In vitro tissue growth and development in fibrin gel remodeled by neonatal smooth muscle cells

A tissue growth and development process occurred in neonatal SMC-fibrin gel constructs when cultured in DMEM supplemented with TGF-β1 and insulin over a 5 week period. These constructs may thus serve as the basis for cardiovascular tissue replacements. Following fibrin gel contraction during week 1, maximum SMC proliferation, collagen production and tropo-elastin production occurred between weeks 1-4. Organized, cross-linked collagen and elastic fibers replaced the degrading fibrin over weeks 3-5 and were manifested as increased mechanical strength. The period of maximum SMC proliferation (weeks 1-2) preceded that for maximum collagen production (weeks 2-4), which was consistent with the 3 week time point of maximum expression of collagen type I and III from qRT-PCR. Insoluble elastin quantification revealed that the majority of elastic fibers were produced by week 4, which was also consistent with the qRT-PCR data showing a dramatic down-regulation of tropo-elastin expression by week 4, indicating elastogenesis occurred during the early stages of tissue growth and development. There was a strong up-regulation of lysyl oxidase expression during weeks 1-3 with a peak in expression at week 3, correlating with the phases of collagen and tropo-elastin production. The down-regulation of lysyl oxidase after week 3 was not as extreme as with tropo-elastin, but more similar to collagen I and III, indicating the potential for an extended cross-linking phase. The expression of the collagenase MMP-1 was strongly down-regulated from its low initial level, suggesting the collagen produced was not being significantly degraded. The increase in gelatinase MMP-2 expression over weeks 1-5 suggested an increase in fibrin degradation as the tissue developed. The remodeling of the initial fibrin gel to SMC-derived ECM was manifested by increasing tensile strength, even doubling over weeks 4-5 when production of collagen and elastic fibers and expression of lysyl oxidase were subsiding. This may have been due in part to the more organized collagen fibrils evident from the histological sections in weeks 3-5. These sections also revealed evidence of a layered ECM and smooth muscle formation. A tubular construct made with this process exhibited a burst pressure exceeding 1100 mm Hg as well as a physiological tensile modulus.