Role of Heterotypic Cellular Interactions in the Regeneration of the Anterior Cruciate Ligament-to-Bone Interface

The anterior cruciate ligament (ACL) inserts into subchondral bone through a fibrocartilage interface with controlled spatial distribution of different cell types and biochemical composition. The function of the interface is to minimize stress concentration and mediate load transfer between the soft and hard tissue. The fibrocartilage interface is however not regenerated post ACL reconstruction surgery using semitendinosus grafts, and the lack of graft-bone integration is one of the primary causes of graft failure. Currently, the mechanism for interface regeneration is not known. To address the challenge of biological fixation and to elucidate the underlying mechanism, our working hypothesis is that interactions between cells derived from soft tissue (fibroblasts) and bone (osteoblast) are important in the fibrochondrogenic differentiation of progenitor cell populations, such as fibroblasts or bone marrow stromal cells (BMSC). To test this hypothesis, we first performed an interface characterization study in order to identify interface-related markers and to establish the baseline parameters for evaluating the fibrochondrogenic differentiation of BMSC. Subsequently, co-culture and tri-culture models mimicking the multi-cellular organization at the native ACL-to-bone interface are designed to determine the effects of heterotypic cellular interactions on the development of fibrocartilage-related markers. These novel multi-culture models allow controlled interactions via paracrine communication as well as cell-cell physical contact. It was observed that co-culture of fibroblasts and osteoblasts lead to phenotypic changes towards the fibrochondrocytic phenotype, and subsequent tri-culture of these two cell types with BMSC induced stem cell differentiation and the expression of fibrochondrocyte-related markers. In addition, the effects of fibroblast-osteoblast interactions are found to be mediated by paracrine communication and are further enhanced by fibroblast-osteoblast physical contact. Furthermore, mechanical loading modulates these cellular interactions in tri-culture. These observations collectively delineate the importance of fibroblast-osteoblast interactions in promoting interface regeneration and the multi-culture models developed in this thesis represent an innovative approach to examine the relevance of heterotypic cellular interactions. Findings from these studies have yielded new insights into the mechanisms governing interface regeneration, and it is anticipated that these critical understandings will facilitate the development of innovative tissue engineering strategies aimed at promoting biological graft fixation and soft tissue-to-bone integration.

