In vitro Biocompatibility Assessment of a Novel PRGD/PDLLA/NGF Composite Material

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1 Journal of Wuhan University of Technology-Mater. Sci. Ed. Dec DOI /s In vitro Biocompatibility Assessment of a Novel PRGD/PDLLA/NGF Composite Material YIN Yixia, YAN Qiongjiao, YAN Yuhua, CHEN Xiaoming, LI Shipu * (Biomaterials and Engineering Research Center, Wuhan University of Technology, Wuhan , China ) Abstract: The objective of this study was to evaluate the degradability and biocompatibility of a novel composite materials which was grafted with RGD and immobilized with NGF(PRGD/PDLLA/NGF). The releasing of NGF, the biodegradability and cell-biocompatibility of PRGD/PDLLA/NGF membrane were evaluated in vitro. The experimental results showed that the NGF release process was prolonged over 30 days. Furthermore, the PRGD/PDLLA/NGF showed a better hydrophilicity, better biodegradation properties and cells affinity than PDLLA, which means a good support to adhesion and proliferate of Schwann cells. Therefore, the novel composite material holds considerable promise as scaffolds in nerve tissue engineering. Key words: PRGD/PDLLA/NGF; schwann cells; biocompatibility; scaffold 1 Introduction Schwann cells play a key role in peripheral nerve growth and regeneration, as well as support axonal regeneration and sprouting by secreting growth factors and providing a permissive surface and matrix molecules [1]. During peripheral nerve regeneration, Schwann cells were found closely associated with growing axons and their presence has been shown to influence axonal migration both in vitro and in vivo [2-4]. Along with the development of tissue engineering, scientists have shown that implanting nerve guide conduits containing Schwann cells can promote the regeneration of the nerve axons [5]. In the other researches, NGF is found necessary to support the survival of the cell bodies and the regeneration of the axons toward specific target organs [6-9]. Although NGF has been shown to improve nerve regeneration under certain conditions, difficulties in manufacture of these large proteins and delivery to target sites are big barriers to therapeutic application [10]. Wuhan University of Technology and SpringerVerlag Berlin Heidelberg 2011 (Received: Oct. 12, 2010; Accepted: Feb. 9, 2011) YIN Yixia( 殷义霞 ): Ph D; yinyx71@hotmail.com *Corresponding author: LI Shipu( 李世普 ): Prof.; lishipu46@126.com Funded by the Key Project of Chinese National Programs for Fundamental Research and Development (No. 2011CB606205)and the Selfdetermined and Innovative Research Funds of WUT (No IV-042) Poly(D,L-lactic acid), a biodegradable polymer, has been approved by FDA as a wide field of applications such as tissue engineering, scaffolds for supporting cells growth. However, it lacks cell biological recognition and a friendly interface with living cells. Recently, some small peptide sequences such as Arg-Gly-Asp (RGD) are being widely used to obtain biomimetic interfaces. Based on these theories and new findings, we immobilized an RGD-based sequence onto poly {(lactic acid)-co-[(glycolic acid)- alt-(l-lysine)]}(plgl) to promote and enhance cells attachment. In our experiment, a cell adhesion peptide, Gly- Arg-Gly-Asp-Tyr (GRGDY) was successfully grafted. The releasing of NGF and the contact angles of the materials was tested, biodegradability of the material were evaluated. Then SCs have been achieved and co-cultured with PRGD/PDLLA/NGF membranes and PDLLA membranes, the cell proliferation and growth on the surface of both PDLLA and PRGD/ PDLLA/NGF membranes culture were observed and characterized. Our results showed that the NGF release process was prolonged over 30 days. Furthermore, the PRGD/PDLLA/NGF shows a better hydrophilicity compared with PDLLA, and has better biodegradation properties and cells affinity than PDLLA, which means a good support to adhesion and proliferate of Schwann cells. Therefore, the novel composite material holds considerable promise as scaffolds in nerve tissue engineering.

