Enhanced osteogenic differentiation of mesenchymal stem cells on poly(L-lactide) nanofibrous scaffolds containing carbon nanomaterials Shun Duan,1 Xiaoping Yang,1,2 Fang Mei,3 Yan Tang,3 Xiaoli Li,4 Yuzhou Shi,1 Jifu Mao,1 Hongquan Zhang,3 Qing Cai1,2 1

State Key Laboratory of Organic-Inorganic Composites, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China 2 Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China 3 School of Basic Medical Sciences, Peking University, Beijing 100191, People’s Republic of China 4 Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057, People’s Republic of China Received 8 April 2014; revised 23 June 2014; accepted 18 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35283 Abstract: Carbon nanomaterials (CNMs), such as carbon nanotube (CNT) and graphene, are highlighted in bone regeneration because of their osteoinductive properties. Their combinations with nanofibrous polymeric scaffolds, which mimic the morphology of natural extracellular matrix of bone, arouse keen interest in bone tissue engineering. To this end, CNM were incorporated into nanofibrous poly(L-lactic acid) scaffolds by thermal-induced phase separation. The CNM-containing composite nanofibrous scaffolds were biologically evaluated by both in vitro co-culture of bone mesenchymal stem cells (BMSCs) and in vivo implantation. The nanofibrous structure itself demonstrated significant enhancement in cell adhesion, proliferation and oseogenic differentiation of BMSCs, and with the incorporation of CNM, the composite nanofibrous scaffolds further promoted osteogenic differentiation of BMSCs significantly. Between the two

CNMs, graphene showed stronger effect in promoting osteogenic differentiation of BMSCs than CNT. The results of in vivo experiments revealed that the composite nanofibrous scaffolds had both good biocompatibility and strong ability in inducing osteogenesis. CNMs could remarkably enhance the expression of osteogenesis-related proteins as well as the formation of type I collagen. Similarly, the graphenecontaining composite nanofibrous scaffolds demonstrated the strongest effect on inducing osteogenesis in vivo. These findings demonstrated that CNM-containing composite nanofibrous scaffolds were obviously more efficient in promoting C osteogenesis than pure polymeric scaffolds. V 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2014.

Key Words: osteogenic differentiation, nanofibrous scaffold, carbon nanotube, graphene

How to cite this article: Duan S, Yang X, Mei F, Tang Y, Li X, Shi Y, Mao J, Zhang H, Cai Q. 2014. Enhanced osteogenic differentiation of mesenchymal stem cells on poly(L-lactide) nanofibrous scaffolds containing carbon nanomaterials. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

Because of trauma, bone tumor and birth defect, patients suffer from various bone defects, which is a serious clinical problem. It is possible to repair the bone defects by tissue engineering method owing to the regenerative feature of bone.1 Scaffold is one of the essential aspects of tissue engineering, whose main function is to support cell proliferation and differentiation.2 The ideal tissue engineering scaffold, on one hand, should mimic the structure of natural extracellular matrix (ECM), which is composed of collagen nanofibers (50–500 nm). Both MC3T3-E1 osteoblasts and mesenchymal stem cells (MSCs)

have been reported attaching better on nanofibers than on solid-wall scaffolds or flat films.3,4 The high specific surface areas of nanofibrous scaffolds enhanced their ability in adsorbing proteins, which were vital for cell anchorage. Additionally, nanofibers could also enhance the osteogenic differentiation of osteoblasts and MSCs, which was suggested via a RhoA-Rock signaling pathway relating to cell adhesion, spreading, and osteogenic differentiation.5–9 To this end, many techniques including electrospinning,10 self-assembly,11 and thermalinduced phase separation (TIPS),12 have been applied to produce nanofibrous scaffolds. Among them, the TIPS technique in

Correspondence to: Q. Cai; e-mail: [email protected] and H. Zhang; e-mail: [email protected] Contract grant sponsor: National Basic Research Program of China; contract grant number: 2012CB933904 Contract grant sponsor: National Key Technology Research and Development Program of China; contract grant number: 2012BAI07B08 Contract grant sponsor: Beijing Municipal Science & Technology Commission; contract grant number: Z121100005212007 Contract grant sponsor: National Natural Science Foundation of China; contract grant number: 51142004 Contract grant sponsor: Program for New Century Excellent Talents in University; contract grant number: NCET-11–0556

