Identification of mesenchymal stem cells and osteogenic factors in bone marrow aspirate and peripheral blood for spinal fusion by flow cytometry and proteomic analysis

Journal of Orthopaedic Surgery and Research

Wen-Jer Chen³  Po-Liang Lai³  Chuen-Yung Yang³  Tai-Long Pan¹  Lih-Huei Chen³  Li-Jen Yuan³  Song-Shu Lin²  Chi-Chien Niu³ 

[1] Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan;Graduate Institutes of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan;College of Medicine, Chang Gung University, Taoyuan, Taiwan

关键词: Lumbar posterolateral fusion;    Proteomics;    Flow cytometry;    Mesenchymal stem cells;    Bone marrow aspirate;   

Others  :  813433
DOI  :  10.1186/1749-799X-9-32

 received in 2014-02-07, accepted in 2014-04-21,  发布年份 2014

【 摘 要 】

Background

An in vivo animal study and a prospective clinical study have indicated that bone marrow aspirate (BMA) augments spinal arthrodesis. However, there is no quantified data to explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion.

Methods

To analyze the proportion of mesenchymal stem cells (MSCs) and osteogenic factors in human BMA and peripheral blood (PB) of the same patient. Autologous BMA and PB from the patients were analyzed by flow cytometry (FACS) using cell markers for MSCs. The osteogenic potential of MSCs was determined by alkaline phosphatase (ALP) activity and calcium level quantification. Proteomics were used for the qualitative and quantitative mapping of the whole proteome from BMA and PB plasma. The mass-to-charge ratio was calculated by time-of-flight mass spectrometry (TOF-MS). The overexpression of protein was confirmed using Western blot analysis.

Results

The proportion of MSCs (CD34⁻/CD29⁺/CD105⁺) was higher in the BMA than that in the PB. Colony-forming cell (CFC) assays suggested that fewer colonies were formed in PB cultures than in BMA culture. There was no significant difference in the osteogenic potential of the MSCs between the PB and BMA. Proteomic mass spectrometry assays suggested that the levels of catalase (osteoclast inhibitor) and glutathione peroxidase 3 (osteogenic biomarker) were higher in the BMA than those in the PB, and this was confirmed by Western blot analysis.

Conclusions

The proportions of MSCs and osteogenic factors were higher in the BMA than in the PB. This may explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion.

