RGD-functionalisation of PLLA nanofibers by surface coupling using plasma treatment: influence on stem cell differentiation
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- Paletta, J.R.J., Bockelmann, S., Walz, A. et al. J Mater Sci: Mater Med (2010) 21: 1363. doi:10.1007/s10856-009-3947-2
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The aim of this study was to functionalize the surface of synthetic poly-(l-lactic) (PLLA) nanofibers with RGD peptide, in order to promote growth and osteogenic differentiation of human mesenchymal stem cells (hMSC) in vitro. The cRGD was coupled onto PLLA nanofibers using oxygen plasma combined with EDC/sulfo-NHS activation. Matrices were seeded with hMSC and cultivated over a period of 22 days under growth conditions and analyzed during the course of cultivation. The plasma activation of PLLA nanofibers resulted in a reduction of hydrophobicity as well as a formation of carboxyl groups on the surface of the fibers. Furthermore, maximum load, but not young’s modulus was influenced by the treatment with oxygen plasma. When hMSC were cultured onto the cRGD functionalized scaffolds, cells showed no increased proliferation or cell density but an induction of genes associated with the osteoblast lineage. In brief, this study indicates that functional peptides of the extracellular matrix can be coupled onto PLLA nanofibers using plasma treatment in combination with EDC/sulfo-NHS treatment. These groups are accessible for the growing cell and mediate probably some osteoinductive properties of collagen nanofibers.
In many native tissues, collagen represents a principal structural element of the extra cellular matrix . It can be isolated from a variety of sources, is highly conserved and relatively non-immunogenic. Therefore, it has been used in a variety of tissue engineering applications.
Collagen can be easily electrospun to a 3-D, non-woven network that supports the growth of keratinocytes , coronary artery smooth muscle cells , dermal fibroblast , endothelial cells  myoblasts  and as well the differentiation of mesenchymal stem cells [7, 8].
Collagen I interacts with the integrin receptor and its interaction is sufficient to induce osteogenic differentiation of human mesenchymal stem cells (hMSC), even in the absence of exogenous soluble stimuli [9, 10]. Since the biologic action of the collagen can be reduced to an RGD motive (R: arginine; G: glycine; D: aspartic acid) numerous materials have been RGD functionalized for medical applications [11–16]. With respect to bone healing, in vitro studies showed evidence that RGD covered surfaces resulted in an increase of both osteoblast cell number and differentiation [17–20]. Furthermore, the presence of signalling peptide allows a favourable micro environment for hMSC to differentiate into osteoblasts [21–23]. Here the direct incorporation of RGD peptides into PLLA nanofibers during electrospinning  mediated the osteogenic potential to some extent, although the incorporated peptides were distributed randomly within the nanofiber.
Hence, we designed this study in order to functionalize PLLA nanofibers with cyclic RGD (cRGD) peptides using plasma activation and surface coupling with EDC/Sulfo-NHS.
2 Materials and methods
2.1 Construction of nanofibers and characterization
The preparation of PLLA nanofibers by electrospinning has already been reported in detail earlier .
In order to incorporate cyclic Arg-Gly-Asp (Cyclo(-RGDfK) AnaSpec, Inc. San Jose, USA) onto the surface, PLLA nanofibers were activated using plasma treatment. Nanofibers were electrospun onto cover slips and subjected to plasma treatment in a power generator (Edwards MPG 137 RF-power generator). The samples were clamped between both electrodes and the chamber was evacuated to 0.0133 mbar and filled with oxygen to a pressure of 0.133 mbar prior to the treatment. Plasma treatment was performed using a grid current of 6 mA, a plate current of 2 A, a forward power of 100 W and reflected power of 30 W over a period of 2 min. The pressure of the plasma chamber was kept at 0.133 mbar by controlling the working gas flow.