C 2014 WILEY PERIODICALS, INC. V

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combination with particle-leaching was proven efficient in preparing 3D macroporous scaffolds with nanofibrous pore walls13 and suggested a proper technique for preparing bone tissue engineering scaffolds.14 The electrospinning and self-assembly technique were good at making nanofibers, but not efficient in producing 3D porous scaffolds that facilitated cell infiltration growth. Another issue about bone tissue engineering scaffold is that the scaffold material should be osteocompatible. To achieve efficient bone regeneration, the scaffold material should provide desired microenvironment for osteoblasts and osteoprogenitor cells adhering, spreading and migrating, as well as differentiating and synthesizing new bone matrix. Toward this approach, various components have been introduced into nanofibrous scaffolds, such as calcium phosphate compounds,15 carbon nanomaterials (CNMs),16 and growth factors.17 These nanocomposite fibers were proven having significantly improved osteoblastic cellular responses in comparison with relative polymeric fibers. CNMs, mainly carbon nanotube (CNT) and carbon nanofiber (CNF), demonstrated great promise in bone tissue engineering.18–20 Some earlier studies found the proliferation of osteoblasts increased on carbon fibers as the fiber diameter decreasing to nanometer,21 and CNFs could selectively increase osteoblast adhesion while at the same time decreased fibroblast adhesion.22 With synthesis technology and purification method of the CNTs being gradually matured, more attentions have been focused on the application of CNTs. Similar to CNFs, CNTs have been reported extensively able to promote the attachment, proliferation, and osteogenic differentiation of osteoblasts and MSCs.23–25 Therefore, one of the most promising applications of CNTs is the fabrication of polymer/CNT composites for bone tissue engineering because of their high mechanical strength and high osteoblasts affinity. On polycaprolactone/multiwall carbon nanotube (MWCNT) composite scaffolds, results showed bone marrow MSCs (BMSCs) differentiated down into osteogenic lineage and expressed high levels of alkaline phosphatase (ALP, a bone marker).26 Collagen scaffold with MWCNT surface-coating was found significantly promoting the ALP activity, calcium deposition and osteopontin (OP) expression of rat primary osteoblasts after 7-day in vitro culture, and stimulating bone formation after implantation in the femur for 8 weeks.27 In comparison with pure poly(lactide-co-glycolide) (PLGA) scaffold, PLGA/CNT composite scaffold demonstrated not only higher mechanical strength but also stronger ability in enhancing the adhesion, proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts.28 For biomedical applications, CNMs were usually purified with acid treatment in advance to remove metallic catalyst residuals, which would cause cytotoxicity.29 CNTs and graphenes (GPs) are both composed of graphene sheet; the only difference between them is that CNTs are hollow structure, while GPs are a kind of unique twodimensional structure. CNTs can be considered as made from graphene sheet rolled into a seamless cylinder that a single graphene sheet results in single-walled CNT (SWCNT), while several graphene sheets make up MWCNT. Graphene-related

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materials have also shown promise in the area of bone tissue engineering.30 The adherence and proliferation of both human osteoblasts and BMSCs were promoted after a single graphene layer being deposited onto SiO2 surfaces.31 The osteogenic differentiation of human BMSCs was also accelerated on graphene sheet, showing differentiation rate comparable with cells cultured in the presence of growth factors.32 More importantly, graphene sheets were able to guide and accelerate the BMSC differentiate toward osteogenic lineage directly, while at the same time suppress the adipogenesis of BMSC.33 As stated above, nanofibrous structured scaffolds and CNMs played positive effects on bone-related cells adhesion, proliferation and differentiation, which were very vital in bone tissue engineering. However, systematic researches are absent in identifying how their combinations will affect cell behaviors, especially the comparison between CNTs and graphenes. The hypothesis was that the sheet-like graphene might have stronger enhancement in regulating osteocompatibility than tubular MWCNT, because the former provided more contacting surface to cells than the latter when they were at the same content. Therefore, composite nanofibrous scaffolds were prepared by using poly(L-lactide) (PLLA) and MWCNT (or graphene) as starting materials. Briefly, acid treated MWCNTs (or graphenes) were added into PLLA/tetrahydrofuran (THF) solutions, and TIPS technique was applied to induce the nanofibrosis of PLLA. To investigate the direct interactions between BMSCs and the PLLA/CNM composite nanofibers, thin sheets without 3D macroporous structure were fabricated in the present study. Mouse primary BMSCs were isolated and cultured on scaffold surface. The adhesion, proliferation and osteogenic differentiation of BMSCs were determined. Animal experiments were performed for the assessment of the ectopic osteogenesis ability of MWCNT (or graphene)-containing PLLA nanofibrous scaffolds in vivo. MATERIALS AND METHODS

