Funktionalisierung von PLLA-Nanofasern mittels integrinbindender RGD-Sequenzen im Rahmen des Tissue Engineering

In clinical practice the adressing of bone defects with less availability of the autologous material are major challenges. The Tissue Engineering offers a sufficient method in order to produce bone substitutes. In this case autologous material from the patient is removed, cultured in laboratory conditions (in vitro) and replanted to the human organism. In previous studies biocompatible scaffolds made of collagen fibre during electrospinning processes were developed and enabled a good approach and differentiation of human mesenchymale stem cells (hMSC). The disadvantage of those Collagen-Scaffolds is a lesser mechanical stability. The results of current research showed that the osteoinductive effect of collagen is due to an amino acid sequence Arginin-Glycin-Asparaginsäure (RGD). The aim of this study was to relate the osteoinductive effect of the collagen with the influence of RGD-sequences on mechanical more stable Poly(lactid)Nanofibres (PLLA). For this, the cell culturing of hMSCs was carried out over a period of 22 days under osteoinductive terms and growth conditions. In this study quantitative determinations of hMSC differentiation markers such as osteocalcin, collagen and alkaline phosphatase over real time PCR and fluorescence microscopy were performed. We compared a linear and cyclic RGD-sequence with each other and investigated various methods of introducing sequences into the fiber. The RGD sequences were introduced by suspension and emulsion into the fiber as well as after plasma treatment of the PLLA scaffolds and linking of EDC (1-ethyl-3 (3dimethylaminopropyl) carbodiimide) and NHS (Nhydroxysulfosuccinimide) to the surface of the fiber. The structural changes within the fiber characteristics of different procedures were evaluated by electron microscopy (assessment of the fiber diameter and contact angle) and by a tensile testing machine (assessing the tear strength). In our study we were able to display that the cyclically arranged RGD sequence has significant higher cell differentiation (p <0.016) compared with the linear variation yet same cell number. The sequences which were introduced by emulsion offered no significant advantage towards the fibers introduced by suspension (p> 0.05). The coupling method using plasma treatment resulted in increased osteoinductivity (increase of gene products from the real time PCR of alkaline phosphatase, osteocalcin and collagen) compared to the PLLA fiber. Moreover, there were no significant changes within the fiber characteristics by different incorporation and coupling methods. Taking all aspects into consideration we could show in this study that all performed methods are suitable for the incorporation of osteoinductive RGD sequences. The cyclic variation of RGD is more inductive than the linear one and increases cell differentiation in particular as a surface contact by plasma treatment of the fiber. However, it is necessary to find a verification procedure which is able to measure the quantity of the RGD sequences in the fiber surface. So the osteoinductive effect will be optimized and the scaffolds will be a possible medium for tissue engineering.