scholar : PubMed/NCBI
12	Manilla P, Rebello T, Afable C, Lu X, Slepushkin V, Humeau LM, Schonely K, Ni Y, Binder GK, Levine BL, et al: Regulatory considerations for novel gene therapy products: A review of the process leading to the first clinical lentiviral vector. Hum Gene Ther. 16:17–25. 2005. View Article : Google Scholar : PubMed/NCBI
13	Sugiyama O, An DS, Kung SP, Feeley BT, Gamradt S, Liu NQ, Chen IS and Lieberman JR: Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo. Mol Ther. 11:390–398. 2005. View Article : Google Scholar : PubMed/NCBI
14	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
15	Baltzer AW and Lieberman JR: Regional gene therapy to enhance bone repair. Gene Ther. 11:344–350. 2004. View Article : Google Scholar : PubMed/NCBI
16	Lieberman JR: Orthopaedic gene therapy. Fracture healing and other nongenetic problems of bone. Clin Orthop Relat Res. (Suppl): S156–S158. 2000. View Article : Google Scholar : PubMed/NCBI
17	Lieberman JR, Ghivizzani SC and Evans CH: Gene transfer approaches to the healing of bone and cartilage. Mol Ther. 6:141–147. 2002. View Article : Google Scholar : PubMed/NCBI
18	Gamradt SC and Lieberman JR: Genetic modification of stem cells to enhance bone repair. Ann Biomed Eng. 32:136–147. 2004. View Article : Google Scholar : PubMed/NCBI
19	Hannallah D, Peterson B, Lieberman JR, Fu FH and Huard J: Gene therapy in orthopaedic surgery. Instr Course Lect. 52:753–768. 2003.PubMed/NCBI
20	Jones AL, Bucholz RW, Bosse MJ, Mirza SK, Lyon TR, Webb LX, Pollak AN, Golden JD and Valentin-Opran A; BMP-2 Evaluation in Surgery for Tibial Trauma-Allgraft (BESTT-ALL) Study Group: Recombinant human BMP-2 and allograft compared with autogenous bone graft for reconstruction of diaphyseal tibial fractures with cortical defects. A randomized, controlled trial. J Bone Joint Surg Am. 88:1431–1441. 2006. View Article : Google Scholar : PubMed/NCBI
21	Vaccaro AR, Whang PG, Patel T, Phillips FM, Anderson DG, Albert TJ, Hilibrand AS, Brower RS, Kurd MF, Appannagari A, et al: The safety and efficacy of OP-1 (rhBMP-7) as a replacement for iliac crest autograft for posterolateral lumbar arthrodesis: Minimum 4-year follow-up of a pilot study. Spine J. 8:457–465. 2008. View Article : Google Scholar
22	Swiontkowski MF, Aro HT, Donell S, Esterhai JL, Goulet J, Jones A, Kregor PJ, Nordsletten L, Paiement G and Patel A: Recombinant human bone morphogenetic protein-2 in open tibial fractures. A subgroup analysis of data combined from two prospective randomized studies. J Bone Joint Surg Am. 88:1258–1265. 2006. View Article : Google Scholar : PubMed/NCBI
23	Blanco Garcia FJ: Catabolic events in osteoarthritic cartilage. Osteoarthritis Cartilage. 7:308–309. 1999. View Article : Google Scholar : PubMed/NCBI
24	Fernandes JC, Martel-Pelletier J and Pelletier JP: The role of cytokines in osteoarthritis pathophysiology. Biorheology. 39:237–246. 2002.PubMed/NCBI
25	Goldring SR and Goldring MB: The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res. 427(Suppl): S27–S36. 2004. View Article : Google Scholar : PubMed/NCBI
26	D'Lima D, Hermida J, Hashimoto S, Colwell C and Lotz M: Caspase inhibitors reduce severity of cartilage lesions in experimental osteoarthritis. Arthritis Rheum. 54:1814–1821. 2006. View Article : Google Scholar : PubMed/NCBI
27	Blanco FJ, López-Armada MJ and Maneiro E: Mitochondrial dysfunction in osteoarthritis. Mitochondrion. 4:715–728. 2004. View Article : Google Scholar
28	Iannone F, De Bari C, Dell'Accio F, Covelli M, Cantatore FP, Patella V, Lo Bianco G and Lapadula G: Interleukin-10 and interleukin-10 receptor in human osteoarthritic and healthy chondrocytes. Clin Exp Rheumatol. 19:139–145. 2001.PubMed/NCBI