The plasma activated scaffolds (carboxyl groups) were incubated in an 0.1 M aqueous 2-(N-morpho)-ethanesulfonic acid solution (pH 5.3) for 30 mins at room temperature by shaking. After this washing step the 0.1 M aqueous 2-(N-morpho)-ethanesulfonic acid solution was removed and the activated nanofiber mashes were incubated with 8.69 mM EDC and 4.35 mM Sulfo-NHS in an 0.1 M aqueous 2-(N-morpho)-ethanesulfonic acid solution (pH 5.3) for 90 mins at room temperature by shaking to activate the carboxylic acid groups into creating a semi-stable amine-reactive sulfo-NHS-ester.
After the reaction the solution was removed and the chemically activated nanofiber meshes were incubated with 0.1 mg/ml cRGD solution (165.8 μM) in 0.1 M PBS for 6 h at room temperature to create cRGD-functionalized fibers by amide bond formation. After 6 h, the cRGD solution was removed and the weakly bound, physically absorbed cRGD was removed by washing with PBS. In some experiments cRGD was replaced by a fluorescein isothiocyanate (FITC)-labeled RGD, in order to analyze the distribution within the nanofibers.
2.2 Contact angles
Static contact angles of water were measured using the sessile drop method with a G10 drops shape analysis system (Krüss, Hamburg, Germany) and calculated using Data Physics SCA20 contact angle analyzer software.
2.3 Scanning electron microscopy
For scanning electron microscopy (SEM), samples were sputter-coated with platinum and examined in a SEM (JSM-7500F, JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 2–5 kV in the SEI mode.
2.4 Specimen preparation and mechanical testing
Mechanical testing was performed using a uni-axial testing machine (zwicki-Line, Zwick GmbH & Co. KG, Ulm, Germany). Test specimens of 10 mm breadth and 50 mm gauge length were prepared from orientated fibres and stretched to failure at a cross head speed of 10 mm/min without any pre-tension or preconditioning. Load (N) and extension (mm) were recorded and tensile strength, strain, young’s modulus were calculated using test expert software (Zwick GmbH & Co. KG, Ulm, Germany).
2.5 Human mesenchymal stem cell isolation, characterization and culture
Human mesenchymal stem cells were obtained from consenting patients with the approval of the institutional review board. The indication for surgery was primary osteoarthritis of the hip with increasing pain, decreased range of motion, and signs of progressive osteoarthritis in radiographs. The patients had no evidence of other bone or auto-immune diseases. The routinely removed bone was obtained from the proximal femur, while preparing the implant bed. Mesenchymal stem cells were isolated and cultured according to the preparation of Pittenger et al. , with minor modifications as described by Brendel et al. . Further treatment was done as described by Schofer et al. .
2.6 Gene expression analysis
RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (PCR) analysis were performed as described earlier . Cycle temperatures and incubation times for human alkaline phosphatase (ALP), osteopontin (OP), collagen I (COLI), osteocalcin (OC), and 18 s rRNA were previously described [8, 27, 28]. Purity of the single PCR products was verified by melting point analysis and expression levels were calculated using ΔΔCT method .
2.7 Immunofluorescence microscopy
Immunefluorescence analysis was carried out as described earlier . Slices were analyzed using a Leica DM5000. Microphotographs of at least three different areas were made. The intensity and area of fluorescence was determined using Quips analysis software. The total cell count of DAPI stained nuclei was obtained. The proliferation index was calculated as a ratio of Ki-67 positive versus total cells.
All values were expressed as mean ± standard error of at least 3 different patients and compared using students’ t-test or ANOVA with Bonferroni as a post hoc test. Values of P < 0.05 were considered to be significant.
3.1 Characterization of cRGD incorporated PLLA nanofibers
3.2 Growth and proliferation of hMSC cultured on nanofibers
3.3 Expression of genes associated with the osteoblast lineage
In order to examine the influence of cRGD coupling on the surface of PLLA nanofibers on osteoblastic differentiation of hMSC, we compared the expression levels of cells grown on PLLA nanofibers with cells grown on PLLA nanofibers after plasma treatment, as well as cRGD coupling using EDC/sulfo-NHS treatment.
