Pericellular Matrix Mechanotransduction Events in Differentiating Human Mesenchymal Stem Cells: Modulating the Pericellular Matrix Through Silencing Type VI Collagen and Decorin

Stem cell therapies are currently being explored for their potential in the regeneration of load bearing tissues, such as cartilage. Current therapies lack the ability to intrinsically overcome a mechanically adverse environment at implantation. To advance the implementation of human mesenchymal stem cells (hMSCs) for cartilage repair, the mechanisms by which cells “feel” and interact with their micromechanical environment need to be understood. Chondrogenic hMSCs develop a thin pericellular matrix (PCM), consisting of type VI collagen (ColVI) and proteoglycans such as decorin (DCN). The PCM is believed to control mechanotransduction events, acting as both a biomechanical and biochemical buffer. This thesis studies the functional role of ColVI and DCN through targeted gene knockdown using shRNA lentiviral vectors complimentary to col6a1 or dcn.

In the first part of the work, the biophysical role of the PCM was determined through comparisons of cellular deformability under uniaxial strain with or without ColVI and DCN knockdown. HMSCs were cultured in alginate scaffolds and were stimulated with transforming growth factor β for 1 to 2 weeks. We found that the PCM with ColVI knockdown lacked the ability to withstand applied compression and with DCN knockdown deformed in a strain-dependent manner. Next we analyzed the mechanosignaling initiation caused by a transient sinusoidal compressive load through studying cytoskeletal kinetics and gene expression. Altering the PCM through ColVI and DCN knockdown caused an increase in actin and vimentin cytoskeletal protein concentration that lacked a dynamic response to load. This lead to a stronger fibroblast growth factor gene expression in ColVI knockdown. DCN also demonstrated direct control over cartilage oligomeric matrix protein gene expression, through a loss of TGF-β regulation. These results were further demonstrated during long term compressive culture. Unconfined sinusoidal compressive culture revealed the highest improvement in material properties in knockdown samples at day 14.

Through these studies, we demonstrated that ColVI and DCN are integral proteins in maintaining the structural microenvironment through protecting the cell from injurious deformation, maintaining cytoskeletal dynamics in response to load, and regulating the differentiation rate through TGF-β signaling. Finally, we demonstrated the ability to manipulate chondrogenic mechanotransduction events using genetic engineering.

Year	Entry
2006	Guilak F, Alexopoulos LG, Upton ML, Youn I, Choi JB, Cao L, Setton LA, Haider MA. The pericellular matrix as a transducer of biomechanical and biochemical signals in articular cartilage. Annals NY Acad Sci. April 2006;1068(1):498-512.
1995	Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB. Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J Cell Sci. April 1995;108(4):1497-1508.
1999	Guilak F, Jones WR, Ting-Beall HP, Lee GM. The deformation behavior and mechanical properties of chondrocytes in articular cartilage. Osteoarthritis Cartilage. January 1999;7(1):59-70.
1994	Freeman PM, Natarajan RN, Kimura JH, Andriacchi TP. Chondrocyte cells respond mechanically to compressive loads. J Orthop Res. May 1994;12(3):311-320.
2007	Choi JB, Youn I, Cao L, Leddy HA, Gilchrist CL, Setton LA, Guilak F. Zonal changes in the three-dimensional morphology of the chondron under compression: the relationship among cellular, pericellular, and extracellular deformation in articular cartilage. J Biomech. 2007;40(12):2596-2603.
2003	Angele P, Yoo JU, Smith C, Mansour J, Jepsen KJ, Nerlich M, Johnstone B. Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. J Orthop Res. May 2003;21(3):451-457.
1995	Guilak F, Ratcliffe A, Mow VC. Chondrocyte deformation and local tissue strain in articular cartilage: a confocal microscopy study. J Orthop Res. May 1995;13(3):410-421.
1994	Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. NEJM. October 6, 1994;331(14):889-895.
2001	Elder SH, Goldstein SA, Kimura JH, Soslowsky LJ, Spengler DM. Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading. Ann Biomed Eng. June 2001;29(6):476-482.
2002	Mauck RL, Seyhan SL, Ateshian GA, Hung CT. Influence of seeding density and dynamic deformational loading on the developing structure/function relationships of chondrocyte-seeded agarose hydrogels. Ann Biomed Eng. September 2002;30(8):1046-1056.
2000	Mauck RL, Soltz MA, Wang CCB, Wong DD, Chao P-HG, Valhmu WB, Hung CT, Ateshian GA. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng. June 2000;122(3):252-260.
2005	Bartlett W, Skinner JA, Gooding CR, Carrington RWJ, Flanagan AM, Briggs TWR, Bentley G. Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg. May 2005;87B(5):640-645.
1998	Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res. January 10, 1998;238(1):265-272.
2001	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods. December 2001;25(4):402-408.
2006	Pritzker KPH, Gay S, Jimenez SA, Ostergaard K, Pelletier J-P, Revell PA, Salter D, van den Berg WB. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage. January 2006;14(1):13-29.
2005	Alexopoulos LG, Williams GM, Upton ML, Setton LA, Guilak F. Osteoarthritic changes in the biphasic mechanical properties of the chondrocyte pericellular matrix in articular cartilage. J Biomech. March 2005;38(3):509-517.
2007	Lima EG, Bian L, Ng KW, Mauck RL, Byers BA, Tuan RS, Ateshian GA, Hung CT. The beneficial effect of delayed compressive loading on tissue-engineered cartilage constructs cultured with TGF-β3. Osteoarthritis Cartilage. September 2007;15(9):1025-1033.
1997	Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol. February 10, 1997;136(3):729-743.
2003	Alexopoulos LG, Haider MA, Vail TP, Guilak F. Alterations in the mechanical properties of the human chondrocyte pericellular matrix with osteoarthritis. J Biomech Eng. June 2003;125(3):323-333.
2004	Huang CC, Hagar KL, Frost LE, Sun Y, Cheung HS. Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells. Stem Cells. May 2004;22(3):313-323.
2000	Lee DA, Knight MM, Bolton JF, Idowu BD, Kayser MV, Bader DL. Chondrocyte deformation within compressed agarose constructs at the cellular and sub-cellular levels. J Biomech. January 2000;33(1):81-95.
2004	Hung CT, Mauck RL, Wang CC-B, Lima EG, Ateshian GA. A paradigm for functional tissue engineering of articular cartilage via applied physiologic deformational loading. Ann Biomed Eng. January 2004;32(1):35-49.

Year

Entry

2006

Guilak F, Alexopoulos LG, Upton ML, Youn I, Choi JB, Cao L, Setton LA, Haider MA. The pericellular matrix as a transducer of biomechanical and biochemical signals in articular cartilage. Annals NY Acad Sci. April 2006;1068(1):498-512.

1995

Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB. Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J Cell Sci. April 1995;108(4):1497-1508.

1999

Guilak F, Jones WR, Ting-Beall HP, Lee GM. The deformation behavior and mechanical properties of chondrocytes in articular cartilage. Osteoarthritis Cartilage. January 1999;7(1):59-70.

1994

Freeman PM, Natarajan RN, Kimura JH, Andriacchi TP. Chondrocyte cells respond mechanically to compressive loads. J Orthop Res. May 1994;12(3):311-320.

2007

Choi JB, Youn I, Cao L, Leddy HA, Gilchrist CL, Setton LA, Guilak F. Zonal changes in the three-dimensional morphology of the chondron under compression: the relationship among cellular, pericellular, and extracellular deformation in articular cartilage. J Biomech. 2007;40(12):2596-2603.