29	Dayer JM: Cytokines and anti-cytokines in inflammatory rheumatism. Rev Rhum Ed Fr. 61:173S–180S. 1994.In French.
30	Isomäki P and Punnonen J: Pro- and anti-inflammatory cytokines in rheumatoid arthritis. Ann Med. 29:499–507. 1997. View Article : Google Scholar
31	Jorgensen C, Apparailly F, Couret I, Canovas F, Jacquet C and Sany J: Interleukin-4 and interleukin-10 are chondroprotective and decrease mononuclear cell recruitment in human rheumatoid synovium in vivo. Immunology. 93:518–523. 1998. View Article : Google Scholar : PubMed/NCBI
32	Gonzalez-Rey E, Chorny A, Del Moral RG, Varela N and Delgado M: Therapeutic effect of cortistatin on experimental arthritis by downregulating inflammatory and Th1 responses. Ann Rheum Dis. 66:582–588. 2007. View Article : Google Scholar
33	Zhou JH, Broussard SR, Strle K, Freund GG, Johnson RW, Dantzer R and Kelley KW: IL-10 inhibits apoptosis of promyeloid cells by activating insulin receptor substrate-2 and phosphatidylinositol 3′-kinase. J Immunol. 167:4436–4442. 2001. View Article : Google Scholar : PubMed/NCBI
34	Bouton LA, Ramirez CD, Bailey DP, Yeatman CF, Yue J, Wright HV, Domen J, Rosato RR, Grant S, Fischer-Stenger K, et al: Costimulation with interleukin-4 and interleukin-10 induces mast cell apoptosis and cell-cycle arrest: The role of p53 and the mitochondrion. Exp Hematol. 32:1137–1145. 2004. View Article : Google Scholar : PubMed/NCBI
35	Rojas M, Olivier M, Gros P, Barrera LF and García LF: TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages. J Immunol. 162:6122–6131. 1999.PubMed/NCBI
36	Bharhani MS, Borojevic R, Basak S, Ho E, Zhou P and Croitoru K: IL-10 protects mouse intestinal epithelial cells from Fas-induced apoptosis via modulating Fas expression and altering caspase-8 and FLIP expression. Am J Physiol Gastrointest Liver Physiol. 291:G820–G829. 2006. View Article : Google Scholar : PubMed/NCBI
37	Wang Y and Lou S: Direct protective effect of interleukin-10 on articular chondrocytes in vitro. Chin Med J (Engl). 114:723–725. 2001.
38	Fischer BA, Mundle S and Cole AA: Tumor necrosis factor-alpha induced DNA cleavage in human articular chondrocytes may involve multiple endonucleolytic activities during apoptosis. Microsc Res Tech. 50:236–242. 2000. View Article : Google Scholar : PubMed/NCBI
39	Aizawa T, Kon T, Einhorn TA and Gerstenfeld LC: Induction of apoptosis in chondrocytes by tumor necrosis factor-alpha. J Orthop Res. 19:785–796. 2001. View Article : Google Scholar : PubMed/NCBI
40	Aigner T and Kim HA: Apoptosis and cellular vitality: Issues in osteoarthritic cartilage degeneration. Arthritis Rheum. 46:1986–1996. 2002. View Article : Google Scholar : PubMed/NCBI
41	Schuerwegh AJ, Dombrecht EJ, Stevens WJ, Van Offel JF, Bridts CH and De Clerck LS: Influence of pro-inflammatory (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and anti-inflammatory (IL-4) cytokines on chondrocyte function. Osteoarthritis Cartilage. 11:681–687. 2003. View Article : Google Scholar : PubMed/NCBI
42	Heyninck K and Beyaert R: Crosstalk between NF-kappaB-activating and apoptosis-inducing proteins of the TNF-receptor complex. Mol Cell Biol Res Commun. 4:259–265. 2001. View Article : Google Scholar : PubMed/NCBI
43	Kühn K, Hashimoto S and Lotz M: IL-1 beta protects human chondrocytes from CD95-induced apoptosis. J Immunol. 164:2233–2239. 2000. View Article : Google Scholar : PubMed/NCBI