Artikel
Fluorinated embroidered PLA/+P[LA-CL]-collagen composites as strong and cytocompatible frameworks for a tissue engineered anterior cruciate ligament
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Veröffentlicht: | 22. Oktober 2019 |
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Gliederung
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Objectives: Reconstruction of ruptured Anterior cruciate ligaments (ACLs) is limited by the availability and donor site morbidity of suitable autografts. Hence, a tissue engineered graft could present an alternative in future. A biomechanically competent ACL scaffold can be realized by an embroidering technique using polylactic acid (PLA) as upper and PLA with polycaprolacton (PCL) as lower threads. Since cell adhesion to untreated PLA is non-satisfying, the embroidered structure can be supplemented with collagen foams mimicking the natural extracellular matrix (ECM) of the ACL to increase not only cytocompatibility but also cell adhesion. The cytocompatibility of PLA as well as adhesion of collagen to PLA can be increased by gas phase fluorination of the material surfaces. To stabilize the collagen/fluorinated scaffold composite an optimized cross-linking strategy must be applied.
The aim of the project is to determine the influence of fluorination and two cross-linking techniques [using either Ethylcarbodiimide (EDC) or Hexamethylen-Diisocyanate (HMDI)] on cell adhesion, growth and ECM production of lapine (L) ACL cells.
Methods: Four scaffolds variations (only collagen, only 10 % fluor, HMDI + 10 % fluor + collagen, EDC + 10 % fluor + collagen) were each colonized with 500000 cells in a dynamic, rotating cell culture system for 7 d and 14 d. Cell viability was assessed by performing a live/dead staining and vitality assay after cultivation time. Based on these results a determination of the colonized area could be done using Image J analysis. Cell numbers were calculated from the DNA content using the CyQuant assay. The quantification of sulfated glycosaminoglycans (sGAG) was carried out by DMMB assay. The metabolic activity of the cells was tested by Alamar Blue assay. Direct cell-material interaction was visualized by the scanning electron microscopy (SEM).
Results and conclusion: None of the four scaffold variations was cytotoxic. All four variations could be colonized in the rotating culture system. The HMDI cross-linker showed the largest colonized area (around 20 % of the scaffold) and a significant higher cell number per scaffold in comparison to the other three variations. LACLs were much more stretched and displayed multiple focal adhesion points on the HMDI-linked composite in comparison to the control, where cells are round shaped. The sGAG production was the highest in the control group (only collagen with no fluor and no cross linking) and low at the HMDI group after 14 d.
In this study we could show, that cells benefit substantially from a functionalization with flour and collagen. The PLA/+P[LA-CL]-collagen composite can be stabilized using HMDI as a suitable cross-linking strategy. In future the colonization will be performed with 3D cell aggregates (spheroids) which allows directed seeding ACL cells with their own pericellular ECM.