Novel System for Engineering Bioartificial Tendons and Application of Mechanical Load

Abstract

Cells cultured in three-dimensional collagen gels express a more native state phenotype because they form a syncytial network that can be mechanically loaded. Moreover, cells remodel their matrix by eliminating water, and by reorganizing and aligning the collagen fibrils. Last, the ability to subject cells to mechanical loading in a native matrix is desirable because cells, in tissues as well as the matrix, bear strains and alter their expression profile consistent with either immobilization, moderate activity, or repetitive loading. This is the first report of a model bioreactor system to fabricate and culture tendon cell-populated, linear, tethered matrix constructs that can be mechanically loaded by a computer-driven, pressure-controlled system. Bioartificial tissues (BATs) as tendon constructs were molded in a novel, rubber bottom Tissue Train culture plate bearing nonwoven nylon mesh anchors at the east and west poles of each culture well. Mechanical loading was achieved by placing an Arctangle loading post (an Arctangle is a rectangle with curved short ends) beneath each well of the six-well culture plate and using vacuum to displace the flexible membrane downward, resulting in uniaxial strain on the BAT. BATs populated with avian flexor tendon cells expressed collagen genes I, III, and XII as well as aggrecan, fibronectin, prolyl hydroxylase, and tenascin, consistent with expression levels of cells grown on collagen-bonded two-dimensional surfaces or in native, whole, avian flexor tendon. Likewise, cells in BATs established a morphology of linearly arranged cells aligned with the principal strain direction as in fasicles of whole tendons. Last, BATs that were mechanically loaded had an ultimate tensile strength that was nearly 3-fold greater than that of nonloaded BATs in the first week of culture. Taken together, these results indicate that tendon cells fabricated in a mechanically loaded, linear collagen gel construct assume a phenotype that is similar to that of a native tendon in terms of appearance and expression and are stronger than nonexercised counterparts yet far weaker than native adult tendons. This technique represents a novel approach to culturing cells in a mechanically active, three-dimensional culture environment that can be readily used for the fabrication of tissue simulates for drug testing or tissue engineering.

The Effects of GDF-5 and Uniaxial Strain on Mesenchymal Stem Cells in 3-D Culture

“…Some approaches involve materials currently used as suture material, including processed silk fibers [1,12] and bioabsorbable polymers [7,34]. Biologic substrates, including collagen gels [2,6,27], hyaluronan [17], and alginate/chitosan polymers [36] have also been used.…”

Mechanoregulation of gene expression in fibroblasts

“…In addition to internally generated tensile stresses, external mechanical loading can also be applied to the FPCGs. A good example is the development of bioartificial tissues of tendon constructs cultured in 3-D collagen gels that can be mechanically loaded by a computer-driven, pressure-controlled system for fabrication of tissue engineered constructs (Garvin et al, 2003).…”

Influence of cyclic strain and decorin deficiency on 3D cellularized collagen matrices

“…The collagen gels were formed in the incubator using the Flexcell ® Tissue Train ® culture system, which has been previously described by Garvin et al [12]. The gels are grown in customized 6-well culture plates that have elastomeric culture surfaces and nylon mesh anchors on two sides of each well.…”

“…Indeed, various engineered tissues such as heart valves, vascular tissues, and cartilage are commonly grown under physiologically-mimicking mechanical conditions in an attempt to achieve native tissue-like properties [9][10][11]. Similarly, collagenous matrices that have been grown under mechanical stimulation demonstrate improvements in cell alignment and collagen fibrillar orientation and packing [12]. In addition, several studies have shown that 2-D cell cultures and native tissues under cyclic strain will increase the synthesis of different matrix molecules, including type I collagen and small proteoglycans (PGs) such as decorin and biglycan [8,13,14].…”