Cell sheets for directed seeding of embroidered scaffolds as an approach for ACL tissue engineering

Objectives Since the anterior cruciate ligament (ACL) has only a limited healing capacity due to low cell content and poor blood supply, the ruptured ACL has to be reconstructed to guarantee knee stability. The use of ACL cell sheets combined with a biomechanically competent scaffold as a carrier might offer a promising strategy for tissue engineering an ACL substitute.

Thermoresponsive polymers allow the temperature-triggered, enzyme-free detachment of cell sheets with an intact extracellular matrix (ECM). In this study, we evaluate the applicability of coatings based on thermoresponsive poly (glycidyl ether)s (PGE) for rapid cell sheet production (within 24 h). Further, the aim of the study was to characterize the phenotype of lapine ACL cells

(L-ACLs) in the harvested cell sheets and in embroidered and functionalized polylactide-co-caprolactone P(LA-CL) scaffolds colonized by cell sheets.

Methods 5-8 x 100000 L-ACLs cm-1 from 3 different donors were seeded on a 12-well cell culture plate coated with PGE (w PGE) for 24 h. As controls, cells were grown on cell culture plates without PGE (wo PGE) coating and as monolayers (ML) (2.4 x 10000 cm-1). After detachment with PBS at various temperatures, the cell sheets were cultivated for 7 days (d) in a 24-well ultra-low attachment plate. In addition, cell sheets were carefully wrapped around an embroidered P(LA-CL) scaffold functionalized with fluorinated collagen foam and cultured for 7 d. Cell vitality (LIVE/DEAD ASSAY), protein (immunohistochemical staining) and gene expression (q-PCR) were determined for the cell sheets (w/wo PGE), scaffolds (w PGE) and the ML at 0 and 7 d. For analysis paired one-way ANOVA with GraphPad Prism was used.

Results and Conclusion Intact cell sheets could be produced within 24 h with cell culture plates w/wo PGE. The time for cell detachment from the plate w PGE compared to those wo PGE was significantly lower. The surface of the cell sheets was extremely significantly smaller after detachment than the original growth area and decreased significantly during the 7-d cultivation period while the cell sheets formed a three-dimensional construct. L-ACLs cell sheets were able to adhere to embroidered scaffolds and cells spread onto the scaffold. The vitality of the sheets and cells on the scaffolds did not decrease significantly after 7 d. Relative gene expression of type I collagen and the ligament transcription factor mohawk decreased in cell sheets (w/wo PGE) significantly compared to the ML after 7 d. However, the relative gene expression of collagen, mohawk, tenascin C and decorin tended to be upregulated in cell sheet colonized scaffolds in comparison to sheets and ML.

It could be shown that PGE coating facilitates the rapid production of cell sheets while cell sheets and colonized scaffolds show no decrease in cell vitality. Although pure cell sheets are not suitable for tissue reconstruction due to the decrease in collagen and mohwak, the cell sheet colonized scaffolds are a promising approach.

Stichwörter cell sheet, ACL, thermoresponsive polymer, embroidered scaffold, tissue engineering

Publication History

Article published online:
15 October 2020