gms | German Medical Science

Objectives: An anterior cruciate ligament (ACL) rupture is one of the most common ligament injuries in the knee joint. Due to poor healing ACL reconstruction is required to avoid consequential damage of the joint cartilage. To prevent implant loosening firm ligament-bone integration is needed. Tissue engineered ACL substitutes attract more and more interest as a future option. The aim of this study was to examine in more detail the enthesis that mediates bone attachment and absorbs mechanical stress at the integration site. For this purpose, osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L)ACL fibroblasts were co-cultured on a triphasic scaffold embroidered from poly(L-lactide-co-caprolactone) and polylactic acid.

Methods: Triphasic embroidered scaffolds (17.8 x 4 x 1 mm) with bone, cartilage, ligament zones were functionalized with 10% gas fluorination and infiltrated with a collagen foam crosslinked with hexamethylene diisocyanate. The scaffolds were colonized with spheroids from LACL fibroblasts precultured for 14 days and hMSCs which were for 14 days osteogenically differentiated and cultivated further for 14 days. As controls, spheroids were used without prolonged pre-cultivation or with similar differentiation as used for colonization. The survival of the cells was tested with a vitality test. Histological staining with picrosirius red and alizarin red were performed. Vimentin associated with human MSCs and collagen type I in human and lapine cells were detected by immunocytochemical staining. The content of DNA and sulfated glycosaminoglycan (sGAG) was measured quantitatively. The relative gene expression of tenascin C, type I and X collagens was analyzed.

Results and Conclusion: Compared to the spheroids from LACL fibroblasts the undifferentiated and differentiated hMSC spheroids adhered better to the scaffold surface and grew faster. Vimentin and type I collagen were mainly expressed by the hMSCs in the bone part, in which Vimentin was increasingly synthesized from the undifferentiated hMSCs. In the ligament part, type I collagen was more expressed in the differentiated LACL fibroblasts. The DNA content was higher in the bone than in the ligament zones and in the undifferentiated parts higher than in the differentiated. The sGAG content was higher for the differentiated parts than for the undifferentiated. The spheroids on the scaffold parts had a lower gene expression than the separate spheroids. The undifferentiated hMSC spheroids displayed a higher gene expression of type I and X collagens than the differentiated hMSC spheroids. The longer pre-cultivated LACL fibroblasts spheroids had a highly upregulated gene expression of type I collagen and tenascin C. Zonal colonization of triphasic scaffolds using the spheroid method is possible within 14 days, offering a novel approach for enthesis tissue engineering.