Fabrication of Novel Scaffolds Containing Collagen-I/Polylactic Acid/Nanohydroxyapatite via Co-electrospinning Methods

Transcription

1 CHEM. RES. CHINESE UNIVERSITIES 2010, 26(4), Fabrication of Novel Scaffolds Containing Collagen-I/Polylactic Acid/Nanohydroxyapatite via Co-electrospinning Methods ZANG Jun-ting, QI Xin, LI Shu-qiang, LI Dong-song, GONG Yu-bao, YANG Chen, XU Feng and LIU Jian-guo * Department of Bone and Joint Surgery, First Hospital of Jilin University, Changchun , P. R. China Abstract A novel scaffold containing collagen-i/polylactic acid(pla)/nanohydroxyapatite(nha) was prepared via co-electrospinning method. Different target substrates were used to improve the collection efficiency of this scaffold. The properties of the novel scaffold were compared with those of conventionally prepared ones. Compared to conventional method, the modified method was more efficient in producing the scaffold. Moreover, the porosity, thickness, and morphology of the novel scaffold were better than those of scaffolds prepared by conventional methods. The properties of collagen-i, collagen-i/pla and collagen-i/pla/nha scaffolds were also compared. Diameters of the electrospun fibers ranged from 180 to 405 nm, and roughness was present on the surface of the fibers due to the deposition of crystals of nha along the long axis of the fibers. The fibers of the collagen-i/pla/nha scaffold and the fibers of natural bone tissue had similar structure. Keywords Electrospinning; Collagen-I; PLA; Nanohydroxyapatite Article ID (2010) Introduction Electrospinning is a simple method that can be used to spin organic and inorganic molecules into nanofibers and scaffolds with high surface areatovolume ratio and porosity [1 3]. Such a technique can be used to manufacture scaffolds that are similar to those that occur naturally in the body. Nanofibers [4] in natural bone tissue form from the triple helices of collagen molecules and the tiny crystals of hydroxyapatite(ha). HA crystals are crucial to mineralization within the collagen fibers [5], and they are arranged regularly along the long axis of the nanofibers. Perumal et al. [6] described the molecular structure of collagen fibril and its structural and organizational framework for skin, bone, tendon and other tissues. Understan- ding of the ultrastructure of collagen fibers in natural bone tissue provides a basis for the electrospinning process of collagen-i and nha. Mimicking the ultrastructure of natural biological tissue, artificial scaffolds that are co-electrospun from organic polymers and inorganic crystals can provide an excellent setting for proliferation, migration and differentiation of cells into specific tissues. Such artificial scaffolds can be designed to act as temporary extracellular matrix [2,7 10]. Over the last decade, thin mats, which were too thin to be used as filling materials in tissue engineering, were fabricated via a single electrospinning process. In order to increase the thickness of scaffolds more efficiently, we compared two different methods. In conventional method, only thin mats could be prepared, so the thickness of the scaffold is increased by folding and pressing the multiple layers of electrospun mats of collagen(with or without nha). However, during this process the fibers collapse easily, and the ultrastructure of these scaffolds is destroyed and the porosity is decreased. In order to increase the thickness without decreasing the porosity of the scaffold, we used modified target substrates. In this study, we compared the ultrastructure and porosity of these novel scaffolds to those created by the method of folding and pressing the multiple layers. To mimic the biological constituents of natural bone tissue, Matthews et al. [1] electrospun collagen-i and nha, but the resulting fibers had poor morphology. In contrast, McCullen et al. [11] and our preliminary experiments reveal that electrospun fibers of PLA had regular morphology. To obtain electrospun fibers with regular morphology that mimic the arrangement of *Corresponding author. jgliu.2005@yahoo.com.cn Received December 10, 2009; accepted April 30, Supported by the Scientific and Technological Developing Scheme of Jilin Province, China(No ) and the Fund of Jilin Provincial Science and Technology Department, China(No ).