2 1060 Vol.26 No.6 YIN Yixia et al: In vitro Biocompatibility Assessment of a Novel PRGD/P 2 Experimental 2.1 Isolation and purification of SCs Schwann cells were harvested from 3-day old neonatal Wister rats according to Grothe et al [11]. In brief, the sciatic nerves were harvested under microscope, and the epineurium was separated then dissected into a 1mm 2 discrete fascicle. Fascicles were enzymatically dissociated using 0.03% type I collagenase (Sigma) and 0.25% trypsin in 10 ml of PBS for 60 min at 37, and the suspension was filtered, then centrifuged (1500 r/min, 10 min), and placed in a T-25 flask overnight. Later 0.05 ml of 10 μm Ara-C solution was added to the culture medium for fibroblast removal and incubated for 4 days. The culture was plated on poly-l-lysine-coated T-25 flasks in 5% CO 2 incubator at 37 and cultured with DMEM medium /10% PBS / PS. The SCs were identified with the S-100 immunostaining, the purity was calculated. 2.2 Preparation of PDLLA, PRGD/ PDLLA/NGF membrane The PRGD/PDLLA/NGF was synthesized as previously described [12] : PRGD was dissolved in ethyl acetate at a concentration of 10wt%. Both PRGD and PDLLA conduits were prepared using the solvent evaporation method. PRGD conduits were soaked in 80 ml EDAC solution with a concentration of 1mg/ ml for 24 h at 4. Unbound and excess EDAC was removed by washing the conduits with double-distilled water. The conduits were then transferred to 80ml NGF solution with a concentration of 60 ng/ml for 24 h at 4. The unbound NGF was removed by washing with PBS. The PRGD/PDLLA/NGF membrane was prepared using the solvent evaporation method. The same method was applied in PDLLA membrane. The membranes were sterilized with ultraviolet light for about 30min for subsequent experimentation. 2.3 Contact angle measurement The change of the hydrophilicity of PDLLA and PRGD/PDLLA/NGF membranes was characterized by measuring the contact angles in air using a JC2000A (Shanghai Zhongchen, China) contact angle meter. A drop of water (5 μl) was introduced on the prepared films, and the contact angle was measured immediately from the image of the water drop. 2.4 NGF in vitro release and quantification by ELISA PRGD/PDLLA/NGF membranes (100 mg approximately, n=5) were immersed in 3 ml of PBS buffer and incubated at 37 on an orbital shaker (60 r/min). For daily release of NGF assay, supernatant was taken every day, and the liquid was replaced with an equal volume of fresh buffer. For total release of NGF, supernatant was collected at the end of the experiment. The concentration of NGF in the supernatant samples was determined by ELISA kit (Promega, USA) following the manufacturer s protocol. 2.5 Biodegradation of the membranes Both PDLLA and PRGD/PDLLA/NGF membranes were dried to constant weight, and then incubated in PBS at 37. The membranes were rinsed with distilled water after 2, 4, 6, 8, 10 and 12 weeks and finally vacuum-dried and weighed respectively. 2.6 Proliferation of SCs on the surface of membranes Schwann cells viability on the PDLLA and PRGD/PDLLA/NGF membranes surface was detected by MTT assay. After culturing for 2 days, 4 days, 6 days, respectively, the upper liquid was removed. 20μl of MTT solution (5 mg/ml) was added to each culture well and incubated at 37 under 5% CO 2 conditions for 4 h. The upper liquid was removed and DMSO was added to each culture well. The absorbance at 560 nm was recorded by an automatic enzyme scanner (Thermo Lab systems, Finland). Five parallels for every sample were averaged. 2.7 Scanning electron microscopy of membranes co-cultured with SCs Scanning electron microscopy (SEM) was used to visualize their surface feature of cocultured membranes. SCs were fixed for 1h in 2.5% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M PBS buffer (ph 7.4). Followed washing with buffer, cultures were post-fixed for 1h with 1% osmium tetraoxide. Following another buffer wash, they were dehydrated in an ascending series of ethanol washes, at which point they were critical point dried by Freon. Surface coating of specimens with a layer of gold was performed using a sputter coater, and examined with SEM. 2.8 Data analysis The statistical significance of the studies was analyzed using Student s t test. Differences with P values of <0.05 are considered significant. The P values were corrected for multiple testing procedures and to control type I error rates. 3 Results 3.1 Primary culture and immunocytochemical characterization of Schwann cells

3 Journal of Wuhan University of Technology-Mater. Sci. Ed. Dec Four days later, Schwann cells increased and bestrewed on the surface of the culture chamber (Fig.1 a). The cells had oval nuclei, neurites appeared typical bi-or tripolar morphology. After stained by S-100 and counterstained by hematoxylin, the body of Schwann cells was shown brown, and the nuclei was shown blue. But the body of fibroblasts were not stained (Fig.1b, pointed by arrow). Three random fields were chosen from six slides and cells were counted from each field (n 800 cells). The positive rate of Schwann cells was more than 95%. 3.2 Amino acid analysis of composite material The results shows that the density of GRGDY peptide bond is µmol/g materials. It has been found [13] that a minimum GRGDY density of 3.1 µmol/ g materials is sufficient to promote cell spreading. Theoretically, the novel materials will have fine cell affinity for the contents of RGD. prolonged over at least 30 days as our previous described [14]. Briefly, the burst releasing happened at the first day, 9.51±0.76 ng per mg conduit was released. The releasing rate was slower afterward; 0.14±0.02 ng NGF per mg conduit was released within the 7 th day. After a week, the NGF was released in a relatively steady manner (about 0.1 ng/mg conduit), and the release continued until the end of the experiment. In conduit preparation, 4.8 ug±0.4 of NGF was embedded in each conduit. However, the cumulative amount of released NGF (as measured after 30 days) was 1.97±0.50 ug per conduit or 41.4% of the added amount (Fig.2) 3.5 Biodegradation analysis After incubated in PBS 2, 4, 6, 8, 10, 12 weeks, the rate of weight loss of PRGD/PDLLA/NGF was 8.5%, 14.57%, 19.7%, 22.6%, 24.4%, 26.8%, respectively. But the rate of weight loss of PDLLA is 4.1%, 7.2%, 10.1%, 13.9%, 14.4%, and 15.6%, respectively. The PRGD/PDLLA/NGF membrane degraded faster than PDLLA during the incubate process. 3.3 Water contact angle of membranes The contact angle values are calculated by averaging three measurement values in different sites of membranes surface. From Table 2, it can be easily seen that the water contact angle of the PDLLA films is 81.75±2.5, which is larger than that of PRGD/PDLLA/ NGF films 55.67±1.6. Compared with PDLLA, PRGD/ PDLLA/NGF showed a better hydrophilicity. 3.4 NGF releasing curve In vitro, NGF release from the NGC was 3.6 Proliferation of SCs on the surface of membranes The increase of the MTT level corresponded to an increase of viable cell number. Fig.4 shows the OD values of SCs on PRGD/PDLLA/NGF membranes were higher than PDLLA culture for 2 days, 4 days, 6 days, 8 days, respectively. Thus, SCs showed a higher proliferation rate on PRGD/PDLLA/NGF membranes.