Materials PLLA (Mn 5 100,000) was purchased from Sigma-Aldrich. MWCNTs (purity: 95%; Nanoharbor, China) or graphenes (Angstron Materials) were dispersed in mixed H2SO4/HNO3 (3/1, v/v), kept at 80 C for 80 min. The acid-oxidized MWCNTs or graphenes were filtered, washed with deionized water, and vacuum dried for further use. All other chemicals used in producing scaffolds were of analytically pure grade, purchased from Beijing Chemical Plant (China). Scaffolds preparation The proposed MWCNTs (or graphenes)-containing PLLA nanofibrous scaffolds were prepared as follows.34 Briefly, acid-oxidized MWCNTs (or graphenes) were ultrasonically dispersed in THF. Subsequently, PLLA (5% wt) was added into the suspension and the system was thermoset at 60 C for 6 h until PLLA dissolved completely. The warm solution was immediately transferred into freezer (221 C), and kept there for over 2 h until gel formation. The gel was extracted with cold ethanol (precooled at 221 C) and dH2O and

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freeze-dried. The content of MWCNTs (or graphenes) in composite scaffolds was 1% wt or 3% wt relating to PLLA component. Similarly, pure PLLA nanofibrous scaffolds were prepared except the addition of CNM. The scaffolds were named P0 (pure PLLA), P1C and P3C (containing 1 or 3% wt of MWCNTs), and P1G and P3G (containing 1 or 3% wt of graphenes), respectively. Characterizations of scaffolds Morphologies of scaffolds were observed by scanning electron microscope (SEM, Hitachi S-4700, Japan). Before the observation, samples were fractured in liquid nitrogen and sputter-coated with gold for 30s by a sputter coater (Polaron E5600). SEM images were obtained at an accelerating voltage of 20 kV. Diameters and unit lengths of nanofibers were measured by using image analysis software (Image J). For each sample, at least 100 fibers were chosen randomly from several SEM images and measured. The hydrophilicity of scaffolds was evaluated by water contact angle measurement using a video contact angle instrument (JC2000C1, China). For each sample, three randomly chosen areas were measured and averaged. Biological property evaluation Cell culture and seeding. BMSCs were isolated from mouse. Two-month-old Balb/c mice were sacrificed and the femurs were collected. Then the two ends of femur were cut and the marrow in the cavity was flushed by BMSC basal culture medium (Cyagen) supplemented with 10% fetal bovine serum (FBS, Cyagen), 100 IU/mL penicillin (Sigma) and 100 mg/mL streptomycin (Sigma). The cell suspension was centrifuged, re-suspended and moved into 25 cm2 culture flasks, and the flasks were put into an incubator (Sanyo, Japan) thermoset at 37 C with 5% CO2 and saturated humidity supply. After 48 h culture, the suspended cells were removed by rinse of phosphate buffer saline (PBS, Hyclone), and the adhered cells were cultured for another 1 week. When grew to confluence of 80%, the cells were digested by 0.25% trypsin (Sigma) and 0.02% ethylene diaminetetraacetic acid (EDTA). The fifth passage of BMSCs was used in the further study. Scaffolds were cut into square sheets with the size of 10 mm 3 10 mm and placed into 24-well culture plates (Corning). Before cell seeding, the samples were sterilized by UV light for 2 h, rinsed by PBS for three times and soaked by culture medium overnight. The cell number on each sample was 5 3 103 for adhesion, proliferation assay and morphology observation, 1 3 105 for osteogenic differentiation assay, and 4 3 105 for animal experiment. For in vitro experiments, tissue culture polystyrene (TCPS) was taken as control. For osteogenic differentiation assay, 0.05 mmol/L vitamin C (Sigma), 10 mmol/L b-sodium glycerophosphate (Sigma), and 1 3 1028 mol/L dexamethasone (Sigma) were added to culture medium. The culture medium and osteogenic inductive medium were changed every 3 days. Cell adhesion. After 2 h and 4 h of cell seeding, the culture medium of each well was aspirated, and the samples were

