Biocomposites
Cell-Assembled Graphene Biocomposite for Enhanced Chondrogenic Differentiation Wong Cheng Lee, Candy Haley Lim, Kenry , Chenliang Su, Kian Ping Loh,* and Chwee Teck Lim*
Graphene-based
nanomaterials are increasingly being explored for use as biomaterials for drug delivery and tissue engineering applications due to their exceptional physicochemical and mechanical properties. However, the twodimensional nature of graphene makes it difficult to extend its applications beyond planar tissue culture. Here, graphene–cell biocomposites are used to preconcentrate growth factors for chondrogenic differentiation. Bone marrow-derived mesenchymal stem cells (MSCs) are assembled with graphene flakes in the solution to form graphene-cell biocomposites. Increasing concentrations of graphene (G) and porous graphene oxide (pGO) are found to correlate positively with the extent of differentiation. However, beyond a certain concentration, especially in the case of graphene oxide, it will lead to decreased chondrogenesis due to increased diffusional barrier and cytotoxic effects. Nevertheless, these findings indicate that both G and pGO could serve as effective pre-concentration platforms for the construction of tissue-engineered cartilage and suspension-based cultures in vitro.
1. Introduction Dr. W. C. Lee, Kenry, Prof. C. T. Lim Department of Biomedical Engineering and Department of Mechanical Engineering National University of Singapore Singapore 117575, Singapore E-mail: [email protected] Dr. C. H. Lim, Dr. C. Su, Prof. K. P. Loh Department of Chemistry National University of Singapore Singapore 117543, Singapore E-mail: [email protected] Prof. C. T. Lim Mechanobiology Institute National University of Singapore Singapore 117411, Singapore Dr. C. H. Lim, Kenry, Dr. C. Su, Prof. K. P. Loh, Prof. C. T. Lim Graphene Research Centre National University of Singapore Singapore 117546, Singapore Dr. W. C. Lee, Prof. K. P. Loh, Prof. C. T. Lim NUS Graduate School of Integrative Sciences and Engineering National University of Singapore Singapore 117456, Singapore DOI: 10.1002/smll.201401635 small 2014, DOI: 10.1002/smll.201401635
Human bone marrow derived mesenchymal stem cells (hMSCs) provide a promising cell source for engineering cartilage tissues or for cell-based treatments of osteochondral defects owing to their multipotency, ease of availability and immunosuppressive properties.[1] Articular cartilage is a highly organized avascular tissue composed of chondrocytes embedded within an extracellular matrix of collagen, proteoglycans and noncollagenous proteins.[2] Cartilage damage and associated joint dysfunction affects a growing number of both young and elderly individuals. Due to the low mitotic activity of chondrocytes and the avascular nature of cartilage tissues, chondral defects are difficult to heal.[3] Thus, surgical interventions such as microfracture technique and autologous chondrocyte transplantations are frequently required to repair cartilage defects and restore joint function.[4] However, these techniques are limited by their clinical efficacy and the lack of supply of chondrocytes for clinical use has led to a call for new regenerative tissue engineering approaches to facilitate the differentiation of hMSCs into mature chondrocytes to augment the repair process. Using graphene (G) as a supporting scaffold for stem cell differentiation have received significant attention in recent years. Owing to their excellent mechanical stability and
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physicochemical properties,[5] graphene based tissue engineering approaches offered new possibilities for the restoration of damaged or lost tissues. To enhance stem cell growth and differentiation, graphene has been used as a planar culture platform for various types of stem cells and differentiation lineages. Graphene and related materials have been investigated for osteogenic,[6,7] adipogenic,[8] myogenic[9] and neural differentiation.[10] Our previous work indicated that osteogenic differentiation is enhanced on G due to its ability to pre-concentrate growth factors and chemical inducers via electron clouds π–π bonding.[6] However, this approach is limited to planar cell growth and is difficult to translate to applications that require suspension cultures for three-dimensional tissue formation. Specifically, during carti- Figure 1. Schematic of experimental design. (A) Serum proteins and differentiation factors lage formation, hMSCs are usually grown were pre-incubated with graphene solutions (G, GO or pGO) overnight before MSCs at in a pellet or on a micromass[11] due to passage 5 were added into the mixture. hMSCs fold and self-assemble into a bio-composite the phenotypic instability of monolayer pellet after 24 hours of incubation (B) 4 h, (C) 8 h, (D) 12 h, (E) 20 h and (F) 24 h. Pellets were subsequently differentiated in chondrogenic induction medium for 4 weeks. culture for chondrocyte maturation.