Claudia Fischbach1, Jörg Tessmar1, Andrea Lucke1, Edith Schnell2, Georg Schmeer2, Torsten Blunk1, Achim Göpferich1
1Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany
2Department of Physical and Theoretical Chemistry, University of Regensburg, 93040 Regensburg, Germany
Surf. Sci. 491 (2001), pp. 333-345
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 hours 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 hours 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. AFM 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 indicates that sterilization using UV irradiation for 2 hours 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.
Introduction
Biodegradable polymers such as poly(a-hydroxyesters) are well established biomaterials in the field of tissue engineering. Their use as membrane or scaffold material providing 2-D and 3-D matrices for cell adhesion and subsequent development of transplantable tissues is commonly acknowledged [1]. A major challenge in guided tissue regeneration is to control the interactions between polymeric carrier and cells [2]. Polymer bulk and surface properties, e.g., chemical composition, hydrophobicity, and topography, determine cell adhesion [3-6] and may also influence cell differentiation [3], which in turn may decisively influence tissue development. A key prerequisite for the use of polymeric cell carriers both in vitro and in vivo is sterility. Therefore an effective and non-destructive sterilization method is needed that maintains the characteristics of the carrier. Because of the susceptibility of many biodegradable polymers to degradation and deformation of highly porous polymer scaffolds at high temperature and pressure, steam autoclave and heat sterilization cannot be applied [7]. Ethylene oxide (ETO) or g-irradiation are used for sterilization in some tissue engineering approaches, however, also with these methods significant changes in properties of biodegradable poly(a-hydroxyesters) such as degradation and shrinking have been observed [5,8]. Furthermore, the potential toxicity of residual ETO [9] has to be considered. An established alternative sterilization method is UV irradiation which is used in many tissue engineering laboratories. The treatment with UV light represents a simple and cheap but effective procedure [10]. However, UV light has also been reported to degrade polymers such as polyethylene or polypropylene [11,12]. Therefore, it appears logical to test polymers and cell carriers for their susceptibility to changes in polymer properties generated by exposure to UV irradiation.
Previously, in an approach to control cell-polymer interactions we have synthesized a range of biocompatible, biodegradable diblock copolymers consisting of a hydrophilic, water-soluble poly(ethylene glycol)-monomethyl ether (Me.PEG) block and a hydrophobic, biodegradable poly(D, L-lactic acid) (PLA) block (Me.PEGx-PLAy) [13]. It was demonstrated that increasing PEG content reduced unspecific protein adsorption from culture medium and subsequently resulted in decreased unspecific cell adhesion and a shift in cell morphology from a spreaded to a more rounded cell shape [3]. Furthermore, changes in copolymer composition showed an additional impact on cell differentiation of rat marrow stromal cells towards the osteogenic lineage [3]. The composition of copolymers could even
be altered in such a way that cell adhesion on polymeric membranes was completely suppressed [3], which may be used in certain tissue engineering applications. Additionally, their protein adsorption-reducing properties may make these polymers candidates for the use in controlled protein drug delivery, where unintentional protein adsorption to carrier material is a major obstacle [14,15]. For any of these applications, maintenance of surface properties is an indispensable prerequisite.
In this study, we investigated effects of UV sterilization on properties of Me.PEG-PLA diblock copolymers. For this purpose Me.PEG-PLA films were prepared and exposed to UV light for varying periods of time. Specifically, first we examined the effects on interactions of polymeric films with biological elements, i.e., cell adhesion and protein adsorption.
Subsequently, effects on polymer film topography and copolymer composition, especially possible cleavage of PEG chains, were investigated in order to find possible causes for the observed alterations in interactions with the biological environment. The study was expected to shed more light on the suitability of UV irradiation as sterilization method for newly developed biomaterials without compromising relevant polymer properties.
Materials and methods Materials:
Me.PEGx-PLAy diblock copolymers (Fig. 1) were synthesized and their composition and molecular weight were determined as previously described [13]. In case of these polymers, x and y represent the weight-average molecular weight (MW) in kDa of the Me.PEG and PLA block, respectively. Me.PEG5-PLA20, for example, consists of a Me.PEG block of MW 5,000 covalently bound to a PLA block of MW 20,000. The two different diblock copolymers used in this study were Me.PEG5-PLA20 and Me.PEG2-PLA20.
Resomer R202 (PLA17), an endcapped poly(D,L-lactic acid) with equal amounts of D- and L-lactic acid units and MW 17,000 was a gift from Boehringer Ingelheim, Ingelheim, Germany. The water used for the investigations was double-distilled. All other chemicals were used in analytical grade or higher.
Fig. 1:
Structure of Me.PEGx-PLAy: m is the number of ethylene glycol units in Me.PEG, n is the number of lactic acid units of the
PLA part of the copolymer. O
H
For investigation of cell adhesion 3T3-L1 preadipocytes were obtained from ATCC (American Type Culture Collection), Rockville, MD/USA. Stock cultures used for experiments were grown as described [16]. Newborn calf serum (NCS), Trypsin (1:250) and DMEM were purchased from Biochrom, Berlin, Germany; PBS and penicillin-streptomycin solution were from Gibco, Life Technologies, Karlsruhe, Germany. Cell culture materials were from Sarstedt, Nümbrecht, Germany, and Corning Costar, Bodenheim, Germany.