Development of self-assembled, tissue-engineered ligament from bone marrow stromal cells.

The human anterior cruciate ligament is ruptured 200,000 times per year in the United States, resulting in medical costs of $1 billion. The standard treatment is patellar tendon autograft, but this treatment is suboptimal because of lengthy recovery time, arthritis, donor site morbidity, and degenerative joint disease. This study aimed to engineer scaffold-free ligament analogs from a clinically relevant cell source and to examine mechanical and histological properties of the resulting engineered tissue. Porcine bone marrow stromal cells were seeded on laminin-coated substrates with silk suture segments as anchor points. Cells developed into monolayers that subsequently delaminated and self-organized into cohesive rod-like tissues that were held in tension above the substrate. After 14 days of maturation, scanning electron microscopy revealed a well-organized extracellular matrix, aligned collagen fibers, and a collagen fibril diameter of 51.1+/-77 nm. Histological evaluation showed that constructs were composed of approximately 60% collagen. During tensile tests to failure, constructs had a stress of 2.11 +/- 0.13 MPa, a strain of 28.8 +/- 0.95%, a force of 0.26 +/- 0.02 N, and a tangent modulus of 15.4+/-1.04 MPa. Mechanically and histologically, engineered ligament resembled native embryonic connective tissue and had an ultimate stress approximately 15% of native adult mouse tissue.

[1]  F. Noyes,et al.  The strength of the anterior cruciate ligament in humans and Rhesus monkeys. , 1976, The Journal of bone and joint surgery. American volume.

[2]  J. Faulkner,et al.  Functional development of engineered skeletal muscle from adult and neonatal rats. , 2001, Tissue engineering.

[3]  Anthony Atala,et al.  Methods Of Tissue Engineering , 2006 .

[4]  T. Chvapil,et al.  Collagen fibers as a temporary scaffold for replacement of ACL in goats. , 1993, Journal of biomedical materials research.

[5]  R G Dennis,et al.  Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines. , 2001, American journal of physiology. Cell physiology.

[6]  Glen A. Livesay,et al.  Development of Ligament-Like Structural Organization and Properties in Cell-Seeded Collagen Scaffolds in vitro , 2006, Annals of Biomedical Engineering.

[7]  Cato T Laurencin,et al.  Three-dimensional, bioactive, biodegradable, polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro. , 2003, Journal of biomedical materials research. Part A.

[8]  D L Butler,et al.  Comparison of material properties in fascicle-bone units from human patellar tendon and knee ligaments. , 1986, Journal of biomechanics.

[9]  R. F. Closkey,et al.  Viability of fibroblast‐seeded ligament analogs after autogenous implantation , 1998, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[10]  Karl Grosh,et al.  Engineering of functional tendon. , 2004, Tissue engineering.

[11]  J. Kohn,et al.  Preliminary development of a novel resorbable synthetic polymer fiber scaffold for anterior cruciate ligament reconstruction. , 2004, Tissue engineering.

[12]  Robert G. Dennis,et al.  Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro , 2000, In Vitro Cellular & Developmental Biology - Animal.

[13]  David L Kaplan,et al.  Tissue engineering of ligaments. , 2004, Annual review of biomedical engineering.

[14]  F A Auger,et al.  A truly new approach for tissue engineering: the LOEX self-assembly technique. , 2002, Ernst Schering Research Foundation workshop.

[15]  Joseph W Freeman,et al.  Anterior cruciate ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies. , 2005, Biomaterials.

[16]  Raphael C. Lee,et al.  Mechanisms and dynamics of mechanical strengthening in ligament-equivalent fibroblast-populated collagen matrices , 1993, Annals of Biomedical Engineering.

[17]  J. B. Liesch,et al.  Development of fibroblast-seeded ligament analogs for ACL reconstruction. , 1995, Journal of biomedical materials research.

[18]  A. Tria,et al.  Anterior cruciate ligament reconstruction using a composite collagenous prosthesis , 1992 .

[19]  F. Silver,et al.  Structural and mechanical assessment of developing chick tendon , 1988 .

[20]  S L Woo,et al.  The effects of strain rate on the properties of the medial collateral ligament in skeletally immature and mature rabbits: A biomechanical and histological study , 1990, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[21]  Ivan Martin,et al.  Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering. , 2002, Journal of biomechanical engineering.

[22]  M. Dunn,et al.  Effect of chemical treatments on tendon cellularity and mechanical properties. , 2000, Journal of biomedical materials research.

[23]  L. Blankevoort,et al.  Augmentation in anterior cruciate ligament reconstruction—a histological and biomechanical study on goats , 2004, International Orthopaedics.

[24]  Pierre Boher,et al.  A transmission electron microscopy study of low‐temperature reaction at the Co‐Si interface , 1990 .

[25]  R. Shadwick,et al.  Elastic energy storage in tendons: mechanical differences related to function and age. , 1990, Journal of applied physiology.

[26]  F A Auger,et al.  A completely biological tissue‐engineered human blood vessel , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[27]  E. Pennisi Tending Tender Tendons , 2002, Science.

[28]  L. Soslowsky,et al.  Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice. , 2005, Journal of biomechanical engineering.