2 No.4 ZANG Jun-ting et al. 663 HA crystal on the fibers found in natural bone tissue, we co-electrospun a mixture of PLA, collagen-i, and nha and then characterized the novel scaffold using scanning electron microscopy(sem), Fourier transform infrared spectroscopy(ftir), and X-ray diffraction(xrd) techniques. 2 Experimental 2.1 Materials Collagen-I(Lyophilized sample), 1,1,1,3,3,3-hexafluoro-2-propanol(HFP), and nha were purchased from Sigma-Aldrich Chemical Company(St. Louis, USA). All other reagents used were of analytical grade. 2.2 Methods Collagen-I and collagen-i/pla(mass ratio 1:1) were dissolved in HFP to obtain a viscous solution(40 mg/ml). The solution was loaded into a 5.0 ml syringe mounted on a syringe pump and electrospun at a high voltage(20 kv). The positive lead from a high voltage supply was attached via an alligator clip to the external surface of the metal syringe needle. The grounded target substrate was placed at a distance of 10 cm from the needle tip. The syringe pump was set to deliver the source solution at a rate of 2 ml/h. The scaffolds were kept in a vacuum drier overnight to remove residual HFP. To make the composite fibers of collagen-i, PLA, and nha, a desired amount of nha powder(mass fraction 20%) was dispersed in the solution of collagen-i/pla via a magnetic stirrer. The solution was electrospun at 20 kv as described earlier. The scaffolds were kept in a vacuum drier overnight to remove residual HFP. The fibers were observed by SEM, and the diameters of the fibers were calculated with Photoshop(Adobe systems incorporated, USA). To increase the thickness of the scaffolds more efficiently and improve the collection efficiency, we modified the targets. Targets composed of different materials were made into circular discs with diameters ranged from 1 cm to 5 cm. However, deposition outside the target substrate increased with the reduction of surface area. To minimize this problem, stainless steel, copper, and aluminum target substrates were used. The scaffolds were kept in a vacuum drier overnight at 24 C. The thickness of the dried scaffolds was measured with Vernier calipers. We also prepared electrospun mats using conventional methods [12] in order to compare our modified method with other method of generating scaffolds. Then, we folded and pressed the multiple layers of electrospun mats to increase the thickness of the scaffolds rapidly. The electrospun collagen-i/pla/nha mats were cut into 1 cm 1 cm squares, folded, and pressed into 3D scaffolds. These scaffolds were characterized via SEM, FTIR, and XRD. The apparent density and porosity of all electrospun scaffolds(generated using the modified and conventional techniques) were calculated via the following equations [2] : Porosity of scaffold(%)=(1 Apparent density of scaffold)/(bulk density the material) 100% Apparent density of Scaffold=(Mass of electrospun matrix)/(thickness area). 3 Results and Discussion The fibers of collagen-i prepared in this study had diameters ranged from 83 to 416 nm. Similar results were reported by Powell et al. [13] and Shabani et al. [14]. However, the size and shape of the electrospun collagen-i fibers were irregular[fig.1(a)], and most of the fibers were oblate. The mats of collagen-i were brittle, which means that handling them would be difficult. Diameters of collagen-i/pla fibers ranged from 127 to 350 nm[fig.1(b)]. These fibers exhibited regular size and shape and were well Fig.1 SEM images of electrospun collagen-i fibers(a) and collagen-i/pla fibers(b) (A) Diameters of the fibers range from 83 to 416 nm; (B) diameters of the fibers range from 127 to 350 nm.