rinsed by PBS softly to remove the nonadhered cells. Then 200 lL of culture medium and 20 lL of Cell Counting Kit-8 (CCK-8, Beyotime, China) were added into each well. CCK-8 is a kind of yellow solution that can be reduced to orange by active cells, whose absorbance is directly proportional to cell number. After 4 h of incubation, the OD value of the liquid in each well was measured by a microreader (Bio-rad 680) at the wavelength of 490 nm. The cell number was proportional to the OD value. Cell proliferation. Cell proliferation was tested by CCK-8 method. Briefly, at days 1, 3, 5, and 7 after cell seeding, 20 lL of CCK-8 solution was added into each well and incubated for 4 h, and then the OD value was measured by a microreader at 490 nm. The cell number was proportional to the OD value. Cell morphology. After 1, 3, 5, and 7 days of cell seeding, samples were retrieved from the culture plate, rinsed by PBS for three times and fixed by 2.5% glutaraldehyde (Beijing Chemical Plant, China). The fixed samples were airdried at room temperature, followed by being scatter-coated with gold and observed by SEM. ALP activity. BMSCs/matrix complexes were incubated in osteogenic medium for 3, 7, 14 and 21 days. Then they were retrieved from the culture plate and rinsed by PBS for three times. Cells were lysated by 1% Triton X-100, and the ALP activity of lysate was tested by p-nitrophenyl phosphate (p-NPP, Amerisco). The aliquots of cell lysate were incubated with 5 mmol/L p-NPP in 0.1 mol/L glycine-KOH solution (pH 5 10.5) for 30 min at 37 C, and the reaction was stopped by KOH solution. OD values were measured by a microreader at 405 nm. The relative ALP activity was represented as OD value/cell number ratio. Calcium content. BMSCs/matrix complexes were incubated in osteogenic medium for 3, 7, 14, and 21 days. Then they were retrieved from the culture plate and rinsed by deionized water. The mineralized Ca deposition was dissolved by HCl (1 mol/L). Then the samples reacted with o-cresolphthaleincomplexone (Genmed) solution for 5 min. OD values were measured by a microreader at 570 nm. The calcium content was calculated basing on a standard curve made by a series of concentrations of Ca21 solutions. Type I collagen content. BMSCs/matrix complexes were incubated in osteogenic medium for 3, 7, 14, and 21 days. Then they were removed from the culture plate and rinsed by PBS for three times. The cell-scaffold complexes were homogenized with PBS by sonication in ice bath. Then the samples were tested by a type I collagen ELISA kit (R&D) following the manufacturer’s instruction. The OD value was measured by a microreader at 450 nm. The content of type I collagen was calculated basing on a standard curve made by a series of concentrations of standard solutions. Animal experiment Four-week-old male nude mice were used in this animal experiment. All animal experimental procedures followed

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FIGURE 1. Scaffold morphology. Macroscopical and SEM observations of PLLA nanofibrous scaffolds containing MWCNTs or graphenes: (a) macroscopical appearance; (b) P0 scaffold; (c) P3C scaffold (white arrows indicating the presence of MWCNTs); and (d) P3G scaffold (black arrow pointing to the graphene sheet).

the guidelines of the Department of Laboratory Animal Science, Health Science Center, Peking University, China. Implantation of the scaffold. BMSCs were seeded onto P0, P3C, P3G scaffolds and cultured in osteogenic medium for 1 week before implantation. The nude mice were anesthetized with pentobarbital sodium by intraperitoneal injection. The cell-seeded scaffolds were cut into small patches (approximately 2 mm 3 2 mm) and implanted into the muscle pouch. At 2, 4, and 8 weeks after surgery, the implants were harvested with the surrounding tissues. The retrieved implants were fixed by 4% paraformaldehyde solution (Cellchip, China), and then dehydrated by gradient concentrations of ethanol, embedded by paraffin, and cut into histological slices. Six animals were used for each group.

cal staining were visualized by light microscope (Optec BK5000, China). In order to observe type I collagen, Masson staining was performed. The histological slices were deparaffinized and hydrated with distilled water. The hydrated slices were stained by Masson staining kit (Senbeijia, China) following the instruction of the manufacturer. The results of staining were observed by light microscope. Statistical analysis The experiments of biological property evaluation were performed in triplicate (n 5 3) and repeated for three times. The results were presented as mean 6 SD. Statistical analysis was made by t-test between two groups and the differences were considered as significant for p  0.05. RESULTS