4 1062 Vol.26 No.6 YIN Yixia et al: In vitro Biocompatibility Assessment of a Novel PRGD/P 3.7 Scanning electron microscopy of membranes co-cultured with SCs As shown in Fig.5, cell numbers on PRGD/ PDLLA/NGF (b) films was significantly higher than on PDLLA (a). Primary SCs were interlaced with each other on PRGD film and maintained the spindle resembling morphology as primary culture. 4 Discussion Among several of biomaterials, PDLLA has been widely used for its biodegradability and biocompatibility as drug delivery systems, nerve tissue scaffold and so on. However, as the other synthetic polymers, it lacks a friendly interface with living cells and cell biological recognition. In this experiment, we developed biomimetic approaches to immobilize RGD and NGF onto synthetic materials in order to obtain better cells affinity, growth and proliferation. This study used several tests to investigate the biocompatibility of PDLLA and PRGD/PDLLA/NGF membranes. In vitro, we compared hydrophilicity and biodegradation of PDLLA and PRGD/PDLLA/NGF membranes. It was found that PRGD/PDLLA/NGF membrane has better hydorphilicity and degradability. NGF has been shown to increase regeneration after nerve injury. In this experiment, the release of NGF was quantified using an ELISA method, and found that the sustained NGF release process was prolonged over 30 days, which can act as a suitable local growth factor delivery system whilst providing a scaffold to support regeneration across a nerve gap. In order to examine the biocompatibility between nerve tissues and the two membranes, we tried the coculture of the never cells with membranes. It is well known that SCs play a pivotal role in the regeneration of the peripheral nervous system. They can secrete bioactive molecules such as nerve growth factor, brainderived neurotrophic factor, laminin, fibronectin and collagen IV among others. Neonatal Wistar rats were choose to isolate Schwann cells from peripheral nerve, including sciatic and brachial nerve so that more Schwann cells will be acquired. Based on the different attachment rates of SCs and fibroblasts, Schwann cells were purified and applied onto coated plates [15]. In a quantitative measure of cell viability (MTT) of this study, which is chiefly chosen method of the cytotoxic test in vitro on evaluation of biocompatibility of biomaterials at present, the results of the cultured SCs, a kind of normal neuroglial cell in the peripheral nerve system, explain whether PDLLA membranes or PRGD/PDLLA/NGF membranes have better biocompatibility. In vitro experiments showed that when co-cultured with PRGD/PDLLA/NGF membranes, SCs showed normal adhesion, survival, migration and proliferation. At the same time, SEM revealed better attachment and spreading of Schwann cells on the surface of PRGD/PDLLA/NGF than that of PDLLA. Due to the combination of RGD and biomimetic materials, which is suitable for the cohesion of extracellular adhesive molecular, such as laminin, fibronectin and collagen IV, which promote cells to adhere, migrate and differentiate. These factors provide a good microenvironment for nerve regeneratation. 5 Conclusion In conclusion, the experiment results shows that compared with PDLLA, PRGD/PDLLA/NGF membrane has better biocompatibility for SCs. The current study provides evidence that PRGD/PDLLA/ NGF membranes have excellent glial cells affinity, and they can promote the attachment, proliferation, and improve the livability of SCs. They are promising materials for nerve repair and could be improved by incorporation with neurotrophic factors and neural cells. Therefore, by introducing NGF and RGD into orientated PDLLA, the PRGD/PDLLA/NGF membrane in conjunction with SCs may provide a better approach for nerve injury treatment.

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