Michaela Schulz

Basic fibroblast growth factor enhances PPARγ ligand-induced adipogenesis of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are capable of differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and differentiation factors. It is widely acknowledged that basic fibroblast growth factor (bFGF) modulates chondrogenic and osteogenic differentiation of MSCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate on the cellular and molecular level that supplementation of bFGF in different phases of cell culture leads to a strong enhancement of adipogenesis of MSCs, as induced by an adipogenic hormonal cocktail. In cultures receiving bFGF, mRNA expression of peroxisome proliferator‐activated receptor γ2 (PPARγ2), a key transcription factor in adipogenesis, was upregulated even prior to adipogenic induction. In order to investigate the effects of bFGF on PPARγ ligand‐induced adipogenic differentiation, the thiazolidinedione troglitazone was administered as a single adipogenic inducer. Basic FGF was demonstrated to also strongly increase adipogenesis induced by troglitazone, that is, bFGF clearly increased the responsiveness of MSCs to a PPARγ ligand.

Poly(D,L-lactic acid)-Poly(ethylene glycol)-Monomethyl Ether Diblock Copolymers Control Adhesion and Osteoblastic Differentiation of Marrow Stromal Cells

Biodegradable polymers, such as poly(lactic acid) (PLA) and poly(lactic-coglycolic acid) (PLGA), are attractive materials for tissue engineering because of their degradative and mechanical properties, which permit scaffolds to be tailored to the individual requirements of different tissues. Although these materials support tissue development, their chemical properties offer no control of cell adhesion or function because their surfaces become immediately masked by adsorbing serum proteins when the materials come into contact with body fluids. Furthermore, adhesion proteins undergo conformational changes and a decrease in bioactivity when adsorbed to hydrophobic materials, such as PLA. To overcome these limitations, we modified the properties of PLA by synthesizing a diblock copolymer with poly(ethylene glycol) (PEG), which is known to reduce the amount of adsorbed proteins and to modify their conformation. By altering the PEG content of these diblock copolymers we were able to control the adsorption of adhesion proteins and, because cell adhesion takes place only in the presence of serum proteins, to control cell adhesion and cell shape. Marrow stromal cell differentiation to the osteoblastic phenotype was strongly improved on PEG-PLA compared with PLA, PLGA and tissue culture polystyrene and led to a 2-fold increase in alkaline phosphatase activity and mineralization.

Towards biomimetic scaffolds: Anhydrous scaffold fabrication from biodegradable amine-reactive diblock copolymers

The development of biomimetic materials and their processing into three-dimensional cell carrying scaffolds is one promising tissue engineering strategy to improve cell adhesion, growth and differentiation on polymeric constructs developing mature and viable tissue. This study was concerned with the fabrication of scaffolds made from amine-reactive diblock copolymers, N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid), which are able to suppress unspecific protein adsorption and to covalently bind proteins or peptides. An appropriate technique for their processing had to be both anhydrous, to avoid hydrolysis of the active ester, and suitable for the generation of interconnected porous structures. Attempts to fabricate scaffolds utilizing hard paraffin microparticles as hexane-extractable porogens failed. Consequently, a technique was developed involving lipid microparticles, which served as biocompatible porogens on which the scaffold forming polymer was precipitated in the porogen extraction media (n-hexane). Porogen melting during the extraction and polymer precipitation step led to an interconnected network of pores. Suitable lipid mixtures and their melting points, extraction conditions (temperature and time) and a low-toxic polymer solvent system were determined for their use in processing diblock copolymers of different molecular weights (22 and 42 kDa) into highly porous off-the-shelf cell carriers ready for easy surface modification towards biomimetic scaffolds. Insulin was employed to demonstrate the principal of instant protein coupling to a prefabricated scaffold.

Insulin in tissue engineering of cartilage: a potential model system for growth factor application.

Investigation of novel experimental application systems for growth factors or other bioactive substances in tissue engineering is often limited by high costs of substances and would benefit from a defined and easily controllable model tissue system. Herein, we demonstrate a potential three-dimensional in vitro system using engineered cartilage as a model tissue and readily available insulin as a model drug. Previously it has been shown that insulin-like growth factor-I (IGF-I) has profound effects on tissue-engineered cartilage in vitro. Insulin is known to bind to the IGF-I receptor and to elicit significant responses in cartilage. In this study, bovine articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured for up to 7 weeks. Exogenous insulin (0.05-50 μg/ml) increased the growth rate and the glycosaminoglycan fraction of tissue-engineered cartilage, decreased the cell number in the tissue constructs, and improved the morphological appearance, with 2.5 μg/ml being the most favorable concentration. The observed effects of insulin were similar to effects of IGF-I (0.05 μg/ml) and were in agreement with the reported binding constants of IGF-I and insulin at the IGF-I receptor. Besides the possibility to employ insulin as a potent substance to improve tissue-engineered cartilage, the presented easily controllable in vitro system may be used in the future to evaluate experimental growth factor application devices using economically favorable insulin as a model protein.