Evaluations The histological slices were stained by hematoxylin and eosin, and observed by a light microscope (Olympus CX-41, Japan). For immunohistochemical analysis, three osteogenic marker proteins including osteopontin (OP), osteonectin (ON), and osteocalcin (OC), were detected by anti-osteopontin (Abcam, UK, cat.# ab91655, 1:200 dilution), anti-osteonectin primary antibody (Abcam, UK, cat.# ab55847, 1:200 dilution) and anti-osteocalcin primary antibody (Abcam, UK, cat.# ab93876, 1:200 dilution), respectively, followed by incubation with twostep IHC detection reagent (ZSGB-Bio, China) and colored by DAB reagent (ZSGB-Bio, China). The nuclei were counterstained by hematoxylin. The specimens of immunohistochemi-

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Characterization of PLLA/CNM composite scaffold Produced by TIPS and finally being freeze-dried, the resulting PLLA/CNM composite scaffolds were observed macroscopically and microscopically (Fig. 1). As shown in Figure 1(a), the addition of MWCNTs or graphenes turned the white PLLA scaffold into dark gray or black. The color distribution was even throughout all the composite scaffolds. From SEM observation [Fig. 1(b-c)], all the samples demonstrated a kind of uniform fibrous structure, independence of compositions. The incorporation of MWCNTs or graphenes did not alter the morphology of PLLA fibers. MWCNTs could be seen combining to PLLA fibers evenly, while graphene pieces were dispersed and entrapped in the PLLA fibrous

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TABLE I. Structure Parameters and Water Contact Angles of Various PLLA Nanofibrous Scaffolds Produced in the Present Study

Sample P0 P1C P3C P1G P3G

Fiber Diameter (nm)

Unit Length (nm)

Water Contacting Angle ( )

120 6 53a 720 6 56b 668 6 51b 915 6 91c 663 6 101d

924 6 94a 3246 6 121b 2688 6 110c 3238 6 141d 2662 6 126e

89.6 6 0.2a 90.9 6 0.3b 91.2 6 0.3b 91.6 6 0.2b 92.9 6 0.2c

Different superscripts in the same column indicated significant difference (p < 0.05, fiber diameter and unit length, n 5 100; water contacting angle, n 5 20), while the same superscript indicated nonsignificant difference.

network. However, their incorporation displayed significant effect on both fiber diameter and unit length (the distance from one crossing of fibers to another; Table I). The average fiber diameter of pure PLLA scaffold was as thin as 120 6 53 nm and the unit length was 924 6 94 nm. The diameters and unit lengths of PLLA/CNM composite scaffolds were much larger than those of PLLA scaffold. They reached several hundreds and thousands nanometers with a decreasing trend as the content of CNM increasing. The water contact angle of the pure PLLA nanofibrous scaffold (P0) was 89.6 6 0.2 . In comparison, PLLA/CNM composite scaffolds were more hydrophobic with their water contact angles increasing slightly. And more the CNMs were incorporated, the higher water contact angles were detected. In vitro evaluation Cell adhesion and proliferation. BMSCs could adhere on the surfaces of all the samples at 2 h after seeding, and the adhesion rate increased over time [Fig. 2(a)]. The adhesion rates of BMSCs on all the nanofibrous scaffolds were higher than those on flat TCPS. In comparison with P0 scaffold, the

incorporation of CNM further increased the cell adhesion rates. The presence of graphenes resulted in the highest cell adhesion rate. The cells could proliferate continuously on all the samples during the 7-day culture [Fig. 2(b)]. The slowest proliferation rate was observed on the flat TCPS surface, while all the other nanofibrous scaffolds demonstrated higher BMSC growth rates. Compared with P0 scaffold, BMSCs proliferated apparently faster on CNM-containing composite scaffolds. The enhancement in cell growth rates displayed a distinct correlation with both the content and the type of CNM. At the same content of CNM, graphenes were more effective than MWCNTs. For the same CNM, higher content of CNM caused faster BMSC proliferation rate. Cell morphology. The morphology of BMSCs on the pure PLLA and CNM-containing nanofibrous scaffolds was observed by SEM (Fig. 3). No significant difference was detected in cell morphology between groups. On all the nanofibrous scaffolds, BMSCs could adhere and spread well on the scaffolds at 1 day after cell seeding. The cells on the nanofibrous scaffolds were in a long and slender shape, presenting a kind of 3D-like morphology. From the third day, the mitosis of BMSCs was active and cell number increased continuously over time. After 7-day culture, a large amount of ECM was secreted and the cells connected with the adjacent ones. The quantities of cells on PLLA/CNM composite scaffolds were significantly more than those on pure PLLA scaffold, which was consistent with the proliferation results of CCK-8 assay. Osteogenic differentiation. The osteogenic differentiation of BMSCs on the pure PLLA and CNM-containing nanofibrous scaffolds was evaluated by ALP activity, type I collagen content and calcium deposition analysis (Fig. 4). ALP activity was an early indicator of osteogenesis. It reached