[12] Although pellet cultures have been used clinically to construct hyaline cartilage and repair chondral applications in tissue engineering.[21–23] The schematic of defects,[13–15] this technique faces limitations such as lack of experimental design is shown in Figure 1A. Briefly, G based controlled release of cytokines for prolonged culture and solutions at different concentrations (10 µg/mL, 25 µg/mL poor process ability.[3] Therefore, a significant need exists for and 50 µg/mL) were incubated in 10% (v/v) fetal bovine a solution based culture technique that can efficiently entrap serum and chondrogenic chemical inducers overnight before cells and sufficiently provide the necessary mechanical and 200,000 hMSCs were added. After the cells were introduced, chemical cues to stimulate new cartilage tissue development. the cells and G (or pGO, G) folded and assembled together Current therapies aimed to regenerate articular cartilage into a composite pellet 24 h later as shown in Figures 1B-F. traumatic injuries are time-consuming and the quality of Cell pellets which were formed and differentiated in the repaired tissues needs improvement. Also, routine surgical absence of G (or pGO, G) (Figures A, B) were used as contechniques used to repair damaged cartilage tissues often trols and the extent of differentiation was assessed through result in significant fibrosis and delayed healing.[16] To date, histochemical staining for alcian blue (Figure 2C) and immuthere has been minimal progress made in tissue engineering nostaining for type II collagen production (Figures 2D-F). techniques to augment surgical strategies for cartilage regenMacroscopic images of the cells were used to assess comeration. Recently, the use of GO film for stem cell differen- posite formation and morphology (Figure 3). After selftiation towards cartilage tissues has been explored but it was assembly, we observed that the microparticles of G and pGO reported that the extent of differentiation decreased with distributed homogeneously within the construct (Figures 3A-C time,[8] as cell-cell interaction was confined to within the 2D and Figure 3G-I). Microparticles of GO appeared larger than G plane. Owing to the exceptional physicochemical properties and pGO and showed a higher propensity to assemble around of G, recent approaches aimed to enhance 3D stem cell dif- the surface of the composite (Figures 3D-F). Similarly, composferentiation using G scaffolds are gaining traction.[7,17] How- ites of GO were significantly larger than those assembled with ever, research on using G and related materials as ‘soluble, G and pGO (Figure 4C). No significant differences in pellet 3-dimensional scaffolds’ are lacking. Therefore, our study aims size were observed between the G and pGO composites. The to develop a solution-supported graphene-based construct to segregation of GO on the outer surface of the composite versus support pellet formation and differentiation of human bone the inner distribution of pGO and G inside the composites may marrow derived hMSCs towards the chondrogenic lineage. be due to the greater hydrophilicity of GO, which favours its interaction with the aqueous-based extracellular environment. Compared to G and pGO, GO has a higher concentration of oxygen functional groups[19] and thus has a tendency to cross2. Results and Discussion link via hydrogen bonding on the hydrophilic cell surfaces to First, we sought to quantitatively evaluate the biocompat- form a thin layer around the exterior of the composite. ibility of G, GO and pGO in vitro. Bone marrow derived To assess the biocompatibility of the G-based micropartihMSCs were used for these experiments because they cles on chondrogenic tissue formation, cell viability was monhave been extensively used for evaluating osteo-chondral itored by measuring the metabolic rates of the composites
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small 2014, DOI: 10.1002/smll.201401635
Cell-Assembled Graphene Biocomposite for Enhanced Chondrogenic Differentiation
Figure 2. Chondrogenesis of hMSCs. (A) hMSCs develop into a micromass 24 h after incubation in chondrogenic induction media. Inset shows monolayer of cells before assembly into pellet. (B) Stereoscopic image of chondrogenic cell pellet in the absence of GO, G or pGO solution. Differentiation toward chondrogenic lineage was assessed by (C) Alcian Blue staining for sulfated mucosubstances and (D-F) type II collagen immunostaining. (Scale bars, 200 µm).