【 授权许可】

   
2014 Niu et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Nather A: Use of allograft in spinal surgery. Ann Transpl 1999, 4:19-22.
• [2]Gibson S, McLeod I, Wardlaw D, Urbaniak S: Allograft versus autograft in instrumented posterolateral lumbar spinal fusion: a randomized control trial. Spine 2002, 27:1599-1603.
• [3]Baramki HG, Steffen T, Lander P, Chang M, Marchesi D: The efficacy of interconnected porous hydroxyapatite in achieving posterolateral lumbar fusion in sheep. Spine 2000, 25:1053-1060.
• [4]Chen WJ, Tsai TT, Chen LH, Niu CC, Lai PL, Fu TS: The fusion rate of calcium sulfate with local autograft bone compared with autologous iliac bone graft for instrumented short-segment spinal fusion. Spine 2005, 30:2293-2297.
• [5]Louis-Ugbo J, Murakami H, Kim HS, Minamide A, Boden SD: Evidence of osteoinduction by Grafton demineralized bone matrix in nonhuman primate spinal fusion. Spine 2004, 29:360-366.
• [6]Cammisa FP Jr, Lowery G, Garfin SR, Geisler FH, Klara PM, McGuire RA, Sassard WR, Stubbs H, Block JE: Two-year fusion rate equivalency between Grft on DBM gel and autograft in posterolateral spine fusion: a prospective controlled trial employing a side-by-side comparison in the same patient. Spine 2004, 29:660-666.
• [7]Tay BK, Le AX, Heilman M, Lotz J, Bradford DS: Use of a collagen-hydroxyapatite matrix in spinal fusion: a rabbit model. Spine 1998, 23:2276-2281.
• [8]Muschler GF, Nitto H, Matsukura Y, Boehm C, Valdevit A, Kambic H, Davros W, Powell K, Easley K: Spine fusion using cell matrix composites enriched in bone marrow-derived cells. Clin Orthop 2003, 407:102-118.
• [9]Becker S, Maissen O, Igor P, Thierry S, Rahn B, Ingo W: Osteopromotion by a β-tricalcium phosphate/bone marrow hybrid implant for use in spine surgery. Spine 2006, 31:11-17.
• [10]Niu CC, Tsai TT, Fu TS, Lai PL, Chen LH, Chen WJ: A comparison of posterolateral lumbar fusion comparing autograft, autogenous laminectomy bone with bone marrow aspirate, and calcium sulphate with bone marrow aspirate. Spine 2009, 34:2715-2719.
• [11]Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craiq S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284:143-147.
• [12]Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E: Minimal criteria for defining multipotent mesenchymal stromal cells: the international society for cellular therapy position statement. Cytotherapy 2006, 8:315-317.
• [13]Ardekani AM, Liotta LA, Petricoin EF III: Clinical potential of proteomics in the diagnosis of ovarian cancer. Expert Rev Mol Diagn 2002, 2:312-320.
• [14]Anderson NL, Anderson NG: The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 2002, 1:845-867.
• [15]Li C, Tan YX, Zhou H, Ding SJ, Li SJ, Ma DJ, Man XB, Hong Y, Zhang L, Li L, Xia QC, Wu JR, Wang HY, Zeng R: Proteomic analysis of hepatitis B virus-associated hepatocellular carcinoma: identification of potential tumor markers. Proteomics 2005, 5:1125-1139.
• [16]He QY, Cheung YH, Leung SY, Yuen ST, Chu KM, Chiu JF: Diverse proteomic alterations in gastric adenocarcinoma. Proteomics 2004, 4:3276-3287.
• [17]Wu CC, Chien KY, Tsang NM, Chang KP, Hao SP, Tsao CH, Chang YS, Yu JS: Cancer cell-secreted proteomes as a basis for searching potential tumor markers: nasopharyngeal carcinoma as a model. Proteomics 2005, 5:3173-3182.
• [18]Hwang TL, Liang Y, Chien KY, Yu JS: Overexpression and elevated serum levels of phosphoglycerate kinase 1 in pancreatic ductal adenocarcinoma. Proteomics 2006, 6:2259-2272.
• [19]Cho SY, Lee EY, Lee JS, Kim HY, Park JM, Kwon MS, Park YK, Lee HJ, Kang MJ, Kim JY, Yoo JS, Park SJ, Cho JW, Kim HS, Paik YK: Efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map. Proteomics 2005, 5:3386-3396.
• [20]Steen R, Egeland T: CD34 molecule epitope distribution on cells of hematopoietic origin. Leuk Lymphoma 1998, 30:23-30.
• [21]D’Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA: Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res 1999, 14:1115-1122.
• [22]Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002, 418:41-49.
• [23]Smiler D, Soltan M, Albitar M: Toward the identification of mesenchymal stem cells in bone marrow and peripheral blood for bone regeneration. Implant Dent 2008, 17:236-247.
• [24]Connolly JF, Guse R, Tiedeman J, Dehne R: Bone marrow injection as a substitute for operative grafting of tibial nonunions. Clin Orthop 1991, 266:259-270.
• [25]Chen CT, Shih YRV, Kuo TK, Lee OK, Wei YH: Coordinated changes of mitochondrial biogenesis and antioxidant enzymes during osteogenic differentiation of human mesenchymal stem cells. Stem Cells 2008, 26:960-968.
• [26]Nihouannen DE, Barralet JE, Fong JE, Komarova SV: Ascorbic acid accelerates osteoclast formation and death. Bone 2010, 46:1336-1343.
• [27]Liu H, Bian W, Liu S, Huang K: Selenium protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation by suppressing oxidative stress and ERK signaling pathway. Biol Trace Elem Res 2012, 150:441-450.
• [28]Smiler D, Soltan M: A histomorphogenic analysis of bone grafts augmented with adult stem cells. Implant Dent 2007, 16:42-47.