FIGURE 2. Cell adhesion and proliferation. Adhesion (a) and proliferation (b) of BMSCs on PLLA nanofibrous scaffolds containing different amounts of MWCNTs or graphenes. Data were presented as mean 6 SD, * p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 3. Cell morphology. SEM images of the morphology of BMSCs cultured on PLLA nanofibrous scaffolds containing different amounts of MWCNTs or graphenes for 1, 3, and 7 days after seeding.

the peak after 14-day-culture in all cases [Fig. 4(a)]. The BMSCs on all the nanofibrous scaffolds showed significantly higher ALP activity than those on flat TCPS surface. The incorporation of MWCNTs or graphenes into PLLA nanofibrous scaffolds could further increase the ALP activity of BMSCs. The enhancement was proportional to the content of CNM. While at the same content, PLLA/graphene composite scaffold demonstrated a stronger ability in inducing the ALP activity expression than PLLA/MWCNT composite scaffold. Among all the nanofibrous scaffolds, the P3G scaffold displayed the highest ALP activity. Calcium salts and type I collagen were the main components of inorganic and organic phase of bone tissue, and they would accumulate along the osteogenic differentiation of BMSCs. The calcium content was determined by the ocresolphthaleincomplexone method. As shown in Figure 4(b), the calcium content increased with culture time in all

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cases. However, the calcium deposition rate was the slowest on TCPS in comparison with other nanofibrous scaffold groups. Among the five nanofibrous scaffold groups, the CNM-containing ones demonstrated significantly higher calcium depositions than the pure PLLA nanofibrous scaffold. For the four CNM-containing PLLA nanofibrous composite scaffolds, the calcium contents were found in an order of P3G > P3C > P1G > P1C, which clearly revealed that the content and the type of CNM could affect the calcium deposition in the osteogenic differentiation of BMSCs. After 21-day culture, the Ca content on P3G could reach about 2.5 times as much as that on TCPS. The content of type I collagen was determined by ELISA method. As shown in Figure 4(c), it distinctly displayed the similar results to those of calcium deposition. After 21-day culture, the type I collagen contents on different substrates were found in an order of P3G > P3C > P1G > P1C > P0 > TCPS.

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FIGURE 4. Cell osteogenic differentiation. Evaluation of the osteogenic differentiation of BMSCs cultured on PLLA nanofibrous scaffolds containing different amounts of MWCNTs or graphenes by determining: (a) ALP activity; (b) Ca deposition; and (c) type I collagen content. Data were presented as mean 6 SD, * p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

In vivo evaluation Histological analysis. To investigate the effects of CNM on inducing osteogenic differentiation in vivo, P3C and P3G nanofibrous scaffolds were implanted into the muscle pouch of nude mice, using P0 as the control. P3C and P3G were selected for in vivo evaluation because they had demonstrated strong ability in enhancing the osteogenic differentiation of BMSCs in vitro. BMSCs were seeded on P0, P3C, P3G scaffolds and cultured for 1 week before implantation. For histological analysis, the morphologies of BMSC/scaffold complexes were visualized by hematoxylin and eosin staining, and the results are shown in Figure 5. At week 2 after implantation, no obvious necrosis, inflammatory response

and fibrous membrane were found in the histological slices for all the three groups. And BMSCs, which were initially seeded on the surface of scaffold, could be seen proliferating and growing into all the scaffolds. At week 4 after implantation, the cell number increased apparently, and more cells had grew into the P0, P3C, or P3G nanofibrous scaffolds with P3G group showing the highest cell density. In addition, vascularization could be detected in the P3G group at this time point. And at 8 week, vascularization was observed rich in the specimens of P3G samples. Expression of OP, ON, and OC. Immunohistochemical staining was utilized in order to characterize the bone formation-related

FIGURE 5. Histological analysis. Histological images of P0, P3C, and P3G after implantation for 2, 4, and 8 weeks. M: muscle; BS: BMSC/scaffold complex; V: vessel. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 6. Immunohistochemical analysis. Immunohistochemical staining of the BMSC/scaffold complexes for osteopontin (a), osteonectin (b), and osteocalcin (c) after being implanted for 2, 4, and 8 weeks. The positive area of immunohistochemical staining was brown. Scale bar 5 40 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

proteins including OP, ON, and OC. For the expression of OP, ON and OC, which are markers of osteogenesis, their immunohistochemical staining results of 2-, 4-, and 8-weekimplantation are shown in Figure 6. Clearly, the brown areas

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showed the presence of OP, ON, and OC in the implants. Although all the three kinds of nanofibrous scaffolds were detected the expression of OP, ON, and OC, the difference in expression extent could be revealed by the area of the staining.