normalized to those formed in the absence of G-based microparticles (Figures 4A, B) at day 24 h and 72 h after composite formation. At day 1 post assembly, the metabolic rates of the composites remained high with increasing concentrations of G, GO and pGO (Figure 4A). However, at day 3, the viability of the cells decreased with increasing concentrations of G and GO despite their low initial cytotoxicity (Figure 4B). In contrast, the viability
of pGO remained high with increasing pGO concentration. Serum proteins and growth factors are essential for cell survival and proliferation. In order to determine whether the observed cell viability differences is due to the capacity of the G-based materials to pre-load growth factors, the protein loading capacity of G and its derivatives was quantified via spectrophotometric measurements. We found that the protein loading capacities of GO and pGO were significantly larger than that of G (Figure 4D). However, this trend did not correlate with the long-term (72 h) viability of the cells since the viability of those assembled with both G and GO decreased while those assembled with pGO remained high (Figure 4B). Therefore, the decrease in the viability of G and GO composites at day 3 and could be attributed to the increased diffusional barrier for the cells embedded in the inner layers of the composite. This effect is more pronounced in GO composites because of the tendency of the GO microparticles to assemble closer to the surface of the construct; thereby creating a thicker diffusion barrier. The higher viability of pGO is related to its porosity, which allows exchange of substances between the cells and its surrounding. After 4 weeks of culture and differentiation, Alcian blue staining was used to assess histological changes and sGAG synthesis. The extent of Alcian blue staining can be correlated to the amount of sGAG Figure 3. Chondrogenesis of hMSCs incubated with different concentrations of (A-C) produced. Composites formed with 10 µg graphene, (D-F) graphene oxide, (G-I) porous graphene oxide. hMSCs self assembled into of G, GO and pGO had uniform staining a graphene-cell biocomposite 24 h after incubation. Inset shows monolayer of cells after graphene solution is introduced. (Scale bars, 200 µm). G and pGO are more homogenously for Alcian blue, which was the characdispersed and distributed within the cell pellet than GO. A thicker GO shell can be seen in the teristic of cartilage matrix. This means that the composites could differentiate case of hMSCs incubated with GO. small 2014, DOI: 10.1002/smll.201401635
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Figure 4. Viability of cells incubated with different graphene solutions was assessed using Alamar Blue metabolic assay at (A) 24 h and (B) 72 h normalized to controls. Cell pellets formed in the absence of G or its derivatives were used as controls. Pellet diameter (C) was measured at 24 h (n = 3). (D) Protein loading capacity of serum proteins on G, GO and pGO was assessed 24h after incubation. After 74 h of culture, cell viability decreased (B) with increasing concentration of GO due to increased diffusional barrier while cell viability remained high for those incubated with pGO. (*p < 0.05; n = 3 for each group).
into chondrocytes and synthesize a cartilaginous matrix (Figures 5A, D and G). For both G and pGO, the extent of staining increased with increasing concentrations of the microparticle during composite formation. Spectrophotometric quantification of the amount of Alcian blue staining revealed that the amount of sGAG produced was highest for 25 µg of G, while sGAG synthesis decreased with increasing concentrations of GO (Figure 6A). To confirm observations from Alcian blue staining and quantification, immunohistochemistry staining of collagen II was carried out. Compared to weakly staining of GO composites when the concentration is increased (Figures 7E, F), staining of collagen II was enhanced when the concentration of pGO is increased (Figures 7G-I). Conversely, intense staining of collagen II was detected in composites with 25µg of G while the production of
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collagen II decreased when the concentration is increased to 50 µg, suggesting that the optimum cells to G ratio for chondrogenic differentiation is 8000 cells/µg of G. We next investigated changes in tissuespecific gene expression to evaluate lineage specificity of the differentiating stem cells in the various composites. Cell pellets formed in the absence of G or its derivatives were used as controls. Gene expression was evaluated after 4 weeks of culture. Cartilage specific markers- type II collagen, Sox9 and aggrecan were up-regulated in both pGO and GO composites compared with bare pellet cultures (Figures 6B-D), indicative of enhanced chondrogenesis. Specifically, collagen II gene was dramatically up-regulated at 25 µg of G but declined when the concentration was increased further. It appears that optimal spatial configuration and cell-cell contacts are provided by the G microparticles at this concentration. However, when the concentration of G was increased further, the extent of chondrogenic differentiation became limited by the decreased cell-cell communication and exchange of oxygen and other nutrients that regulate matrix production and chondrocyte phenotype. Decreased availability of glucose is known to alter the metabolism of cells in a pellet and their susceptibility to apoptosis.[24,25] Moreover, the cartilage markers remained relatively low in all GO composites as the GO shell increased the compactness of the composite which restricted hMSC proliferation and differentiation. In contrast, pGO further enhanced chondrogenic differentiation when the concentration of the microparticles is increased because the porous nature of pGO provides routes for diffusional exchange and sustains the metabolism of the cells in the pellet.