Year	Entry
1999	Buschmann MD, Kim Y-J, Wong M, Frank E, Hunziker EB, Grodzinsky AJ. Stimulation of aggrecan synthesis in cartilage explants by cyclic loading is localized to regions of high interstitial fluid flow1. Arch Biochem Biophys. June 1, 1999;366(1):1-7.
1998	Benjamin M, Ralphs JR. Fibrocartilage in tendons and ligaments: an adaptation to compressive load. J Anat. 1998;193(4):481-494.
1992	Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res. November 1992;10(6):745-758.
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.
1997	Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science. May 30, 1997;276(5317):1425.
1984	Palmoski MJ, Brandt KD. Effects of static and cyclic compressive loading on articular cartilage plugs in vitro. Arthritis Rheum. June 1984;27(6):675-681.
1892	Wolff J. Das Gesetz der Transformation der Knochen. Berlin: Hirschwald; 1892.
1983	Woo SL-Y, Gomez MA, Seguchi Y, Endo CM, Akeson WH. Measurement of mechanical properties of ligament substance from a bone-ligament-bone preparation. J Orthop Res. 1983;1(1):22-29.
1995	Setton LA, Mow VC, Howell DS. Mechanical behavior of articular cartilage in shear is altered by transection of the anterior cruciate ligament. J Orthop Res. July 1995;13(4):473-482.
2003	Mauck RL, Hung CT, Ateshian GA. Modeling of neutral solute transport in a dynamically loaded porous permeable gel: implications for articular cartilage biosynthesis and tissue engineering. J Biomech Eng. October 2003;125(5):602-614.
2003	Mauck RL, Wang CC-B, Oswald ES, Ateshian GA, Hung CT. The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading. Osteoarthritis Cartilage. December 2003;11(12):879-890.
2003	Mauck RL, Nicoll SB, Seyhan SL, Ateshian GA, Hung CT. Synergistic action of growth factors and dynamic loading for articular cartilage tissue engineering. Tissue Eng. August 2003;9(4):597-611.
1994	Guilak F, Meyer BC, Ratcliffe A, Mow VC. The effects of matrix compression on proteoglycan metabolism in articular cartilage explants. Osteoarthritis Cartilage. June 1994;2(2):91-101.
1973	Kempson GE, Muir H, Pollard C, Tuke M. The tensile properties of the cartilage of human femoral condyles related to the content of collagen and glycosaminoglycans. Biochim Biophys Acta. February 28, 1973;297(2):456-472.
1970	Cooper RR, Misol S. Tendon and ligament insertion: a light and electron microscopic study. J Bone Joint Surg. January 1970;52A(1):1-20, 170.
1994	Kim Y-J, Sah RLY, Grodzinsky AJ, Plaas AHK, Sandy JD. Mechanical regulation of cartilage biosynthetic behavior: physical stimuli. Arch Biochem Biophys. May 1994;311(1):1-12.
1999	Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. April 2, 1999;284(5411):143-147.
2003	Ferguson VL, Bushby AJ, Boyde A. Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone. J Anat. August 2003;203(2):191-202.
1988	Currey JD. The effect of porosity and mineral content on the Young's modulus of elasticity of compact bone. J Biomech. 1988;21(2):131-139.
2007	Baker BM, Mauck RL. The effect of nanofiber alignment on the maturation of engineered meniscus constructs. Biomaterials. April 2007;28(11):1967-1977.
2001	Graverand M-PHL, Ou Y, Schield-Yee T, Barclay L, Hart D, Natsume T, Rattner JB. The cells of the rabbit meniscus: their arrangement, interrelationship, morphological variations and cytoarchitecture. J Anat. May 2001;198(5):525-535.
2001	Bonassar LJ, Grodzinsky AJ, Frank EH, Davila SG, Bhaktav NR, Trippel SB. The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-i. J Orthop Res. January 2001;19(1):11-17.
1989	Sah RL-Y, Kim Y-J, Doong J-YH, Grodzinsky AJ, Plass AHK, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res. 1989;7(5):619-636.
1998	Yoo JU, Barthel TS, Nishimura K, Solchaga L, Caplan AI, Goldberg VM, Johnstone B. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg. December 1998;80A(12):1745-1757.
1988	Gray ML, Pizzanelli AM, Grodzinsky AJ, Lee RC. Mechanical and physicochemical determinants of the chondrocyte biosynthetic response. J Orthop Res. November 1988;6(6):777-792.
1979	Junqueira LCU, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histchem J. 1979;11:447-455.
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.
2003	Thomopoulos S, Williams GR, Gimbel JA, Favata M, Soslowsky LJ. Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site. J Orthop Res. May 2003;21(3):413-419.
2006	Mauck RL, Yuan X, Tuan RS. Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage. February 2006;14(2):179-189.
2007	Mauck RL, Byers BA, Yuan X, Tuan RS. Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. Biomech Model Mechanobiol. January 2007;6(1-2):113-125.
1989	Vogel KG, Koob TJ. Structural specialization in tendons under compression. Int Rev Cytol. 1989;115:267-293.
1998	Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. Winter 1998;4(4):415-428.
1990	O'Hara BP, Urban JP, Maroudas A. Influence of cyclic loading on the nutrition of articular cartilage. Ann Rheum Dis. July 1990;49(7):536-539.
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.
1991	Sah RL-Y, Doong J-YH, Grodzinsky AJ, Plaas AHK, Sandy JD. Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants. Arch Biochem Biophys. April 1991;286(1):209-29.
1974	Noyes FR, DeLucas JL, Torvik PJ. Biomechanics of anterior cruciate ligament failure: an analysis of strain-rate sensitivity and mechanisms of failure in primates. J Bone Joint Surg. March 1974;56A(2):236-253.
2000	LeRoux MA, Arokoski J, Vail TP, Guilak F, Hyttinen MM, Kiviranta I, Setton LA. Simultaneous changes in the mechanical properties, quantitative collagen organization, and proteoglycan concentration of articular cartilage following canine meniscectomy. J Orthop Res. May 2000;18(3):383-392.
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.
2004	Almarza AJ, Athanasiou KA. Design characteristics for the tissue engineering of cartilaginous tissues. Ann Biomed Eng. January 2004;32(1):2-17.
1982	Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell. August 1982;30(1):215-224.
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.
1998	Valhmu WB, Stazzone EJ, Bachrach NM, Saed-Nejad F, Fischer SG, Mow VC, Ratcliffe A. Load-controlled compression of articular cartilage induces a transient stimulation of aggrecan gene expression. Arch Biochem Biophys. May 1, 1998;353(1):29-36.

Year

Entry

1999

Buschmann MD, Kim Y-J, Wong M, Frank E, Hunziker EB, Grodzinsky AJ. Stimulation of aggrecan synthesis in cartilage explants by cyclic loading is localized to regions of high interstitial fluid flow1. Arch Biochem Biophys. June 1, 1999;366(1):1-7.

1998

Benjamin M, Ralphs JR. Fibrocartilage in tendons and ligaments: an adaptation to compressive load. J Anat. 1998;193(4):481-494.

1992

Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res. November 1992;10(6):745-758.

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.

1997

Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science. May 30, 1997;276(5317):1425.