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FIGURE 6. (Continued).

P3C and P3G groups obviously displayed stronger and larger staining than the P0 group. Among the three groups, P3G group resulted in the strongest expression of OP, ON, and OC in the early period of osteogenesis. Expression of type I collagen. Masson staining was used to evaluate the formation of type I collagen, and the staining results are shown in Figure 7, in which the collagen was stained into blue area. After 2 weeks of implantation, type I collagen could be seen mainly existing around the implants. As the implantation prolonging, more and more type I collagen was synthesized and prevailing into the implants. Rich type I collagen could be detected in the two CNMcontaining groups after 8 weeks of implantation, in which the expression of type I collagen was much stronger than that in the P0 group. Especially, the P3G group demonstrated the strongest type I collagen staining and the evenly distribution of blue areas throughout the implantation. DISCUSSION

Both nanofiber and CNM can enhance the osteogenic differentiation of osteoblast and BMSC. Their combinations were envisioned good substrates for bone regeneration. CNT and graphene are two common CNMs being used in biomedical applications, but they are in different morphologies. It is interested to know how the nanofibrous CNT and the sheet-like graphene would affect the osteogenic differentiation of BMSC. The PLLA nanofibrous scaffolds used in this study were prepared by a kind of phase separation technique. As shown

in Figure 1, the scaffolds demonstrated perfect nanofibrous network structure. The incorporation of MWCNT or graphene did not cause adverse effect on the nanofibrosis of PLLA; however, their presences changed the fiber diameter and unit length (Table I). The mechanism of nanofibrosis of PLLA by using phase separation was lying on the gelation upon cooling. The addition of black CNMs might change the themoconductive property of PLLA/THF solutions, and result in slower gelation rate in comparison with the pure PLLA/THF solution.35 Accordingly, this might lead to the thicker fibers diameter and the longer unit lengths in PLLA/ CNM nanofibrous composite scaffolds than those in pure PLLA nanofibrous scaffold. In addition, the incorporation of CNMs also slightly increased the hydrophobicity of the composite scaffolds in comparison with pure PLLA scaffold (Table I). The MWCNT and graphene used in preparing the scaffolds were acid-oxidized in advance, which would introduce some hydrophilic groups as hydroxyl and carboxyl groups onto them.36 These groups might interact with the hydroxyl or carboxyl end-groups of PLLA backbone via hydrogen bond interaction. Therefore, it was suggested that the decreased content of hydrophilic polar groups on the surface led to the increased water contact angles. All the nanofibrous scaffolds prepared in this study could be seen resembling the morphology of natural bone ECM, which was mainly composed of collagen nanofibers.37 Compared with TCPS, all the nanofibrous scaffolds demonstrated enhancements in every aspect of biological behaviors of BMSCs both in vitro and in vivo, including cell attachment, proliferation and osteogenic differentiation. The

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FIGURE 7. Masson Staining. Masson staining of BMSC/scaffold complexes at 2, 4, and 8 weeks post-implantation. In the images, type I collagen was stained blue, while skeletal muscle was stained pink and nuclei were stained purple. BS: BMSC/scaffold complex. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

