Jörg Tessmar

Polyanhydride degradation and erosion

It was the intention of this paper to give a survey on the degradation and erosion of polyanhydrides. Due to the multitude of polymers that have been synthesized in this class of material in recent years, it was not possible to discuss all polyanhydrides that have gained in significance based on their application. It was rather the intention to provide a broad picture on polyanhydride degradation and erosion based on the knowledge that we have from those polymers that have been intensively investigated. To reach this goal this review contains several sections. First, the foundation for an understanding of the nomenclature are laid by defining degradation and erosion which was deemed necessary because many different definitions exist in the current literature. Next, the properties of major classes of anhydrides are reviewed and the impact of geometry on degradation and erosion is discussed. A complicated issue is the control of drug release from degradable polymers. Therefore, the aspect of erosion-controlled release and drug stability inside polyanhydrides are discussed. Towards the end of the paper models are briefly reviewed that describe the erosion of polyanhydrides. Empirical models as well as Monte-Carlo-based approaches are described. Finally it is outlined how theoretical models can help to answer the question why polyanhydrides are surface eroding. A look at the microstructure and the results from these models lead to the conclusion that polyanhydrides are surface eroding due to their fast degradation. However they switch to bulk erosion once the device dimensions drop below a critical limit.

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.

The use of poly(ethylene glycol)-block-poly(lactic acid) derived copolymers for the rapid creation of biomimetic surfaces

For many tissue engineering applications biomimetic or bioactive polymers would allow for a more precise control of cell behavior in growing tissues than has so far been possible. For this application recently developed amine reactive diblock copolymers (N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [ST-NH-PEGxPLAy]) were investigated concerning their reactivity in binding model substances. Their ability to covalently immobilize proteins on their surfaces was examined using polymer films with amine reactive surfaces. Furthermore, thiol reactive polymers were obtained by attaching N-succinimidyl 3-maleinimido propionate, a thiol reactive linker to monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [H2N-PEGxPLAy]. This allowed the immobilization of proteins carrying free thiol groups. The amine and thiol reactive polymers were characterized by 1H-NMR spectroscopy and gel permeation chromatography (GPC). Investigation of glass transitions temperatures using modulated differential scanning calorimetry proved suitability for the fabrication of polymeric scaffolds for tissue engineering applications. The functionality of the polymers was demonstrated by investigating their ability to bind model amines, like the fluorescent dye EDANS. Moreover, insulin and somatostatin were covalently attached to the active linker groups via amine and thiol groups. The polymers will permit covalently attaching different bioactive molecules, such as growth and differentiation factors, with fast and gentle procedures securing their biological activity.

Does UV irradiation affect polymer properties relevant to tissue engineering

For most tissue engineering approaches aiming at the repair or generation of living tissues the interaction of cells and polymeric biomaterials is of paramount importance. Prior to contact with cells or tissues, biomaterials have to be sterilized. However, many sterilization procedures such as steam autoclave or heat sterilization are known to strongly affect polymer properties. UV irradiation is used as an alternative sterilization method in many tissue engineering laboratories on a routine basis, however, potential alterations of polymer properties have not been extensively considered. In this study we investigated the effects of UV irradiation on spin-cast films made from biodegradable poly(d,l-lactic acid)–poly(ethylene glycol)–monomethyl ether diblock copolymers (Me.PEG–PLA) which have recently been developed for controlled cell-biomaterial interaction. After 2 h of UV irradiation, which is sufficient for sterilization, no alterations in cell adhesion to polymer films were detected, as demonstrated with 3T3-L1 preadipocytes. This correlated with unchanged film topography and molecular weight distribution. However, extended UV irradiation for 5–24 h elicited drastic responses regarding Me.PEG–PLA polymer properties and interactions with biological elements: Large increases in unspecific protein adsorption and subsequent cell adhesion were observed. Changes in polymer surface properties could be correlated with the observed alterations in cell/protein–polymer interactions. Atomic force microscopy analysis of polymer films revealed a marked “smoothing” of the polymer surface after UV irradiation. Investigations using GPC, 1H-NMR, mass spectrometry, and a PEG-specific colorimetric assay demonstrated that polymer film composition was time-dependently affected by exposure to UV irradiation, i.e., that large amounts of PEG were lost from the copolymer surface. The data indicate that sterilization using UV irradiation for 2 h is an appropriate technique for the recently synthesized Me.PEG–PLA diblock copolymers. However, the study also serves as an example that it is indispensable to control the duration of exposure to UV irradiation for a given biomaterial in order not to compromise polymer properties relevant to tissue engineering purposes.

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.

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.

Biomimetic polymers to control cell adhesion

This review describes the application of customized surface modifications using biomimetic polymers to attempt to reduce unspecific protein adsorption to the materials or promote specific cell adhesion to implants or tissue engineering scaffolds. Various polymers that are suited to suppress almost all unspecific protein interactions and underlying mechanisms of cell adhesion are presented and discussed. Suitable modifications to the inert polymers that can later favor the adhesion of specific cell types are also described. These modifications involve utilizing the binding characteristics of peptide sequences to promote specific cell attachment, and techniques to incorporate these sequences into the polymers are explained. Finally, it is shown that biomimetic surface modification techniques are also of tremendous importance for drug delivery with nanoparticles, and the opportunities and limits associated with these approaches are demonstrated.

Biomimetic Polymers (for Biomedical Applications)

The scope of this chapter is to give an overview on the current developments of biomimetic polymers in biomedical applications. This includes an introduction about cell–biomaterial interactions, applied biomimetic strategies, synthesis and modification steps for selected natural and synthetic polymers, and examples of recent studies using biomimetic polymers in gene delivery and tissue engineering.