Hyalocytes in tissue engineering [Elektronische Ressource] : first steps towards a cell-based vitreous substitute / vorgelegt von Florian Sommer

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• Gesundheit

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 103
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

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Hyalocytes in Tissue Engineering

First Steps Towards a
Cell-based Vitreous Substitute



Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)
der Fakultät Chemie und Pharmazie
der Universität Regensburg










vorgelegt von
Florian Sommer
aus Hawangen
im Oktober 2006

Diese Doktorarbeit entstand in der Zeit von Januar 2003 bis September 2006 am
Lehrstuhl Pharmazeutische Technologie an der Universität Regensburg.



Die Arbeit wurde von Prof. Dr. Achim Göpferich angeleitet.































Promotionsgesuch eingereicht am: 24.10.2006

Mündliche Prüfung am: 16.11.2006

Prüfungsausschuss: Prof. Dr. S. Elz (Vorsitzender)

Prof. Dr. A. Göpferich (Erstgutachter)

PD Dr. C. Framme (Zweitgutachter)

Prof. Dr. J. Heilmann (Drittprüfer)
Hyalocytes in Tissue Engineering
Table of Contents


Chapter 1 Ocular Tissue Engineering .............................................. 5


Chapter 2 Introduction and Goals of the Thesis ............................ 31


Chapter 3 Culture Conditions for Primary Hyalocytes.................. 37


Chapter 4 Analytical Methods to Quantify Extracellular Matrix
Components Accumulated by Hyalocytes .................... 53


Chapter 5 Ascorbic Acid Modulates Proliferation and Extracellular
Matrix Accumulation of Hyalocytes ............................. 69


Chapter 6 Pyruvate Modulates the Effect of Ascorbic Acid on
Hyalocytes.....................................................................91


Chapter 7 Modulation of Hyalocyte Proliferation and ECM
Accumulation via bFGF and TGF- β1 ......................... 109


Chapter 8 Three-Dimensional Hyalocyte Culture Systems ......... 129


Chapter 9 FACS as Useful Tool to Study Distinct Hyalocyte
Populations..................................................................149


Chapter 10 Summary and Conclusions.......................................... 163


Appendix Abbreviations..............................................................171
Curriculum vitae..........................................................173
List of Publications...................................................... 175 Acknowledgements.....................................................179
- 3 - Hyalocytes in Tissue Engineering

- 4 - Chapter 1 Ocular Tissue Engineering
Chapter 1
Ocular Tissue Engineering
Florian Sommer, Ferdinand Brandl, Achim Göpferich
Institute of Pharmaceutical Technology, Department of Chemistry, University of Regensburg,
Universitätsstraße 31, 93040 Regensburg, Germany






