6th International Virtual Congress (IYSC-2020) And Workshop. 10th International Science Congress (ISC-2020).  International E-publication: Publish Projects, Dissertation, Theses, Books, Souvenir, Conference Proceeding with ISBN.  International E-Bulletin: Information/News regarding: Academics and Research

Confluence-Associated Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cell (BMMSCs)

Author Affiliations

  • 1Department of Parasitology and Animal Diseases, Veterinary Research Division, National Research Center, Cairo, Egypt
  • 2Department of Parasitology and Animal Diseases, Veterinary Research Division, National Research Center, Cairo, Egypt
  • 3Department of Physiology, College of Veterinary Medicine, Cairo University, Giza, Egypt

Int. Res. J. Biological Sci., Volume 5, Issue (5), Pages 45-56, May,10 (2016)


In cellular therapy field, the impact of confluence degree to harvest or differentiate BMMSCs and the effect created by cell-to-cell contact remains controversial. Therefore, the impact of 20, 50, 70, 80 and 100% confluences on BMMSCs proliferation properties, ERK and p-ERK proteins expression and glucose consumption rate was studied. Confluence-associated osteogenic differentiation efficiency was identified by determining calcium deposition, alizarin red staining, Alkaline phosphatase activity and osteopontin and osteocalcin genes expression. There was a correlation between confluence% and density. Viability was declined at the lower and higher confluences. The highest CFU-F, Brd-U uptake and population doubling were obtained at 80% confluence. ERK band intensity in 100% confluent BMMSCs was lower. Bands of p-ERK were highly detectable at 70% and 80% confluences. Glucose consumption rate at 70% and 80% confluences were higher than at 20% and 100% confluences. Although higher osteogenic differentiation appeared at 80% confluence, it was also extended at 100% confluence. Osteopontin gene expressed among all confluences while osteocalcin gene was highly expressed in 70% confluence. We concluded that the optimum seeding density for maximal expansion and harvesting purposes is 80% confluence and up to 100% confluence for osteogenic differentiation to trigger the process to be more cost effective.


  1. Prockop DJ, Gregory C and Spees JL. (2003)., One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues., Proc Natl Acad Sci U S A. 100(1), 11917- 11923.
  2. Barry FP. (2003)., Biology and clinical applications of mesenchymal stem cells., Birth Defects Res C Embryo Today. 69, 250–256.
  3. Bittencourt R and Aparecida C. (2006)., Isolation of bone marrow mesenchymal stem cells., Acta ortop. bras. 14(1), 22-24.
  4. Ren J, Huan W, Katherine T, Sara C, Ping J, Luciano C, Ji F, Sergei A K, Pamela GR, Marianna S. and David FS. (2015)., Human bone marrow stromal cell confluence: effects on cell characteristics and methods of assessment., Cytotherapy. 17, 897-911.
  5. Jeong JA, Ko KM, Bae S, Jeon CJ, Koh GY and Kim H. (2007)., Genome-wide differential gene expression profiling of human bone marrow stromal cells., Stem Cells. 25, 994-1002.
  6. Bae S., Ahn J.H., Park C.W., Son H.K., Kim K.S. and Lim N.K. et al. (2009)., Gene and micro RNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts., Cell Tissue Res. 335, 565-573.
  7. Tormin A, Brune JC, Olsson E, Valcich J, Neuman U, Olofsson T, et al. (2009)., Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets., Cytotherapy. 11, 114-128.
  8. Lazarus HM, Koc ON, Devine SM, Curtin P, Maziarz RT, Holland HK, et al. (2005)., Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients., Biol. Blood Marrow Transplant. 11, 389-398. 

  9. Jones E, English A, Churchman SM, Kouroupis D, Boxall SA, Kinsey S, et al. (2010)., Large-scale extraction and characterization of CD271ţ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells., Arthritis Rheum. 62, 1944-1954.
  10. Rosova I, Dao M, Capoccia B, Link D and Nolta JA. (2008)., Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells., Stem Cells. 26, 2173-2182.
  11. Grisendi G, Anneren C, Cafarelli L, Sternieri R, Veronesi E, Cervo GL, et al. (2010)., GMP- manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion., Cytotherapy. 12, 466-477. 

  12. Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, Cazzanti F et al. (2006)., Human mesenchymal stem cells modulate B-cell functions., Blood. 107, 367-372. 

  13. Samuelsson H, Ringden O, Lonnies H and Le Blanc K. (2009)., Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells., Cytotherapy. 11, 129-136.
  14. Mankani MH, Kuznetsov SA, Marshall GW and Robey PG. (2008)., Creation of new bone by the percutaneous injection of hu- man bone marrow stromal cell and HA/TCP suspensions., Tissue Eng Part A. 14, 1949-1958. 

