gms | German Medical Science

Objectives: Ruptures of the anterior cruciate ligament (ACL) are among the most common ligament injuries. In order to avoid consequential damage of joint cartilage, tissue engineered ACL reconstruction is becoming increasingly important. The aim of this study was to evaluate the influence of cyclic stretch on primary lapine ACL (LACL) derived fibroblasts on an embroidered poly(l-lactide-co-caprolactone) and polylactic acid scaffold.

Methods: Scaffolds (30x4x1mm) were non (control)- and functionalized with 10% gas fluorination and infiltrated with a collagen foam cross-linked with hexamethylene diisocyanate. The scaffolds were pre-colonized with LACL fibroblasts with cell suspension- or spheroid-based strategies. For the suspension pre-cultivation the scaffolds were dynamically colonized for one day on a rotatory device. For the spheroid pre-cultivation the scaffolds were colonized statically for 5 days until spheroid adherence. After the pre-cultivation the scaffolds were cyclically stretched (4% stretch, 0.18 Hz) for 3 days. Cell survival was tested with a vitality assay and the migration depth into the scaffold was evaluated. The proliferation marker (ki67) and the myofibroblast marker alpha smooth muscle actin (SMA) were detected by immunocytochemical staining. DNA and sulfated glycosaminoglycan (sGAG) contents were quantitatively measured. Relative gene expression of type I collagen, decorin, tenascin C, tenomodulin, Mohawk and connexin 43 were analyzed.

Results and Conclusion: Stretch induced cell spreading and a more homogeneous cell distribution, especially on the functionalized scaffolds without impairing cell vitality. Stretch led to a significant higher colonized scaffold surface on the functionalized scaffolds seeded with a cell suspension in comparison to the stretched control scaffolds or unstretched functionalized scaffolds. A similar trend could be observed in scaffolds seeded with spheroids. The migration depth into inner scaffold parts was larger in the stretched control scaffolds seeded with spheroids compared to the functionalized and stretched variants. The protein expression of ki67 and SMA was higher in stimulated and functionalized scaffolds irrespectively, whether seeded with spheroids or cell suspension. The DNA content was higher in functionalized and stretched scaffolds. Stretch induced a higher sGAG content in functionalized scaffolds seeded with spheroids in comparison to those colonized with a cell suspension. Stretched functionalized scaffolds seeded with spheroids showed a higher gene expressions of type I collagen, decorin, tenomodulin and Mohawk in comparison to those stretched after pre-seeded with a cell suspension. A scaffold functionalization facilitates cell adherence, spreading and sGAG synthesis. The spheroid colonization method is a valuable tool for ligament tissue engineering. Cyclic stretch induced some distinct effects such as enhanced cell spreading and upregulated tenascin C gene expression. The stretch protocol is to be revised.