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

Abstract

Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.

Co-Culture of Mesenchymal Stem Cells and Ligamentocytes on Triphasic Embroidered Poly(L-lactide-co-ε-caprolactone) and Polylactic Acid Scaffolds for Anterior Cruciate Ligament Enthesis Tissue Engineering

“…However, the initial adhesion of the spheroids on the scaffold needs a static phase of culture. Until now, there has been no clear consensus concerning the cultivation of ligamentous spheroids, and therefore there has been no clear consensus concerning their ECM synthesis and maturation for ligament tissue engineering [ 89 , 90 ]. For this reason, we chose the same pre-cultivation (14 days) as for the hMSC spheroids, and we saw that the long-time pre-cultivation of lACL spheroids was possible, supported by the maintained cell vitality, spheroid roundness and sGAG production.…”

Anatomical Tissue Engineering of the Anterior Cruciate Ligament Entheses

“…In addition, the selection of a cell source, fully capable of generating enthesis zone-specific cell populations in vivo, e.g., ASCs or MSCs, which are intensively researched and are easy to harvest and propagate, remains a challenge. In the case of combining different cell types or precursor cells committed to ACL zone-specific differentiation lineages, directed seeding strategies are needed, e.g., based on cell sheets or spheroids [151,152], and a mechanostimulation profile closely mimicking the unique biomechanical niche of the ACL enthesis. The development of modeling approaches for the human ACL enthesis might be very helpful in future to address some of the above-mentioned open questions, particularly concerning scaffold biomechanics.…”

“…The size of the spheroids could be adapted to the pore size. It is known that ACL ligamentocytes maintain their expression profile in spheroids [147][148][149][150][151]. Using cell sheets is another tool to place cells on a defined position on the scaffolds, leading to cell emigration on them [152].…”

Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc

“…Moreover, the material part of the spheroids could be used to sustainably release bioactive drugs (e.g., GFs) in order to fine-tune the properties of target tissue [ 169 ]. Including biomaterials during spheroid fabrication process was also shown to influence spheroid roundness in ligament tissue engineering [ 170 ].…”