  15. Koc ON, Gerson SL, Cooper BW, Dyhouse SM, Haynesworth SE, Caplan AI, et al. (2000)., Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemo- therapy., J Clin Oncol. 18, 307-316.
  16. Zhukareva V, Obrocka M, Houle JD, Fischer I and Neuhuber B. (2010)., Secretion profile of human bone marrow stromal cells: donor variability and response to inflammatory stimuli., Cytokine. 50, 317-321.
  17. Wolfe M, Pochampally R, Swaney W and Reger RL. (2008)., Isolation and Culture of Bone Marrow-Derived Human Multipotent Stromal Cells (hMSCs)., Methods Mol Biol. 449, 3-25.
  18. Kinnaird T, Stabile E, Burnett MS, et al. (2004)., Marrow- derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms., Circulation Research. 94(5), 678–685.
  19. Gnecchi, M., He, H., Liang, O. D., et al. (2005)., Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells., Nature Medicine. 11(4), 367–368.
  20. Iván C, Naiara T, Jesús D, Ainhoa G, David O, Valerie L and César T. (2008)., ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential., Experimental Cell Research. 314, 1777–1788.
  21. Mandana H, Susanne K, Louise H, Michael G, Poul H, Annette E and Jens K. (2013)., Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion., Stem Cell Rev and Rep. 9, 44–58.
  22. Jean-Claude C., Renaud L. Jacques P., Philippe L. (2007)., ERK implication in cell cycle regulation., Biochimica et Biophysica Acta. 1773, 1299–1310
  23. Palaez D, Arita N and Cheung H. (2012)., Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression., Biochemical and Biophysical Research Communications. 417(4), 1286-1291.
  24. Liu J, Zhao Z, Li J, Zou L, Shuler C, Zou Y, Huang X, Li M, and Wang J. (2009)., Hydrostatic Pressures Promote Initial Osteodifferentiation with ERK 1/2 Not p38 MAPK Signaling Involved., Journal of Cellular Biochemistry. 107(2), 224-232.
  25. Lee J, Suh J, Park H, Bak E, Yoo YJ, and Cha JH. (2008)., Heparin-binding epidermal growth factor-like growth factor inhibits adipocyte differentiation at commitment and early induction stages., Differentiation. 76(5), 478-487.
  26. Roman MS, Robert FK, Mariah KH and George E. P. (2004)., ERK Signaling Pathways Regulate the Osteogenic Differentiation of Human Mesenchymal Stem Cells on Collagen I and Vitronectin., Cell Communication and Adhesion. 11, 137–153.
  27. Claes L, Recknagel S and Ignatius A. (2012)., Fracture healing under healthy and inflammatory conditions., Nat Rev Rheumatol. 8, 133-143.
  28. Chandrasekhar KS, Zhou H, Zeng P, Alge D, Li W, Finney BA, Yoder MC, Li J. (2011)., Blood vessel wall- derived endothelial colony-forming cells enhance fracture repair and bone regeneration., Calcif Tissue Int., 89, 347-357.
  29. Wen JH, Vincent LG, Fuhrmann A, Choi YS, Hribar KC, Taylor-Weiner H, Chen S, and Engler AJ. (2014)., Interplay of matrix stiffness and protein tethering in stem cell differentiation., Nature Materials, 13 (10), 979-987.
  30. Kentaro A, Yong-Ouk Y, Takayoshi Y, Chider C, Liang T, Yan J, Xiao-Dong C, Stan G and Songtao S. (2012)., Characterization of bone marrow derived mesenchymal stem cells in suspension., Stem Cell Research and Therapy. 3, 40.
  31. Cho Y, Shin J, Kim H, Gerelmaa M, Yoon H, Ryoo H, Kim D and Han J. (2014)., Comparison of the Osteogenic Potential of Titanium- and Modified Zirconia-Based Bioceramics., Int. J. Mol. Sci. 15, 4442-4452.
  32. Lilian PE, Renata BR, Isis SO, Paulo OG, Paulo P, Alice TF et al. (2009)., Comparative study of technique to obtain stem cells from bone marrow collection between the iliac crest and the femoral epiphysis in rabbits., Acta Cirúrgica Brasileira. 24(5), 400.
  33. Röntgen V, Blakytny R, Matthys R, Landauer M, Wehner T, Göckelmann M, et al. (2010)., Fracture healing in mice under controlled rigid and flexible conditions using an adjustable external fixator., J Orthop Res. 28, 1456-1462.
  34. Tahrin M and Ping-Chang Y. (2012)., Western Blot: Technique, Theory, and Trouble Shooting., N Am J Med Sci., 4(9), 429–434.
  35. Tain-Hsiung C, Wei-Ming C, Ke-Hsun H, Cheng-Deng K and Shih-Chieh H. (2007)., Sodium butyrate activates ERK to regulate differentiation of mesenchymal stem cells., Biochemical and Biophysical Research Communications. 355, 913–918.
  36. Waters, WR, Palmer MV, Whipple DL, Carlson MP and Nonnecke BJ. (2003)., Diagnostic implications of antigen-induced gamma interferon, nitric oxide and tumor necrosis factor alpha production by peripheral blood mononuclear cells from mycobacterium bovis-infected cattle., Clinical and Diagnostic Laboratory Immunology. 10, 960-966.
  37. Arash Z, Iraj RK, Mohammad B, Azim H, Reza M and Ahmadreza FN. (2008)., Osteogenic Differentiation of Rat Mesenchymal Stem Cells from Adipose Tissue in Comparison with Bone Marrow Mesenchymal. Stem Cells: Melatonin as a Differentiation Factor., Iranian Biomedical J. 12(3), 133-141.
  38. Gregory CA, Gunn WG, Peister A and Prockop DJ. (2004)., An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction., Anal. Biochem. 329, 77-84. 

