Maintenance of Ligament Homeostasis of Spheroid-Colonized Embroidered and Functionalized Scaffolds after 3D Stretch

Abstract

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.

In vivo ligamentogenesis in embroidered poly(lactic-co-ε-caprolactone) / polylactic acid scaffolds functionalized by fluorination and hexamethylene diisocyanate cross-linked collagen foams

“…2b). When calling up a previous study where we determined a mean DNA content of 2.1 ± 1.2 µg/cm 3 in the native LACL (Gögele et al 2021), the DNA contents in the scaffold variants of this study were substantially higher than that of the native LACL.…”

“…The combination of P(LA-CL) with PLA allowed lapine ligamentocyte adherence and a biomechanical competence of the construct comparable to the native lapine ACL (LACL) (Hahn et al 2019). The functionalization of the material combination with gas-phase fluorination and collagen provided promising properties in several in vitro studies (Gögele et al 2020a(Gögele et al , 2021Hahn et al 2019).…”

Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

“…In previous work, we could show that cruciate ligamentocyte spheroids are suitable for the colonization of synthetic polymer scaffolds [18,19,24]. When spheroids [4,22] or cell sheets [32] are seeded onto the scaffolds, cells emigrate and spread onto the scaffolds. However, high numbers of spheroids are required for tissue engineering; hence, high-throughput strategies for spheroid formation are needed.…”

“…With the hanging drop method, around 15 dishes with 9 cm diameters are needed, each containing 50 spheroids to achieve the same number of spheroids. The conventional hanging drop method is widely used for spheroid production [2,4,14,16,19]. However, this culture has some further limitations.…”

“…These cell-cell communications might stabilize the differentiated phenotype of primary cells. Spheroids can be produced using many cell types and techniques [1] including tenocytes and ligamentocytes [2][3][4]. They can be used as 3D tumor models for the screening of agents and strategies effective to inhibit tumorigenesis [1,5].…”