3 664 CHEM. RES. CHINESE UNIVERSITIES Vol.26 distributed. These fibers were not brittle and were easy to handle, possibly due to the blending of PLA with collagen. The diameters of electrospun fibers of collagen-i/pla/nha ranged from 180 to 405 nm, and this range was found to be similar to that found in the collagen fibers of natural bone tissue [15]. The surface morphology of the collagen-i/pla/nha fibers was rough and the diameters were well distributed(figs.2 and 3). Compared to that of the collagen-i/pla fibers, the surface roughness of collagen-i/pla/nha fibers was increased. The tiny crystal of nha, which were arranged regularly along the long axis of the fibers, imparted roughness to the surface. The ultrastructure of the fibers is similar to the schematic view of collagen fibers found in natural bone tissue(fig.4) [4]. In order to improve the scaffold s cell adhesion, migration and proliferation properties, it is necessary to increase the thickness of the scaffold(the properties of cell adhesion, migration and proliferation of the novel scaffold will be reported in future manuscript). The traditional method involves folding multiple layers of the mats of fibers and pressing them together to diminish the gaps between them. However, the resulting morphology of the fibers is irregular and the diameters are not well distributed(fig.5), resulting in poor porosity. To solve this problem and to fabricate a novel scaffold, we prepared modified target substrates and decreased the surface area of stainless steel, copper, and aluminum targets to find the most effective target. However, as surface area decreased, deposition outside the target increased, thus, there was no linear relationship between the rate of the increase in thickness and the decrease in surface area of target substrate. Nevertheless, we observed an increase in the thickness of the scaffolds on targets with diameters ranged from 1 to 5 cm(figs.6 and 7). Fig.2 SEM image of an electrospun fiber of collagen-i/pla/nha Surface roughness on the surface of the fiber is visible due to the presence of nha crystals. Fig.5 SEM image of a traditionally prepared collagen-i/pla/nha scaffold Fig.3 SEM image of an electrospun scaffold of collagen-i/pla/nha Diameters of the scaffold fibers range from 180 to 405 nm. The tiny nha crystals, which are arranged regularly along the long axis of the fibers, form tiny thorns that are 100 to 150 nm high and cause roughness. Fig.6 Change of thickness of scaffolds on the stainless steel(ss), copper(cu) and aluminum(al) targets with time The diameter of target was 2 cm. SS; Cu; Al. t/h: a. 3; b. 6; c. 9; d. 12. Fig.4 Schematic view of collagen fibers from natural bone tissue In natural bone tissue, the diameters of collagen fibers range from 50 to 500 nm. The tiny nha crystals are arranged along the collagen fibers. Fig.7 Change of thickness of scaffolds on copper targets with time 1 cm; 2 cm; 3 cm; 4 cm; 5 cm. t/h: a. 3; b. 6; c. 9; d. 12.

4 No.4 ZANG Jun-ting et al. 665 The experiment reveals that the deposition of fibers was difficult on all targets with a dimeter of 1 cm. Most of the fibers could be deposited on targets with a diameter of 2 cm, and the thickness of the scaffold on the 2 cm copper target was 5 mm after 12 h(these scaffolds were used for further characterization studies). When the diameter was increased to >3 cm, the total amount of deposition on the target increased but the increase in the thickness was less because of the increased surface area(figs.6 and 7). The novel scaffolds prepared via the modified electrospinning process have the advantage of possessing high porosity. Table 1 lists the apparent density and porosity of collagen-i/pla/nha scaffolds prepared on 2 cm diameter copper targets and those of traditional scaffolds. The porosity of the novel scaffold was 96%, which is much higher than that of traditional scaffolds. Table 1 Apparent density and porosity characteristics of novel and conventional collagen-i/ PLA/nHA scaffolds Electrospun scaffold Apparent density/(g cm 3 ) Porosity(%) Novel collagen-i/pla/nha scaffold Conventional collagen-i/ PLA/nHA scaffold The presence of collagen-i, PLA, and nha in the fibers was confirmed by FTIR and XRD analysis. The FTIR spectrum(fig.8) of the electrospun collagen-i/pla/nha fibers shows characteristic peaks for collagen, PLA, and nha. Typical bands, such as N H stretching at cm 1 were found in the spectrum. Peaks also were observed at 1653(C=O), 1541(N H), 1040(C N), and 2854 cm 1 (C H) in the FTIR spectrum of the