  39. Salasznyk RM, Klees RF, Hughlock MK, Plopper GE. (2004)., ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells on collagen I and vitronectin., Cell Commun Adhes. 11, 137–153
  40. Liu BS, Yao CH, Chen YS, and Hsu SH. (2003)., In vitro evaluation of degradation and cytotoxicity of a novel composite as a bone substitute., Journal of Biomedical Materials Research. 67(4), 1163–1169.
  41. Colter D, Class R and DiGirolamo C et al. (2000)., Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow., Proc Natl Acad Sci U S A. 97, 3213–3218.
  42. Sekiya I, Larson BL and Smith JR et al. (2002)., Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality., Stem Cells. 20, 530–541.
  43. Jiang Y, Jahagirdar BN and Reinhardt RL et al. (2002)., Pluripotency of mesenchymal stem cells derived from adult marrow., Nature, 418, 41.
  44. Reyes and M Verfaillie CM. (2001)., Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells., Ann N Y Acad Sci. 938, 231–233.
  45. Song I., Arnold I.C. and James E.D. (2009)., Dexamethasone Inhibition of Confluence-Induced Apoptosis in Human Mesenchymal Stem Cells., Inc. J Orthop Res. 27, 216–221.
  46. Neuhuber B., Sharon A. S., Linda H., Alastair M., and Itzhak F. (2008)., Effects of Plating Density and Culture Time On Bone Marrow Stromal Cell Characteristics., Exp Hematol. 36 (9): 1176–1185.
  47. Gregory CA, Ylostalo J, Prockop DJ. (2005)., Adult bone marrow stem/progenitor cells (MSCs) are preconditioned by microenvironmental “niches” in culture: a two-stage hypothesis for regulation of MSC fate., Sci STKE, 294, 37.
  48. Both SK, van derMuijsenberg AJC, van Blitterswijk CA, de Boer J. and de Bruijn JDA. (2007)., Rapid and efficient method for expansion of human mesenchymal stem cells., Tissue Engineering. 13(1), 3–9.
  49. Lode A, Bernhardt A, and Gelinsky M. (2008)., Cultivation of human bone marrow stromal cells on three-dimensional scaffolds of mineralized collagen: influence of seeding density on colonization, proliferation and osteogenic differentiation., Journal of Tissue Engineering and Regenerative Medicine. 2(7), 400–407.
  50. Fossett E and Khan WS. (2012)., Optimising HumanMesenchymal Stem Cell Numbers for Clinical Application: A Literature Review., Stem Cells International. Article ID 465259.
  51. Bartmann C, Rohde E, Schallmoser K et al. (2007)., Two steps to functional mesenchymal stromal cells for clinical application Transfusion., 47(8), 1426–1435.
  52. Mochizuki T, Muneta T, Sakaguchi Y et al. (2006)., Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: distinguishing properties of mesenchymal stem cells in humans., Arthritis and Rheumatism. 54(3), 843–853.
  53. Haack-Sorensen M, Susanne KH, Louise H, Michael G, Poul H, Annette E and Jens K. (2013)., Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion., Stem Cell Rev and Rep. 9, 44–58
  54. Gnecchi M, He H and Liang OD. et al. (2005)., Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells., Nature Medicine. 11(4), 367–368.
  55. Lu H, Guo L, Wozniak MJ, Kawazoe N, Tateishi T and Zhang X et al. (2009)., Effect of cell density on adipogenic differentiation of mesenchymal stem cells., Biochem. Biophys. Res. Commun. 381, 322-327.
  56. Nakahara H, Goldberg VM and Caplan AI. (1991)., Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo., J. Orthop. Res. 9, 465-476.
  57. Seghatoleslami MR and Tuan RS. (2002)., Cell density dependent regulation of AP-1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells., J. Cell. Biochem. 84, 237-248.
  58. Kilian KA, Bugarija B, Lahn BT and Mrksich M. (2010)., Geometric cues for directing the differentiation of mesenchymal stem cells., Proc. Natl. Acad. Sci. USA. 107, 4872-4877.
  59. Gao L, McBeath R and Chen CS. (2010)., Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin., Stem Cells. 28, 564-572.
  60. Sotiropoulou P, Perez S, Salagianni M et al. (2006)., Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells., Stem Cells. 24, 462–471.
  61. Kuznetsov SA, Mankani MH, Robey PG. (2000)., Effect of serum on human bone marrow stromal cells: ex vivo expansion and in vivo bone formation., Transplantation. 70, 1780-1787.
  62. Balint R, Stephen MR and Sarah HC. (2015)., Low-density subculture: a technical note on the importance of avoiding cell-to-cell contact during mesenchymal stromal cell expansion., J Tissue Eng Regen Med., 9, 1200–1203.