In ‘Tissue Engineering’, Adv Exp Med Biol, Fisher J P (ed.), 585 (2006), in press as a review
- 5 - Chapter 1 Ocular Tissue Engineering
Introduction
In the early 1990s, tissue engineering emerged as a new concept to overcome the problem of
tissue and organ failure. It proposed to supply engineered, yet biological, organ and tissue
substitutes. It was anticipated that this technology would soon allow us to overcome donor
shortages and graft rejection, the major limitations of tissue and organ transplantation. Tissue
engineering approaches that were developed on the basis of this paradigm relied on the use of
cells and stem cells, preferably of autologous origin, the application of growth factors and
1, 2cytokines, the design of biodegradable scaffolds and bioreactor technology .
Over the past decades, there has been tremendous progress towards the regeneration of tissues
3 4 5 6such as bone , heart valves , myocardial tissue and cartilage . While these examples
impressively show that tissue engineering technology holds great promise for the manufacture
of tissue grafts, even more diverse applications have emerged in recent years. Tissue
7constructs have been used to investigate cellular and molecular mechanisms , are used for in
vitro drug screening and can be expected to reduce the number of time and cost intensive in
8vivo experiments in drug development . Despite this success, one may still question, why
tissue engineering has not progressed even faster and further.
Obviously, we underestimated some of the obstacles on the way towards the development of
functional tissue-engineered grafts. Frequently, the host tissue fails to support the integration
of engineered tissue. In many cases wound healing processes leading to scar formation
dominate over the intended tissue repair and biodegradable scaffolds frequently raise concerns
9due to the risk of inflammatory responses . With increasing size, engineered tissues also
suffer from insufficient nutrient availability and limited metabolic waste removal by passive
diffusion, resulting in cell death and necrosis. A rapid and adequate vascularization of an
implanted tissue has, therefore, been identified as an essential prerequisite for its survival and
integration. Induction of angiogenesis is recognized as one of the most critical factors to the
10, 11success of tissue engineering . Although growth factors, such as vascular endothelial
growth factor (VEGF) or basic fibroblast growth factor (bFGF), are potent angiogenic factors,
their use is associated with problems spanning from limited in vivo stability to an abnormal
12, 13growth of blood vessels resembling the vascularization of tumor tissue .
For the reasons outlined above, it would be advantageous to focus our tissue engineering
efforts on systems that display less complexity. With these role models, it would be possible
to gather experience that helps in the future to solve problems related to the regeneration of
more complex tissues. Ocular tissues seem an ideal candidate for this strategy. Most of them,
such as the corneal epithelium or the retinal pigment epithelium (RPE), are not vascularized
- 6 - Chapter 1 Ocular Tissue Engineering
and resemble more sheet-like than three-dimensional structures. Nutrients and oxygen are
sufficiently supplied by diffusion from adjacent tissues and, finally, parts of the eye enjoy an
immune privilege that adds additional degrees of freedom with respect to the choice of
materials and cells.
Altogether, ocular tissues seem to be predestined for regeneration using tissue engineering
approaches. But besides the scientific and strategic incentive for reconstructing ocular tissues,
there is also a tremendous need for novel therapeutic options for treating numerous eye
diseases related to tissue failure. Age-related macular degeneration (ARMD), glaucoma and
diabetic retinopathy (DR) are leading causes of blindness. The prevalence of these diseases
14among persons aged over 50 is between 3 and 10 % , illustrating the significance of the
problem. Despite the tremendous medical progress made in recent years, especially in
ophthalmology, the prevalence of age-related blindness is still increasing, spurred by
15, 16demographic trends , outlining the need for alternative treatments.
This article will review the state of the art in ocular tissue engineering. The goal is to illustrate
the progress already made and the strides still necessary to create clinically relevant tissue
substitutes.
Corneal Tissue Engineering
The cornea is the transparent barrier between the eye and the environment, protecting the eye
from pathogenic microbes and dryness. The cornea is comprised of three major cellular
layers: an outermost stratified squamous epithelium, a stroma with corneal fibroblasts
17(keratocytes), and an innermost monolayer of specialized endothelial cells (Figure 1). In
severe diseases of the cornea, their transparency is no longer maintained, usually due to a
malfunction of only one of the three parts of the cornea. Therefore, tissue engineering
developments focus on the reconstruction of the damaged part to restore transparency of the
whole cornea. These strategies, especially the regeneration of the corneal epithelium, will
probably be clinically approved in the near future.
Corneal Epithelium
The corneal epithelium consists of five cell layers in the tissue center and about ten layers on
its periphery. It shows a distinct physiological turnover; the cells are constantly renewed by
18, 19proliferating cells of the basal epithelium, often termed transient amplifying cells . These
20cells can divide only a limited number of times and are themselves replaced by slowly
21proliferating stem cells of the limbus . The limbus is surrounding the cornea; it was
- 7 - Chapter 1 Ocular Tissue Engineering
demonstrated to be a reservoir of corneal epithelial stem cells, cells that are, therefore, also
termed limbal stem cells. If these corneal epithelial stem cells are completely absent due to
limbal disorders from severe trauma (for example thermal or chemical burns) or eye diseases
(for example St