Table of Contents:
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
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
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.