Research interests of the Interface Chemistry and Biomaterials Group
Institute of Physical and Theoretical Chemistry, University of Regensburg

Three-dimensional scaffolds derived from natural polymers for cartilage tissue engineering

Esterified hyaluronan-gelatin polymer composites with improved cell binding and chondrogenic differentiation characteristics of mesenchymal stem cells

Peter Angele, Detlef Schumann, Carsten Englert, Johannes Zellner, Michael Nerlich and Richard Kujat, Department of Trauma Surgery, University Hospital of Regensburg
Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg
Brian Johnstone and Jung Yoo, Department of Orthopedic Surgery, Oregon Health and Science University at Portland (USA)
Joachim Hammer and Johann Fierlbeck, Department of Biomechanics, University of Applied Sciences Regensburg

The project was supported by the Bavarian State Ministry of Sciences, Research and the Arts within the program "High Tech Offensive Bayern"

Published in Journal of Biomedical Materials Research Part A 2009, 91, 416-427.

Abstract
Esterified hyaluronan (HY)-gelatin (G) composite scaffolds were manufactured with variable component compositions (HY100%, HY95%/G5%; HY70%/G30%). The goals of the study were to analyze the produced composite scaffolds using physical and chemical methods, e.g., scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation and chondrogenic differentiation of human mesenchymal stem cells. The composite scaffolds showed significantly higher cell attachment and proliferation compared with 100% hyaluronan scaffolds (p<0.003). In composite scaffolds, a significantly greater cartilaginous extracellular matrix was deposited after 28 days in culture (glycosaminoglycan: p<0.001; collagen: p<0.001) when compared with 100% hyaluronan scaffolds. The mechanism by which gelatin influences these parameters was examined. The effects were inhibited by collagenase treatment of the composites or by addition of a5ß1-integrin blocking antibodies to the cell suspension. These results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation.

Figure 1: Biological performance of hyaluronan benzylic ester-gelatin composite scaffolds. (A) Adhesion and proliferation of MSC on scaffolds of different compositions as determined by the XTT assay (A) and cell morphology (B) clearly demonstrate the positive influence of the gelatin constituent on cell behavior.

Stabilization of hyaluronan ester-gelatin composite scaffolds by chemical cross-linking

Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg
Torsten Blunk and Hatem Sarhan, Institute of Pharmaceutical Technology, University of Regensburg
Richard Kujat, Carsten Englert, Michael Nerlich and Peter Angele, Department of Trauma Surgery, University Hospital of Regensburg
Johann Fierlbeck and Joachim Hammer, Department of Biomechanics, University of Applied Sciences Regensburg

The project was supported by the Bavarian State Ministry of Sciences, Research and the Arts within the program "High Tech Offensive Bayern"

Publication in submission

Abstract
Hyaluronic acid and collagen are main constituents of the natural connective tissue and, therefore, they are considered as promising components for tissue engineering scaffolds. To decelerate biodegradation and to increase biomechanical strength, scaffolds consisting of hyaluronic acid benzylester and gelatin were stabilized by means of chemical cross-linking using glutaraldehyde, carbodiimide, genipin, or cyanamide. It was found that cyanamide did not enhance the stability of the hybrid material. Carbodiimide and genipin cross-linked sponges were superior in tensile strength whereas glutaraldehyde and genipin cross-linking displayed the highest resistance against enzymatic degradation. Human mesenchymal progenitor cells adhered well on carbodiimide or genipin stabilized polymer composites while glutaraldehyde cross-linking caused lower cell adhesion. Carbodiimide as well as genipin cross-linked hyaluronan-gelatin sponges enabled adequate development of cartilaginous tissue in a 3-D in vitro culture. Carbodiimide cross-linked hyaluronan-gelatin sponges showed inhibited in vivo degradation during 21 days of subcutaneous implantation in either an empty state or after loading with human mesenchymal progenitor cells.

