International Journal of Hematology-Oncology and Stem Cell Research 2017. 11(2):121-132.

The Impact of Confluence on Bone Marrow Mesenchymal Stem (BMMSC) Proliferation and Osteogenic Differentiation
Faten Abo-Aziza Mohamed Abo-Aziza, Abd El-Kader Ahmed Zaki


Background: In the field of cellular therapy, the impact of confluence degree on harvesting or differentiation of BMMSCs and the effect of cell-to-cell contact remain controversial. Therefore, the effect of confluence on properties of BMMSCs was studied and efficiency of confluence-associated osteogenic differentiation was identified.

Materials and Methods: The impact of 20, 50, 70, 80 and 100% confluences on proliferation properties of BMMSCs, expression of ERK and p-ERK proteins and glucose consumption rate was studied. Efficiency of confluence-associated osteogenic differentiation was identified by determining calcium deposition, Alizarin Red staining, ALP activity and expression of osteopontin and osteocalcin genes.

Results: There was a correlation between confluence % and BMMSCs 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 compared to other confluences. Bands of p-ERK were highly detectable in 70% and 80% confluences. Glucose consumption rate of 70% and 80% confluences in the last days were higher than 20% and 100% confluences. Although higher osteogenic differentiation was estimated at 80% confluence using calcium deposition, Alizarin Red staining and ALP activity, it was also extended at 100% confluence Osteopontin gene was expressed among all confluences including 100% confluence, while osteocalcin gene was expressed highly in 70% confluent cells.

Conclusion: We concluded that the optimum seeding density for maximal expansion and harvesting purposes is 80% confluence and for osteogenic differentiation up to 100% confluence is also acceptable.


BMMSCS; Confluence; Osteogenic Differentiation; Proliferation

Full Text:



Prockop DJ, Gregory C and Spees JL. One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci U S A. 2003; 100 (1): 11917- 11923.

Barry FP. Biology and clinical applications of mesenchymal stem cells. Birth Defects Res C Embryo Today. 2003; 69(3): 250–6.

Bittencourt R, Aparecida C. Isolation of bone marrow mesenchymal stem cells. Acta Ortop Bras. 2006; 14 (1): 22-24.

Ren J, Huan W, Katherine T, et al. Human bone marrow stromal cell confluence: effects on cell characteristics and methods of assessment. Cytotherapy. 2015; 17(7): 897-911.

Jeong JA, Ko KM, Bae S, et al. Genome-wide differential gene expression profiling of human bone marrow stromal cells. Stem Cells. 2007; 25(4): 994-1002.

Bae S, Ahn JH, Park CW, et al. Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts. Cell Tissue Res. 2009; 335(3): 565-573.

Tormin A, Brune JC, Olsson E, et al. Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets. Cytotherapy. 2009; 11(2): 114-28.

Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biology of Blood and Marrow Transplantation. 2005; 11(5): 389-398. 

Jones E, English A, Churchman SM, et al. Large-scale extraction and characterization of CD271þ 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. 2010; 62: 1944-1954.

Rosova I, Dao M, Capoccia B, et al. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells. 2008; 26(8): 2173-82.

Grisendi G, Anneren C, Cafarelli L, et al. GMP- manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion. Cytotherapy. 2010; 12(4): 466-77. 

Corcione A, Benvenuto F, Ferretti E, et al. Human mesenchymal stem cells modulate B-cell functions. Blood. 2006; 107(1): 367-72. 

Samuelsson H, Ringden O, Lonnies H, et al. Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells. Cytotherapy. 2009; 11:129-136.

Mankani MH, Kuznetsov SA, Marshall GW, et al. Creation of new bone by the percutaneous injection of hu- man bone marrow stromal cell and HA/TCP suspensions. Tissue Eng Part A. 2008; 14(12): 1949-1958. 

Koc ON, Gerson SL, Cooper BW, et al. 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. 2000; 18: 307-316.

Zhukareva V, Obrocka M, Houle JD, et al. Secretion profile of human bone marrow stromal cells: donor variability and response to inflammatory stimuli. Cytokine. 2010; 50(3): 317-321.

Wolfe M, Pochampally R, Swaney W, et al. Isolation and Culture of Bone Marrow-Derived Human Multipotent Stromal Cells (hMSCs). Methods Mol Biol. 2008; 449: 3-25.

Kinnaird T, Stabile E, Burnett MS, et al. 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. 2004; 94 (5): 678–85.

Gnecchi M, He H, Liang OD, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nature Medicine. 2005; 11(4): 367–8.

Iván C, Naiara T, Jesús D, et al. ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential. Experimental Cell Research. 2008; 314(8): 1777–1788.

Mandana H, Susanne K, Louise H, et al. Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion. Stem Cell Rev and Rep. 2013; 9: 44–58.

Chambard JC, Lefloch R, Pouysségur J, et al. ERK implication in cell cycle regulation. Biochimica et Biophysica Acta. 2007; 1773: 1299–1310

Palaez D, Arita N, Cheung H. Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression. Biochemical and Biophysical Research Communications. 2012; 417 (4): 1286-1291.

Liu J, Zhao Z, Li J, et al. Hydrostatic Pressures Promote Initial Osteodifferentiation with ERK 1/2 Not p38 MAPK Signaling Involved. Journal of Cellular Biochemistry. 2009; 107 (2): 224-232.

Lee J, Suh J, Park H, et al. Heparin-binding epidermal growth factor-like growth factor inhibits adipocyte differentiation at commitment and early induction stages. Differentiation. 2008; 76 (5): 478-487.