Effects of Hedgehog Proteins on Tissue Engineering of Cartilage in Vitro

The effects of three derivatives of the N-terminal signaling domain of hedgehog proteins on cartilage engineered in vitro were investigated, with specific focus on the ability to increase tissue growth rate and concentrations of major extracellular matrix components, that is, glycosaminoglycans (GAG) and collagen, and on the effects on morphological appearance of the tissue. Bovine articular chondrocytes were cultured on biodegradable polyglycolic acid (PGA) scaffolds with or without the addition of dipalmitoylated sonic hedgehog (dp-shh), dipalmitoylated indian hedgehog (dp-ihh), or sonic hedgehog dimer (shh-dimer) to medium with either 1% or 10% fetal bovine serum (FBS). All three hedgehog proteins dose-dependently increased construct weights (by up to 1.95-fold, dp-shh at 1,000 ng/mL) and the fraction of GAG over 4 weeks (by up to 2.7-fold, dp-shh at 1,000 ng/mL), as compared to control constructs. Dp-shh and dp-ihh elicited similar responses; a 10-fold higher concentration of nonacylated shh-dimer was necessary to reach comparable results. Positive hedgehog effects were more pronounced in medium containing 1% FBS than in medium containing 10% FBS; however, at either FBS concentration, cartilaginous tissues grown in the presence of hedgehog proteins appeared morphologically more mature. Hedgehog derivatives thus appear as promising candidates to improve the development and composition of engineered cartilage.

Toward the Development of Biomimetic Polymers by Protein Immobilization: PEGylation of Insulin as a Model Reaction

Many current tissue-engineering investigations aim at the rational control of cell adhesion and tailored composition of biomaterial surfaces by immobilizing various protein and peptide components, such as growth factors. As a step on the way to develop polymers that allow for such surface modifications, water-soluble polymers were used as model substances to examine reactions with proteins containing amine groups. Consequently, the uncommon PEGylation of insulin in aqueous buffers was used to characterize reaction products and simulate the intended immobilization step for surface modification. Amine reactive poly(ethylene glycol)s were synthesized and characterized by (1)H nuclear magnetic resonance and gel-permeation chromatography. Furthermore, the model protein insulin was characterized concerning its accessible amino groups, using a fluorescent dye (TAMRA-SE). The resulting reaction products were identified by reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. After PEGylation with hydrolytically stable poly(ethylene glycol) succinimidyl ester, the obtained PEGylated insulin was investigated by gel filtration chromatography, indicating successful attachment of the hydrophilic polymer chains. Application of an aqueous PEGylation scheme opens the door to the immediate investigation of various growth factors in cell culture, allowing for direct assessment of biological activity after forming the polymer-protein constructs with regard to later immobilization on surfaces.

PEGylation Does Not Impair Insulin Efficacy in Three-Dimensional Cartilage Culture: An Investigation toward Biomimetic Polymers

A major goal in tissue engineering is the controlled application of growth factors. As a novel application system, we are currently developing biomimetic polymers that are processed into three-dimensional scaffolds. Bioactive proteins will be covalently bound to the polymers via a poly(ethylene glycol) (PEG) linker. Of paramount importance is the maintenance of the biological activity of the protein after PEGylation and covalent binding to the polymer. Therefore, within this study, insulin used as a model protein was PEGylated with an active succinimidyl ester of poly(ethylene glycol) (SS-NH-PEG) (MW ~2000) and biological effects of the protein-PEG conjugate were monitored in comparison with unmodified insulin. No significant differences in chondrocyte proliferation were observed in a conventional proliferation assay after treatment with insulin or PEGylated insulin. In a complex three-dimensional cartilage-engineering model the effects of insulin and PEGylated insulin were investigated over a wide concentration range (0.025-25 microg/mL). Insulin and PEGylated insulin at equivalent concentrations resulted in cartilaginous tissue constructs exhibiting identical wet weight, cell number, biochemical composition of the extracellular matrix, and histological appearance, both compounds significantly improving tissue quality as compared with control constructs. In conclusion, the presented study demonstrates that PEGylation of insulin using SS-NH-PEG did not change the activity of the protein in a complex biological environment and is regarded as a step toward the development of biomimetic polymers.