biomimetic topographic structure was one contribution.38 Cell morphology played an important role in the expression of cell functions.39 The cells on nanofibrous scaffolds were in elongated and slender shape, and the cells connected to the nanofibers and neighboring cells by filopodia. This morphology was beneficial to the expression of the cell phenotype. Due to the nanofibrous structure, the significantly increased specific surface area of the scaffolds raised the surface roughness and improved the protein absorption ability, which were believed the main contribution to the enhanced biological behaviors.40 It was reported that a network of pathways consisting of FAK, TGF-b, Wnt signaling, and MAPK was involved in the osteogenic differentiation of BMSCs on PLLA nanofibers.8 The random nanofibers could trigger nonspecific and multilevel responses in BMSCs via mechanotransduction without osteogenic supplement. The incorporation of MWCNT or graphene further promoted the enhancement in cell biological behaviors. In all cases, it could be seen clearly that the BMSCs proliferated faster, expressed ALP activity higher, produced bone formation-related proteins richer as the contents of CNMs in the composite scaffolds increasing. It was reported the electric property of CNMs played an important role in this promotion.23 CNMs demonstrated a strong ability of absorbing proteins because the p electron cloud of graphite structure could intact with the hydrophobic part of protein.33 And they could specifically absorb the proteins relating to cell proliferation, such as albumin and fibronectin.41 In addition, the bioactive components in the osteoinductive culture

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medium are dexamethasone, b-glycerophosphate disodium, and ascorbic acid. Dexamethasone had aromatic structure which could interact with the graphite structure by p-p bond.42 The absorbed dexamethasone could promote the differentiation of BMSC and mineralization. Ascorbic acid and b-glycerophosphate could also be absorbed by CNMs via hydrogen bond interaction between polar functional groups, and had a synergy with dexamethasone to increase the ALP activity of BMSCs.43 Therefore, the existence of MWCNT or graphene in PLLA nanofibrous scaffolds further improved the biological properties of PLLA nanofibrous scaffolds. Although the MWCNT and graphene had the similar graphite structure and electric property, the sheet-like graphenes displayed even stronger enhancement in cell behaviors than the nanofibrous MWCNTs. From Figure 1, it could be seen the tiny tubular MWCNTs were partly embedded by PLLA nanofibers, while most of the sheet-like graphenes were only entrapped in the nanofibrous scaffold. Theoretically, the BMSCs were more likely exposed to sheet-like graphenes than to nanofibrous MWCNTs in cell culture. What’s more, the sheet-like graphenes have more defects along their edges than the tubular MWCNTs, which would result in more hydroxyl and carboxyl groups being introduced in the acidic treatment. Therefore, graphenes could exert stronger effects on biological properties of the PLLA scaffolds than MWCNTs via the above mentioned mechanisms. Accordingly, the PLLA composite scaffolds containing 3% wt of acid-oxidized graphenes (P3G) distinctly showed the

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ORIGINAL ARTICLE

highest levels of proliferation, ALP activity, calcium deposition and type I collagen synthesis in vitro. By implanting the BMSC/scaffold complexes in vivo, both the pure PLLA scaffold and PLLA/CNM composite scaffolds demonstrated excellent biocompatibility. No obvious necrosis, inflammatory response and fibrous membrane were found in the histological slices for all groups. From 2 weeks to 8 weeks, cells gradually migrated into the nanofibrous scaffolds, and the immigration rates were faster in the PLLA/CNM scaffold groups than in the pure PLLA scaffold group (Fig. 5). This was suggested due to their enlarged unit length and the effect of CNMs. Cell proliferation was faster on PLLA/CNM scaffolds than on pure PLLA scaffold. Vascularization was first detected on P3G nanofibrous scaffold. The immunohistochemical staining results further confirmed the function of CNMs in upregulating the express of bone formation-related proteins as OP, ON, and OC in vivo (Fig. 6). CNMs, especially the graphenes, demonstrated a significant ability in promoting type I collagen synthesis in vivo (Fig. 7). These results were well coincident with those in vitro data. It was envisioned the PLLA/CNM nanofibrous composite scaffolds were able to provide a beneficial microenvironment for the osteogenic differentiation of BMSCs, which categorized them promising substrates for bone regeneration applications. CONCLUSION

By incorporation CNT or graphene with PLLA, the PLLA/ CNM nanofibrous composite scaffolds with the similar structure of natural EMC were prepared by TIPS, and they could support the cell adhesion, proliferation and differentiation both in vitro and in vivo. It demonstrated that CNMs could improve the osteocompatibility of PLLA nanofibrous scaffold by promoting the osteogenic differentiation of BMSCs. These effects were closely related to the content of CNM, in which higher content of CNM resulted in stronger effect. The sheet-like graphene displayed even stronger enhancement than the tubular CNT when they were at the same content. The PLLA/CNM nanofibrous composite scaffolds were biocompatible and able to provide suitable microenvironment for the proliferation and differentiation of BMSC. The present study proposed a potential strategy of using PLLA/CNM nanofibrous composite scaffolds for stem cell application in bone tissue engineering.

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