Regardless of whether the cells were cultured under growth or osteoinductive conditions, the plasma treatment of PLLA nanofibers resulted in an increased expression of genes associated with osteoblast linkage.
Scaffolds made of PLLA nanofibers represent an appropriate matrix for osteoblast growth and osteogenic differentiation of hMSC in principle [30–32]. However, we reported earlier that during the differentiation of hMSC the initial gene expression of osteoblast marker genes decreased. In order to overcome this disadvantage, a functionalization of PLLA nanofibers using bioactive motives of collagen, the main component of bone, might be a suitable tool. Indeed it has been shown that collagen nanofibers support growth and differentiation of hMSC [7, 8] even in the absence of osteogenic stimuli. Therefore, the fibers mediate, at least in part, the osteoinductive potential of collagen [9, 10]. In addition, PLLA nanofibers can be improved by either coating  or blending either with gelatin  or collagen . These properties of collagen were mediated by the RGD sequences within the polymer chain. Furthermore, a direct incorporation of RGD peptides into PLLA nanofibers mediated, at least in part, the osteogenic potential of collagen (determined as the expression and deposition of collagen and osteocalcin) to some extent .
The direct coupling using the plasma EDC/sulfo-NHS system represents a method of coupling RGD sequences on the surface of nanofibers.
In contrast to other physical or chemical surface modifications the plasma action is limited to 10 nm below the surface and does not affect the bulk properties of the material [36, 37]. Nevertheless, these changes seem to be sufficient to weaken the PLLA nanofiber scaffolds (as demonstrated by maximum load measurements) and to decrease the hydrophobicity of the scaffold, presumably due to the induction of polar chemical groups [38, 39]. It is not clear, however, which of these groups is most effective in the cell-material interaction, although there is some evidence that hydroxyl groups are involved in cell adhesion . However, in this study we found no effect on the proliferation and cell densities of hMSC. With respect to this, it should be realized that the effects of plasma treatment on polylactide scaffolds can be transient and/or permanent. The transient effects, such as surface activation and the formation of free radicals, disappear during storage, and will not be perceived by the cells in culture or in vivo . On the other hand, we found some impact of plasma treatment on the differentiation of hMSC as demonstrated by an increased gene expression of genes associated with the osteoblast lineage, which might lead to the speculation that different groups on the surface may elicit different cellular response.
However, the formation of polar chemical groups was sufficient to couple RGD peptides on the surface of the nanofiber mash using a combination of EDC and sulfo-NHS comparable to Noga et al. .
Nevertheless, the presence of cRGD on PLLA nanofiber surfaces promoted the differentiation of hMSC to some extent. Therefore, the functionalisation of nanofibers by RGD has similar effects to the functionalisation of other surfaces by RGD peptides [46–48]. We interpret this finding to mean that the RGD is coupled on the fiber surface in a bioactive form in principal. However, as compared to the gene expression elicited by collagen nanofibers  or collagen blended PLLA nanofibers  the observed effect was small and limited to the expression of osteocalcin. One explanation here might be that the osteoinductive effect of RGD functionalized scaffolds depends on the concentration of RGD on the surface , and other bioactive sequences in the collagen. Within this context, further studies are needed in order to optimize the density of RGD on the surface of the nanofibers.
Taken together, this study clearly indicates that functional peptides of the extra cellular matrix can be coupled onto PLLA nanofibers using plasma treatment. These groups are accessible for the growing cell and mediate the expression of osteocalcin, indicating that the osteoblast linkage of hMSC is promoted to some extent.
This work was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation, Grant No. BO 3065/1-1). We would like to thank M. Hellwig for SEM and Dr. F. Grolig for confocal fluorescence microscopy measurements.