3. Conclusions We have demonstrated the ability of G, GO and pGO to support pellet formation and chondrogenic differentiation of hMSCs. These G microparticles serve as ‘growth factor factories’ for the formation and maturation of chondrogenic tissues. The loading amount of GO and its derivatives in the composite accelerates chondrogenic differentiation initially, but excessive amounts affect the viability of the cell composites. Our results show that engendering porosity in G allows a higher loading of GO and further enhances the differentiation rate of the cells. Overall, we envision that these novel synthetic cell-assembled composites can provide an easy and effective way to construct cartilage tissues for various regenerative applications.
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small 2014, DOI: 10.1002/smll.201401635
Cell-Assembled Graphene Biocomposite for Enhanced Chondrogenic Differentiation
Figure 5. Representative images of Alcian blue staining of the cross section of cells incubated with G (A-C), GO (D-F) and pGO (G-I) after 4 weeks of culture. Inset shows the respective biocomposite pellet. (Scale bars, 200 µm). The extent of Alcian blue staining increased when the concentration of G and pGO were increased while the extent of staining decreased when the concentration of GO was increased.
Figure 6. Biochemical and gene expression analysis of in vitro chondrogenesis. (A) Alcian blue dye was extracted from the biocomposites and its optical density measured and normalized to pellets in the absence of graphene solutions. Real-time PCR analysis of markers for cartilage normalized to cell pellets formed in the absence of G or its derivatives (B-D). Data are presented as average ± SD (n = 3). Gene expression analysis of in vitro chondrogenesis corroborates with histological and immunological staining. Greatest extent of chondrogenic differentiation was observed for cells incubated with 25 µg of G. Differentiation decreased when the concentration of GO is increased because the amount of chemical factors becomes limiting due to increased diffusional barrier. In contrast, differentiation increased with the concentration of pGO. This can be ascribed to the porous nature of pGO which facilitates diffusional exchange even when the concentration of pGO is increased. small 2014, DOI: 10.1002/smll.201401635
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Figure 7. Representative images of type II collagen immunostaining of the cross section of cells incubated with G (A-C), GO (D-F) and pGO (G-I) after 4 weeks of culture. Blue = DAPI, Green = type II collagen. (Scale bars, 200µm). Cells incubated with 25 µg of G produced the largest amount of type II collagen (B) while the amount of type II collagen produced decreased with increasing concentration of GO (D-F). In contrast, the extent of type II collagen produced increased with increasing concentration of pGO (G-I).
4. Experimental Section Cell Culture and Composite Formation: Bone marrow derived hMSCs were isolated from the mononuclear fraction of a bone marrow aspirate obtained from a commercial source (Lonza) based on their attachment to tissue culture plastics, and expanded in high glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% Pen-Strep. Passage four (P4) hMSCs were used in composite pellet formation. Briefly, 1 mL of the supplemented DMEM containing 100 nM dexamethasone, 50 µg/mL ascorbic acid-2-phosphate (Sigma), 10 ng/mL of transforming growth factor-beta 3 (TGF-β3) 100 µg/mL sodium pyruvate (Sigma), 40 µg/mL of proline (Sigma), 1% insulin, transferrin and sodium selenite (ITS-mix, Gibco) was incubated with 10 µg, 25 µg or 50 µg of G, GO or pGO overnight before 200,000 hMSCs were added. The mixture was centrifuged at 1000g for 10 min. Composite pellet spontaneously formed after 24 h of incubation and was subsequently cultured at 37 °C for 4 weeks in 5% carbon dioxide atmosphere and the medium was replaced every 3 days. Macroscopic images of pellets (n = 3) under each condition were used to measure the pellet diameters. Pellets formed in the absence of G or its derivatives were used as controls. Preparation of G, GO, and p-GO: GO was synthesized by the conventional Hummer's method.[18] Porous graphene oxide (pGO) was synthesized by subjecting the as-prepared GO to sequential base and acid treatment. Such treatments had been verified to produce nanoscale porosity on the GO sheets as verified by scanning tunnelling microscopy, as well as resulting in its chemical reduction to produce reduced GO.[19] G, which was made of few layer graphene flakes, was prepared from the electrochemical exfoliation of
