Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This given study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or gas fluorination combined with the foam (F+coll). Cell free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, biomechanical properties, cell vitality and content, histopathology of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed.

Implantation of the scaffolds did not negatively affect mice weight development and organs, indicating biocompatibility. All scaffolds maintained their size and shape for the duration of the implantation. A high cell viability was detected in the scaffolds prior to and following implantation. Coll or F+coll scaffolds seeded with cells yielded superior macroscopic properties when compared to the controls. Mild signs of inflammation (foreign-body giant cells, hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable in vivo, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in the in vivo compared to the samples cultured 1 week in vitro, but did not differ between scaffold subtypes, except for a higher maximum force in F+coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering.

(shared first authorship)

Acknowledgement: The study was supported by DFG grants SCHU1979/9-1 and SCHU1979/14-1.

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.

Cell sheets are manufactured from a high-density cell layer stabilized by its own freshly produced extracellular matrix (ECM). They could serve as versatile scaffolds for tissue repair. Unfortunately, their production often remains time-consuming requiring weeks of culturing. Ligament cell sheets are so far barely available. Regarding musculoskeletal tissues exposed to high repetitive biomechanical forces, the stability of cell sheets is insufficient. It could help to combine them with a biomechanical competent scaffold e.g. produced by an embroidering technique. Hence, we wanted to (1) develop a very rapid strategy to produce ACL ligamentocyte sheets within 24 h by using a thermoresponsive polymer surface, (2) use the sheets for scaffold seeding and (3) reflect the fibrocartilaginous transition zone of an ACL enthesis by combining sheets of ligamentocytes with chondrocytes or chondrogenic precursor cells as a strategy for directed seeding of two cell types on topologically different scaffold areas.

Different cell numbers of lapine ACL ligamentocytes (L-ACLs), lapine articular chondrocytes (L-ACs) and human mesenchymal stromal cells (H-MSCs) were used for sheet formation. Experiments were performed with novel, self-assembled poly(glycidyl ether) (PGE) brushes based on random glycidyl methyl ether and ethyl glycidyl ether copolymers on polystyrene 12-well cell culture plates, which allow rapid sheet formation within 24 h. Uncoated plates served as controls. Temperature-triggered detachment was performed by 10 min incubation with PBS at ambient temperature before treatment with fresh warm PBS for 5 min at 37 degrees Celsius. Harvested cell sheets were transferred on polyglycolic acid (PGA) or embroidered poly-lactic acid / poly-co-caprolactone (PLA/P[LA-CL]) scaffolds, functionalized with collagen foam and fluorine gas treatment (prepared at the IPF in Dresden and the FILK in Freiberg). Cell distribution, growth, vitality and synthesis of ECM components were monitored up to 7 days. Cell numbers required for sheet preparation (3.9 cm2) depended strongly on the cell type (L-ACLs: 0.395 mio/cm2, L-AC: 0.342 mio/cm2, H-MSCs: 0.131 mio/cm2) and was highest for L-ACLs. The majority of cells survived sheet assembly, detachment, transfer onto the scaffolds and culturing. Cells migrated from the sheets into the scaffolds and spread through the scaffolds. L-ACLs and L-ACs produced ECM and maintained their phenotypes (type II collagen and sulfated glycosaminoglycans in L-AC sheets, decorin and tenascin C in L-ACL sheets). The presence and distribution of two cell types in scaffold cocultures (L-ACLs and H-MSCs) was proven by anti-human vimentin labeling. Hence, the PGE brush surface allows rapid formation (24 h) of cell sheets.

Ligament fibroblasts must be mechanosensitive and possess sufficient adaptability to a novel mechanomilieu ensuring the permanent load capacity of the tissue. Once mechanoreceptors are activated, the fibroblasts react with a specific signal transmission (mechanotransduction), which ultimately leads to an adaption of their cytoskeletal organization and protein synthesis. However, the cellular response of anterior cruciate ligament (ACL) fibroblasts to cyclic mechanical stretching is still unclear. Hence, this study should allow a deeper understanding of the reaction profile of mechanically stretched ACL cells in two- (2D) and three-dimensional (3D) biomaterial-free and biomaterial cultures with respect to cell survival, size, orientation, migration and distribution. For the 2D approach consisting of monolayers with 6000 lapine (L) ACL cells per cm2 and for the 3D cultures using preformed LACL cell spheroids (2.5–4/cm2) with 25.000 cells per spheroid, silicone chambers were coated with geltrex and statically colonized with the LACL cells for 24 h before cyclically stretched for 48 h (14 percent uniaxial stretch). A second approach using 3D scaffold cultures was performed which were seeded dynamically for 24 h with LACL cells before cyclically stretched in a novel custom-made mechanostimulator. The scaffolds [polylactic acid (PLA) and polycaprolactone (PCL)] were functionalized with 10 percent gas fluorination and a collagen foam. Scaffolds (120 mm2) were precolonized dynamically with an LACL cell suspension (1 mio cells/mL) for 24 h before stretched for 72 h (4 percent uniaxial stretch). Cell vitality and numbers were monitored. The cytoskeleton orientation was shown by cytochemistry (F-actin) and evaluated (ImageJ). Cell proliferation, based on the DNA content was measured. Cell viability in stretched samples (2D, 3D and scaffold) remained above 90 percent. Stretching on the silicone chambers led to increased cell counts, length and significantly higher colonized areas than in unstretched controls. Higher numbers of LACL cells migrated out of the 3D spheroids under stretching conditions. In response to intermittent stretching, cells oriented in a 70 degrees' angle against the stretch direction in silicone chambers, whereas cell arrangement was more compact on the threads of the scaffolds than in unstretched cultures. In summary, stretching induced a rapid (48 h) cell and cytoskeletal alignment in 2D as well as in 3D cultures. The natural ACL is characterized by a strongly uniaxial cell and extracellular matrix organization which might be achieved in tissue engineered constructs by a suitable cyclic stretching protocol in future.

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.