Establishment of a 3D-Tissue-Engineering-Model to Fabricate Connective Tissue

Target: Tissue engineering is a biomedical technology, which has the potential to both repair damaged tissue and to create tissues de novo. There are various different complex methods, which have been previously described to generate a three-dimensional (3D) tissue; however, no simple methods have been presented yet for the reconstruction of connective tissue for dental treatment. The aim of this present study was to establish a simple method to fabricate connective tissue within a 3D tissue engineering model. Parameters including size, thickness, vitality and self-production of collagen fibers were assessed in order to characterize the fabri-cated tissues. Materials and Methods: Gingival probes obtained from 5 different patients were extracted during parodontal surgery and brought into cell culture. The cultivated fibroblasts were firstly used to produce cell-collagen suspensions (2.5 x 10^6 cells/ml), which was then transferred into our novel designed Teflon chambers. This procedure was repeated three times for three consecutive days, while the cell suspensions were cultivated in the presence of ascorbic acid rich medium. The tissues were incubated for a total of six days and thereafter subsequently fixed and removed out of the chambers for histological assessment. The tissues were assessed for: 1) thickness and homogeneity of fibroblast arrangement in the collagen scaffold via Hematoxylin and Eosin (H.E.) staining; 2) cellular apoptosis with Transferase Uridyl Nick End Labeling (TUNEL) assay; and 3) self-fabricated collagen with Sirius Red staining. Results: All 5 tissue probes demonstrated a compact and multilayered connective tissue re-production. An average tissue thickness of 1.7mm and surface area of 1.69cm2 were obtained respectively. Histological analyses showed a dense collagen scaffold with varying fibroblast arrangements (homogenous up to accumulated to one tissue terminal), while the vast of the tissue was vital. In all tissues a self-production of collagen was detected. Conclusion: With our novel 3D tissue engineering model connective tissue reconstructions can be fabricated easily and reproducible. This method offers a good foundation for following researchers with the aim to produce connective tissue for in vivo usage for dental applications.2015-09-2