Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts.

These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed.

Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition.

Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering.

Cultured primary cells have a limited life span and undergo dedifferentiation. Tissue engineering (TE) approaches require high cell numbers, but availability of human derived cells is limited and animal cells show inter-species differences. The advantages of immortalized cells are delayed senescence and phenotypic stability. The present study was undertaken to validate key properties of immortalized human anterior cruciate ligament (ACL) fibroblasts in direct comparison with non-immortalized cells from the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40 years old female donor) were either immortalized using repeated transient transfection with a simian virus SV40 plasmid or remained untreated. Both cell populations were analyzed for cell survival, DNA content, tendon marker, extracellular matrix (ECM) and cytoskeletal protein expression. Cell spheroids of both populations were seeded on scaffolds embroidered either from polylactic acid (PLA) threads alone or combined PLA- and PLA-co-caprolacton-(P(LA-CL)) threads, functionalized with fluor treatment and collagen foams. Cell survival on the scaffolds was monitored for up to 5 weeks. In contrast to non-immortalized ligamentocytes, immortalized cells reflected some chaotic and incomplete cell divisions, higher DNA content, numbers of dying cells and nucleoli, reduced vimentin and vinculin-associated focal adhesions. Analysed markers, other cytoskeletal and ECM components were similarly expressed. Compared to the non-immortalized ligamentocytes immortalized formed instable spheroids and died on the scaffolds after 21 d. Both cell populations reflected superior growth on the PLA-P(LA-CL) compared with PLA scaffolds. Immortalized cells share crucial properties with their non-immortalized counterparts, but TE is only possible for limited culturing periods.