Claudia Fischbach, Jörg Tessmar, Michael Hacker, Sigrid Drotleff, Torsten Blunk, Achim Göpferich
Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany
Introduction
In tissue engineering, polymer matrices are frequently used to guide new tissue formation [1]. The generation of appropriate constructs for different fields of application can be optimized at three different levels: A) the cells, B) the polymer scaffolds, and C) the methodologies employed for construct assembly [2]. On the scaffold level, advances in polymer chemistry as well as polymer processing have led to the development of tailored 3-D matrices allowing to distinctly modify the characteristics of the created tissues. Not only are the physical and mechanical properties of the constructs tunable, for example by adjusting the porosity and degradation rate of the construct, but it is also being increasingly recognized that the polymer itself may influence cellular features including cell adhesion and differentiation.
In order to investigate the impact of the polymer with relatively simple experimental procedures, many groups culture cells in 2-D on various polymer films. Using this approach, surface roughness and surface energy have been demonstrated as being critical to direct cell adhesion [3-5]. Nevertheless, cellular characteristics are known to be modulated through the chemical composition of the polymers. For instance, an increase in the hydrophobicity of the biomaterials leads to an enhanced cell adhesion and proliferation [6,7], caused by an increased adsorption of adhesion-mediating serum proteins such as fibronectin onto the polymer surface [8,9]. However, depending on the type and conformation of the adsorbed proteins, the adherent cells interact differently depending on the polymer surface and, hence, may adopt dissimilar phenotypes [9]. This can be explained, in part, by the fact that the adsorbed adhesive proteins dictate via which integrin receptors the cells attach to the substrate.
Integrins are known to affect cellular differentiation by mediating cytoskeletal reorganization [10] and, thereby, allowing for changes in cell morphology. In turn, cell shape is reported to impact the differentiation of many cell types [11,12]. To conclude, one can, therefore, hypothesize that specific protein adsorption, controlled by tailored polymer constructs, may facilitate the differentiation of cells into the desired phenotype and, thus, guide the formation of the desired tissue.
Currently, polyglycolic acid (PGA), polylactic acid (PLA), and their respective copolymers (PLGA) are the most widely used polymers in tissue engineering [2,13]. This is due, in part, to their reported biocompatibility, as proven by their long-standing use as suture materials in humans, as well as their favorable properties with regard to processing and degradation [2]. These materials are not suitable for guiding specific tissue formation,
however, because of unspecific protein adsorption, which allows for the infiltration of undesired cell types, such as fibroblasts, into the tissue engineered construct. With the aim of preventing such unspecific protein adsorption, we synthesized poly(D,L-lactic acid)-poly(ethylene glycol)-monomethyl ether (Me.PEG-PLA) diblock co-polymers by attaching hydrophilic poly(ethylene glycol) blocks to poly(lactic acid) blocks [14]. Subsequently, unspecific protein adsorption was demonstrated to be reduced with increasing PEG content [14,15]. In terms of cell behavior, the resulting polymers have recently been described to further the differentiation of marrow stromal cells towards the osteoblastic phenotype [16,17].
Furthermore, the novel biomaterials have been shown to substantially influence the adhesion of 3T3-L1 preadipocytes; although fewer preadipocytes attached to Me.PEG-PLAy films as compared to pure PLA films, the adhered cells exhibited a more rounded cell shape [15,18].
As adipose differentiation is accompanied by cytoskeletal reorganization to cells exhibiting a spherical appearance [12,19], this observation prompted us to hypothesize that Me.PEG-PLA may promote 3T3-L1 differentiation into adipocytes.
Therefore, the focus of this study was to address this hypothesis and to evaluate if the block-co-polymer is suitable for guiding adipose differentiation. To this end, we processed polymer films for 2-D cell culture and examined the typical features of adipose differentiation. Specifically, adipogenesis was investigated on hydrophilic films made from Me.PEG2PLA20 (MW (PEG): 2 kDa; MW (PLA): 20 kDa) and on hydrophobic films prepared from PLGA (molar ratio of PLA:PGA was 75:25). Conventional tissue culture polystyrene (TCPS) served for comparison. Adipose conversion was triggered by treatment with a hormonal cocktail according to protocols evaluated in preceding experiments (see Chapter 2).
Following a differentiation phase of 9 days, adipocyte properties were analyzed. In detail, intracellular lipid accumulation was investigated. Furthermore, expression of typical fat cell genes was examined on both the protein and the mRNA level. In order to assess the functionality of the cells on different surfaces, the lipolysis rate of the adipocytes was measured under different conditions. Finally, the polymers were used in the form of 3-D porous scaffolds in order to investigate their impact on 3-D adipose differentiation, as compared to the polyglycolic acid (PGA) fiber meshes used so far.
Materials and methods Materials:
For the fabrication of hydrophilic polymer films, poly(D,L-lactic acid)-poly(ethylene glycol)-monomethyl ether (Me.PEG2PLA20) diblock copolymers were synthesized in our lab as previously described [14]. The polymers consist of a hydrophilic, water-soluble poly(ethylene glycol)-monomethyl ether (Me.PEG) block of 2 kDa and a hydrophobic, biodegradable poly(D, L-lactic acid) (PLA) block of 20 kDa, as indicated by the subscripted numbers (Fig. 1A).
CH3 m
A) Me.PEG2PLA20 B) PLGA
Fig. 1:
Structure of the investigated polymers:
m and n is the number of the respective monomer units used for synthesis of copolymers A) m = ethylene glycol, n = lactic acid; B) m = glycolic acid, n = lactic acid
To prepare hydrophobic polymer films, PLGA (Resomer®, RG 756) featuring a molar ratio of 75:25 (P(D,L)LA:PGA) was used (Fig. 1B). It was kindly provided by Boehringer Ingelheim (Ingelheim, Germany). Polypropylene sheets employed to prepare carriers for film manufacture were a kind gift from Dr. Stricker, Targor Application Technology (Ludwigshafen, Germany).
For cell experiments 3T3-L1 preadipocytes were obtained from ATCC (Manassas, VA, USA). Stock cultures were grown as described [20]. Fetal bovine serum (FBS), and trypsin (1:250) were purchased from Biochrom KG Seromed (Berlin, Germany); phosphate buffered saline (PBS) and penicillin-streptomycin solution were from Life Technologies (Karlsruhe, Germany). MEM (alpha-modification), corticosterone, indomethacin, and oil red O were purchased from Sigma-Aldrich (Deisenhofen, Germany). 3-isobutyl-1-methylxanthine (IBMX) was from Serva Electrophoresis GmbH (Heidelberg, Germany). Insulin was a gift from Hoechst Marion Roussel (Frankfurt a. M., Germany). Cell culture materials were obtained from Sarstedt AG & Co. (Nuembrecht, Germany) and BD Biosciences Labware (Heidelberg, Germany). Polyglycolic acid (PGA) non-woven fiber meshes (12-14 µm fiber
diameter; 96% porosity; 62 mg/cm3 bulk density) were purchased from Albany Int. Research Co. (Mansfield, MA, USA) and die-punched into discs 5 mm in diameter and 2 mm thick.