Cell orientation determines the alignment of cell-produced collagenous matrix.

In healing ligaments and tendons, the cells are not aligned and collagen matrix is not organized as in normal tissues. In addition, the mechanical properties of the tissues are abnormal. We hypothesized that the lack of alignment of the collagen matrix results from random orientation of the cells seen in the healing area. To test this hypothesis, a novel in vitro model was used in which the orientation of cells could be controlled via microgrooves, and alignment of the collagen matrix formed by these cells could be easily observed. It is known that cells align uniformly along the direction of microgrooves; therefore MC3T3-E1 cells, which produce large amounts of collagen, were grown on silicone membranes with parallel microgrooves (10 microm wide x 3 microm deep) in the surface. As a control, the same cells were also grown on smooth silicone membranes. Cells on both the microgrooved and smooth silicone surfaces produced a layer of readily visible collagen matrix. Immunohistochemical staining showed that the matrix consisted of abundant type I collagen. Polarized light microscopy of the collagen matrix revealed the collagen fibers to be parallel to the direction of the microgrooves, whereas the collagen matrix produced by the randomly oriented cells on the smooth membranes was disorganized. Thus, the results of this study suggest that the orientation of cells affects the organization of the collagenous matrix produced by the cells. The results also suggest that orienting cells along the longitudinal direction of healing ligaments and tendons may lead to the production of aligned collagenous matrix that more closely represents the uninjured state. This may enhance the mechanical properties of healing ligaments and tendons.

[1]  P. Friedl,et al.  Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44. , 1997, Cancer research.

[2]  T. Matsuda,et al.  Tissue engineered skeletal muscle: preparation of highly dense, highly oriented hybrid muscular tissues. , 1998, Cell transplantation.

[3]  L. Quarles,et al.  Distinct proliferative and differentiated stages of murine MC3T3‐E1 cells in culture: An in vitro model of osteoblast development , 1992, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[4]  E. Grood,et al.  Cell orientation response to cyclically deformed substrates: experimental validation of a cell model. , 1995, Journal of biomechanics.

[5]  J. Jansen,et al.  Contact guidance of rat fibroblasts on various implant materials. , 1999, Journal of biomedical materials research.

[6]  E S Grood,et al.  Alignment and proliferation of MC3T3-E1 osteoblasts in microgrooved silicone substrata subjected to cyclic stretching. , 2000, Journal of biomechanics.

[7]  P. Canham,et al.  Demonstration of quantitative fabric analysis of tendon collagen using two-dimensional polarized light microscopy. , 1991, Matrix.

[8]  G. Lee,et al.  Cell surface receptors transmit sufficient force to bend collagen fibrils. , 1999, Experimental cell research.

[9]  Peter Friedl,et al.  Cell migration strategies in 3‐D extracellular matrix: Differences in morphology, cell matrix interactions, and integrin function , 1998, Microscopy research and technique.

[10]  D. Stamenović,et al.  Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells. , 2002, American journal of physiology. Cell physiology.

[11]  D. Brunette,et al.  The effects of the surface topography of micromachined titanium substrata on cell behavior in vitro and in vivo. , 1999, Journal of biomechanical engineering.

[12]  S L Woo,et al.  Medial collateral ligament healing , 1983, The American journal of sports medicine.

[13]  S. Woo,et al.  The effects of increased tension on healing medial collateral ligaments , 1991, The American journal of sports medicine.

[14]  E Bell,et al.  Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R T Tranquillo,et al.  A methodology for the systematic and quantitative study of cell contact guidance in oriented collagen gels. Correlation of fibroblast orientation and gel birefringence. , 1993, Journal of cell science.

[16]  M Eastwood,et al.  Tensional homeostasis in dermal fibroblasts: Mechanical responses to mechanical loading in three‐dimensional substrates , 1998, Journal of cellular physiology.

[17]  S L Woo,et al.  The biomechanical and morphological changes in the medial collateral ligament of the rabbit after immobilization and remobilization. , 1987, The Journal of bone and joint surgery. American volume.

[18]  M Eastwood,et al.  Effect of precise mechanical loading on fibroblast populated collagen lattices: morphological changes. , 1998, Cell motility and the cytoskeleton.

[19]  S L Woo,et al.  Evaluation of a new injury model to study medial collateral ligament healing: Primary repair versus nonoperative treatment , 1991, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[20]  Robert M. Nerem,et al.  Dynamic Mechanical Conditioning of Collagen-Gel Blood Vessel Constructs Induces Remodeling In Vitro , 2000, Annals of Biomedical Engineering.

[21]  E. K. Kline.  The Microtomist's Formulary and Guide , 1955 .

[22]  A Curtis,et al.  Topographical control of cells. , 1997, Biomaterials.

[23]  G. Stein,et al.  MOLECULAR MECHANISMS MEDIATING DEVELOPMENTAL AND HORMONE-REGULATED EXPRESSION OF GENES IN OSTEOBLASTS: An Integrated Relationship of Cell Growth and Differentiation , 1993 .

[24]  D. Birk,et al.  Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation , 1986, The Journal of cell biology.

[25]  R L Trelstad,et al.  Tendon collagen fibrillogenesis: intracellular subassemblies and cell surface changes associated with fibril growth. , 1979, Developmental biology.

[26]  HighWire Press,et al.  American journal of physiology. Cell physiology , 1977 .

[27]  野田 政樹.  Cellular and molecular biology of bone , 1993 .

[28]  M. Kumegawa,et al.  Selective inhibition of type I collagen synthesis in osteoblastic cells by epidermal growth factor. , 1984, Endocrinology.

[29]  S. Woo,et al.  Immobilization of the knee joint alters the mechanical and ultrastructural properties of the rabbit anterior cruciate ligament , 1995, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[30]  B. Coulomb,et al.  Production of ordered collagen matrices for three-dimensional cell culture. , 2002, Biomaterials.

[31]  D. Brunette,et al.  The use of micromachined surfaces to investigate the cell behavioural factors essential to osseointegration. , 2008, Oral diseases.

[32]  S. Woo,et al.  Mechanical properties of tendons and ligaments. II. The relationships of immobilization and exercise on tissue remodeling. , 1982, Biorheology.

[33]  M. Dembo,et al.  Cell movement is guided by the rigidity of the substrate. , 2000, Biophysical journal.

[34]  T. V. van Kooten,et al.  Influence of silicone (PDMS) surface texture on human skin fibroblast proliferation as determined by cell cycle analysis. , 1998, Journal of biomedical materials research.