Biodegradable hyaluronic acid ester-gelatin composite matrix for osteochondral differentiation of mesenchymal progenitor cells

Peter Angele, Richard Kujat, Hubert Faltermeier, Detlef Schumann and Michael Nerlich, Department of Trauma Surgery, University Hospital of Regensburg
Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg

The project was supported by the Bavarian State Ministry of Sciences, Research and the Arts within the program "High Tech Offensive Bayern"

Published in: BIOmaterialien 2003, 4, 11-18.

Abstract
The aim of the study was the comparison of commercially available collagen matrices with a new series of composite matrices, containing esterified hyaluronic acid and gelatin, for their potential to serve as a suitable tissue engineering scaffold for cartilage repair. The ultrastructure of collagen matrices and composite sponges were analyzed with scanning electron microscopy. Furthermore, the macroscopic changes in matrix morphology after cell loading were assessed. In contrast to collagen martrices, which showed early contraction, composite matrices revealed form stability. Both matrix types were analyzed for their potential to allow chondrogenic differentiation of culture-expanded, bone marrow-derived mesenchymal progenitor cells. In vitro, the collagen matrices showed a cartilage-like extracellular matrix production throughout the scaffold. In vivo, the collagen matrices were completely degraded after 3 weeks in contrast to the composite matrices, which were still detectable after 6 weeks. Non cell-loaded composite matrices showed complete filling with fibrous tissue whereas matrices loaded with mesenchymal progenitor cells revealed osteochondrogenic cell differentiation. The rsults suggest that commercially available collagen matrices are less suitable scaffolds for the repair of cartilage defects. Especially the change in matrix size after cell loading and culturing, the variability in extracellular matrix deposition in vitro and the quick degradation rate in vivo made it necessary to construct new biomaterials for tissue engineering. The hyaluronan ester-gelatin composite matrices meet these requirements especially for tissue engineering of cartilage, because of achieving a suitable milieu for optimized chondrogenic differentiation of mesenchmyal progenitor cells.

Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices

Peter Angele, Richard Kujat, Hubert Faltermeier, Detlef Schumann, Michael Nerlich, Bernd Kinner and Carsten Englert, Department of Trauma Surgery, University Hospital Regensburg
Jochen Abke and Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg
Zbigniev Ruszczak and Robert Mehrl, Innocoll GmbH, Saal an der Donau (Germany)

The project was supported by the Bavarian Research Foundation and the Bavarian State Ministry of Sciences, Research and the Arts within the program FORBIOMAT II

Published in: Biomaterials 2004, 25, 2831-2841.

Abstract
Collagen based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the species-related differences of collagen scaffolds with and without EDC/NHS-crosslinking. Resistance against collagenase digestion, swelling ratio, amino acid sequence, shrinkage temperature, ultrastructural matrix morphology, crosslinking density and stress-strain characteristics were determined to evaluate the physico-chemical properties of equine and bovine collagen based scaffolds. Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and time (p=0.0001) on degradation of the collagen samples by collagenase treatment. Crosslinked equine collagen samples showed a significantly reduced swelling ratio compared to bovine collagen samples (p<0.0001). The amino acid composition of equine collagen revealed a higher amount of hydroxylysine and lysine. Shrinkage temperatures of non-crosslinked samples showed a significant difference between equine (60°C) and bovine collagen (57°C). Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and matrix condition (p=0.0001) on rupture strength measured by stress-strain analysis. The ultrastructure, the crosslinking density and the strain at rupture between collagen matrices of both species showed no significant differences. For tissue engineering purposes, the higher enzymatic stability, the higher form stability, as well as the lower risk of transmissible disease make the case for considering equine based collagen. This study also indicates that results obtained for scaffolds based on a certain collagen species may not be transferable to scaffolds based on another, because of the differing physico-chemical properties.

Last update on 17.11.2009 - For more informations please contact Dr. Rainer Müller