Roman MS, Robert FK, Mariah KH, et al. ERK Signaling Pathways Regulate the Osteogenic Differentiation of Human Mesenchymal Stem Cells on Collagen I and Vitronectin. Cell Communication and Adhesion. 2004; 11: 137–153.

Claes L, Recknagel S, Ignatius A. Fracture healing under healthy and inflammatory conditions. Nat Rev Rheumatol. 2012; 8: 133-143.

Chandrasekhar KS, Zhou H, Zeng P, et al. Blood vessel wall- derived endothelial colony-forming cells enhance fracture repair and bone regeneration. Calcif Tissue Int. 2011; 89(5): 347-357.

Wen JH, Vincent LG, Fuhrmann A, et al. Interplay of matrix stiffness and protein tethering in stem cell differentiation. Nature Materials. 2014; 13 (10): 979-87.

Kentaro A, Yong-Ouk Y, Takayoshi Y, et al. Characterization of bone marrow derived mesenchymal stem cells in suspension. Stem Cell Research & Therapy. 2012; 3(5):40.

Cho Y, Shin J, Kim H, et al. Comparison of the Osteogenic Potential of Titanium- and Modified Zirconia-Based Bioceramics Int. J. Mol. Sci. 2014; 15: 4442-4452.

Lilian PE, Renata BR, Isis SO, et al. 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. 2009; 24 (5): 400-404.

Röntgen V, Blakytny R, Matthys R, et al. Fracture healing in mice under controlled rigid and flexible conditions using an adjustable external fixator. J Orthop Res. 2010; 28(11): 1456-62.

Tahrin M and Ping-Chang Y. Western Blot: Technique, Theory, and Trouble Shooting. N Am J Med Sci. 2012; 4 (9): 429–434.

Tain-Hsiung C, Wei-Ming C, Ke-Hsun H, et al. Sodium butyrate activates ERK to regulate differentiation of mesenchymal stem cells. Biochemical and Biophysical Research Communications. 2007; 355(4): 913–918.

Waters WR, Palmer MV, Whipple DL, et al. 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. 2003; 10: 960-966.

Arash Z, Iraj RK, Mohammad B, et al. 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 Journal. 2008; 12 (3): 133-141.

Gregory CA, Gunn WG, Peister A, et al. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem. 2004; 329(1): 77-84. 

Salasznyk RM, Klees RF, Hughlock MK, et al. ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells on collagen I and vitronectin. Cell Commun Adhes. 2004; 11(5-6): 137–53

Liu BS, Yao CH, Chen YS, et al. In vitro evaluation of degradation and cytotoxicity of a novel composite as a bone substitute,” Journal of Biomedical Materials Research. 2003; 67 (4): 1163–1169.

Colter D, Class R, DiGirolamo C, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A. 2000; 97(7): 3213–8.

Sekiya I, Larson BL, Smith JR, et al. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells. 2002; 20(6): 530–41.

Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002; 418(6893):41-9.

Reyes M Verfaillie CM. Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells. Ann N Y Acad Sci. 2001; 938: 231–3.

Song I, Arnold IC, James E. D. Dexamethasone Inhibition of Confluence-Induced Apoptosis in Human Mesenchymal Stem Cells. Inc. J. Orthop Res. 2009; 27: 216–221.

Neuhuber B, Sharon A S, Linda H, et al. Effects of Plating Density and Culture Time On Bone Marrow Stromal Cell Characteristics. Exp Hematol. 2008; 36 (9): 1176–1185.

Gregory CA, Ylostalo J, Prockop DJ. 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. 2005; 294: 37.

Both SK, van der Muijsenberg AJC, van Blitterswijk CA, et al. A rapid and efficient method for expansion of human mesenchymal stem cells. Tissue Engineering. 2007; 13 (1): 3–9.

Lode A, Bernhardt A, Gelinsky M. 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. 2008; 2(7): 400–7.

Fossett E and Khan WS. Optimising Human Mesenchymal Stem Cell Numbers for Clinical Application: A Literature Review. Stem Cells International. 2012; 2012. Article ID 465259.

Bartmann C, Rohde E, Schallmoser K, et al. Two steps to functional mesenchymal stromal cells for clinical application. Transfusion. 2007; 47 (8): 1426–35.

Mochizuki T, Muneta T, Sakaguchi Y, et al. 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. 2006; 54 (3): 843–853.

Haack-Sorensen M, Susanne K H, Louise H, et al. Mesenchymal stromal cell phenotype is not influenced by confluence during culture expansion. Stem Cell Rev. 2013; 9(1): 44–58

Gnecchi M, He H, Liang, OD, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nature Medicine. 2005. 11(4): 367–368.

Lu H, Guo L, Wozniak MJ, et al. Effect of cell density on adipogenic differentiation of mesenchymal stem cells. Biochem Biophys Res Commun. 2009; 381:322-7.

Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J Orthop Res. 1991; 9(4): 465-476.

Seghatoleslami MR, Tuan RS. Cell density dependent regulation of AP-1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells. J Cell Biochem. 2002; 84: 237-248.

Kilian KA, Bugarija B, Lahn BT, et al. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci USA. 2010; 107(11): 4872-4877.

Gao L, McBeath R, Chen CS. Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin. Stem Cells. 2010; 28(3): 564-72.

Sotiropoulou P, Perez SA, Salagianni M, et al. Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells. 2006; 24(2): 462–71.

Kuznetsov SA, Mankani MH, Robey PG. Effect of serum on human bone marrow stromal cells: ex vivo expansion and in vivo bone formation. Transplantation. 2000; 70(12): 1780-7.

Balint R, Stephen MR, Sarah HC. 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. 2015; 9(10): 1200–1203.


  • There are currently no refbacks.

Creative Commons Attribution-NonCommercial 3.0

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.