collagen-i/pla/nha scaffold. The band regions of collagen ranging from 1652 cm 1 to 1215 cm 1 are directly related to the polypeptide conformation. The characteristic peak at 1653 cm 1 (C=O of collagen), which is directly related to the stretching vibrations on carbonyl groups along the polypeptide backbone, is a sensitive marker of Fig.8 FTIR spectrum of novel collagen-i/ PLA/nHA scaffold polypeptide secondary structure. The presence of this peak indicates that the secondary structure of the polypeptide has not been destroyed. The characteristic peak at 1757 cm 1 (C=O of PLA) represents the stretching vibration of the carbonyl groups in PLA. Peaks at 2997( CH 3 stretching vibration), 1456( CH 3 bending vibration), 2942( CH stretching vibration), 1382 ( CH bending vibration), and 1184 and 1130 cm 1 (C O stretching vibration) indicate the presence of PLA in the scaffold fibers. The characteristic peak at 1091 cm 1 (PO 4 3 stretching vibration), the tiny peak at 960 cm 1 (P O vibration), and the bending bands between 569 cm 1 and 601 cm 1 are due to the presence of nha. Fig.9 shows the reflection planes corresponding to the characteristic XRD spectral peaks of the novel scaffold. The characteristic peaks of nha at 25.84, 31.70, 32.18, 32.88, 34.00, 39.74, 46.62, and were observed in both the diffraction patterns of the collagen-i/pla/nha scaffold and the pure nha particles, but they were not observed in the diffraction pattern of the collagen-i/pla scaffold. Compared to the peaks of pure nha particles, the diffraction peaks of collagen-i/pla/nha scaffold were weaker, however, the base of the peaks was wider, which corresponds to a greater number of tiny particles and a lower degree of crystallinity. Fig.9 XRD patterns of nha powder(a), novel scaffold of collagen-i/pla/nha(b) and collagen-i/pla(c) 4 Conclusions Modifying the target substrates and co-electrospinning collagen-i, PLA and nha, we successfully fabricated novel scaffolds. The structure and morphology of the novel fibers are regular and the diameters of the fibers are well distributed. The diameters of collagen-i/pla/nha electrospun fibers range from 180 nm to 405 nm. By blending nha into the solution, the surface roughness of the fibers is increased. The

5 666 CHEM. RES. CHINESE UNIVERSITIES Vol.26 ultrastructure of the novel scaffolds is similar to that of the collagen fibers found in natural bone tissue. Compared to traditionally prepared scaffolds, the novel scaffolds have higher porosity and increased surface roughness, which contributes to cell adhesion, proliferation, and even gene expression. The purpose of co-electrospinning collagen-i and nha, which are normal constituents of natural bone tissue, was to mimic natural bone tissue, and this goal was achieved to a certain extent. The increased thickness of the novel scaffolds makes them good candidates for application in tissue engineering, especially in cell culture. Acknowledgment The authors would like to thank Professor YANG Bai(Jilin University, China) for fruitful collaboration and discussions. References [1] Matthews J. A., Wnek G. E., Simpson D. G., Bowlin G. L., Biomacromolecules, 2002, 3(2), 232 [2] Thomas V., Dean D. R., Jose M. V., Mattew B., Chowdhury S., Vohra Y. K., Biomacromolecules, 2007, 8(2), 631 [3] Huang H. M., Li Z. Y., Yang F., Wang W., Wang C., Chem. J. Chinese Universities, 2007, 28(6), 1200 [4] Taton T. A., Nature, 2001, 412(6846), 491 [5] Olszta M. J., Materials Science & Engineering. R, Reports, 2007, 58, 77 [6] Perumal S., Antipova O., Orgel J. P., Proc. Natl. Acad. Sci. USA, 2008, 105(8), 2824 [7] Bashur C. A., Shaffer R. D., Dahlgren L. A., Guelcher S. A., Goldstein A. S., Tissue Eng. Part A, 2009, 15, 1 [8] Baker B. M., Mauck R. L., Biomaterials, 2007, 28(11), 1967 [9] Christopherson G. T., Song H., Mao H. Q., Biomaterials, 2009, 30(4), 556 [10] Goldberg M., Langer R., Jia X., J. Biomater. Sci. Polym. Ed., 2007, 18(3), 241 [11] McCullen S. D., Steven D. R., Roberts W. A., Clarke L. I., Bernacki S. H., Gorga R. E., Loboa E. G., Int. J. Nanomedicine, 2007, 2(2), 253 [12] Christenson E. M., Anseth K. S., Beucken J. J., Chan C. K., Ercan B., Jansen J. A., Laurencin C. T., Li W. J., Murugan R., Nair L. S., Ramakrishna S., Tuan R. S., Webster T. J., Mikos A. G., J. Orthop. Res., 2007, 25(1), 11 [13] Powell H. M., Boyce S. T., Tissue Eng. Part A, 2009, 15, 1 [14] Shabani I., Haddadi-Asl V., Seyedjafari E., Babaeijandaghi F., Soleimani M., Biochem Biophys Res. Commun., 2009, 382(1), 129 [15] Elsdale T., Bard J., J. Cell. Biol., 1972, 54(3), 626