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.

Alveolar Rhabdomyosarcoma (RMA) are characterised by chromosomal translocations fusing the PAX3 or PAX7 gene with FKHR in ~85%. Previous studies have suggested that PAX3/7-FKHR fusion types are related to prognosis. In order to prove these findings we performed a retrospective analysis of the PAX-FKHR fusion status and its relation to outcome in patients treated in the CWS trials.

Between 1986 and 2004, out of 446 RMA patients treated in four consecutive CWS trials (CWS-86, -91, -96 or -2002-P), tumor samples from 121 patients with adequate quality for analysis of PAX-FKHR fusion status by RT-nested PCR were available. Survival analysis depending on clinical risk factors and fusion status was performed using the Kaplan-Meier Method, the log rank test and the Cox regression model.

There were no major differences in distribution of known risk factors in the analysed cohort of 121 patients compared to all patients enrolled in the CWS trials. PAX-FKHR fusions were detected in 83%: 72 PAX3-, 29 PAX7-FKHR fusions. Patients with PAX3-FKHR positive tumors more often showed a pattern of adverse clinical risk factors (age > 10 years, primary metastases, lymph node involvement) than the PAX7-FKHR positive group. The 5-year event free survival rate of patients with initially metastatic tumors positive for either of the two fusion transcripts was significantly lower compared with the fusion transcript negative cohort and the non-analysed RMA patients. There was no significant outcome difference between patients with PAX3-FKHR and PAX7-FKHR positive tumors in uni- and multivariate analysis.

In the present analysis, which is to our knowledge the largest reported so far, PAX-FKHR fusion type was no significant predictor for prognosis, thus not supporting results of previous studies.