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graphite powder in organic solvent (lithium perchlorate and propylene carbonate), as reported previously.[20] According to analysis by atomic force microscope, the typical flake sizes of GO and G are between 1–2 µm, while pGO is usually sub-micron in sizes. Cell Viability Test: Viability of composite pellets was determined based on the metabolic rate of the hMSCs 24 h and 72 h after composite formation. Cell pellets formed in the absence of G or its derivatives were used as controls. Briefly, pellets were washed twice with PBS and incubated in supplemented DMEM with 10% of Alamar Blue (Thermo Scientific) for 3 h. After incubation, the fluorescence of 100 µL of the solution was measured with excitation wavelength at 530 nm and emission wavelength at 590 nm. The percentage reduction of Alamar Blue reagent of each composite was calculated and normalized to the control. Histology and Immunostaining: After 4 weeks of culture, the composites were fixed overnight in 10% formalin solution and stored in 70% ethanol. The fixed composites were embedded in paraffin, sectioned and stained with Alcian Blue. For immunostaining, sections were permeabilized for 10 min in 0.1% Triton X in PBS, washed and blocked with 1% BSA for 30 min before incubating in rabbit polyclonal antibody against type II collagen (Abcam) using a 1:100 dilution factor overnight. After extensive washing, fluorescei isothiocyanate-conjugated anti-rabbit antibodies (Molecular Probes) were used for fluorescent detection of type II collagen. Finally, 4′,6-diamidino-2-phenylindole (DAPI) solution (0.1 mg/mL, Millipore) was applied to dye cell nucleus as counter staining. Confocal microscopy (LSM 510 Meta Confocal Microscope, Zeiss) was performed to collect images. Spectrophotometric Determination of Protein Loading Capacities: Protein loading capacities of G, GO and pGO were determined
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small 2014, DOI: 10.1002/smll.201401635
Cell-Assembled Graphene Biocomposite for Enhanced Chondrogenic Differentiation
as previously described.[6] Briefly, G, GO and pGO at 1 mg/mL in PBS were sonicated for 1 h prior to incubation with 10% (v/v) of fetal bovine serum in DMEM to ensure the formation of a homogeneous dispersion. The mixtures were vortexed in a shaker for equilibration. After 24 h of incubation, the mixture was centrifuged at 14,000 rpm for 10 min and the supernatant collected for spectrophotometric measurement at 280 nm. The amount of FBS adsorbed was determined from the change in protein adsorption before and after the addition of G, GO and pGO using a UV-vis spectrophotometer (NanoDrop 2000, Thermo Scientific). Real-Time Quantitative RT-PCR Analysis: Total RNA was extracted from pellets using RNeasy Mini Kit (Qiagen) according to the vendor’s protocol. RNA concentration was determined by using a spectrophotometer (NanoDrop 2000, Thermo Scientific), and 200 ng of RNA was used to synthesize cDNA with an Iscropt cDNA synthesis kit (Biorad laboratories). Quantitative real-time PCR reactions were carried out and monitored with a Stratagene Mx3000P system. QuantiTect SYBR Green PCR kit (Qiagen) was used to quantify the transcription level of collagen II, aggrecan and sox 9 genes. Primers for GAPDH, collagen II were synthesized as reported.[6] One µL of cDNA from each sample was then mixed with 10 µL of QuantiTect SYBR Green PCR master mix, 0.25 µL of each primer, and 8.5 µL of RNase-free water. Real-time PCR reactions were performed at 95 °C for 15 s and an extension step at 60 °C for 1 min. The transcription levels were normalized to GAPDH and were calculated using the 2ΔCt formula with reference to the undifferentiated hMSCs. Statistical Analysis: All sample values were expressed as the mean ± standard deviation (SD), and the data was analyzed using Prism software. Statistically significant values were defined as p < 0.05 (95% confidence interval) based on one-way analysis of variance (ANOVA).
Acknowledgements K. P. Loh acknowledges NRF-CRP Grant NRF-CRP-9-2011-03 “Towards Commercialization of Graphene Technologies (R-143-000-546-281).”
[2] J. A. Buckwalter, H. J. Mankin, A. J. Grodzinsky, Instr. Course Lect. 2005, 54, 465. [3] H. Fan, C. Zhang, J. Li, L. Bi, L. Qin, H. Wu, Y. Hu, Biomacromolecules 2008, 9(3), 927. [4] N. S. Kalson, P. D. Gikas, T. W. Briggs, Int. J. Clin. Pract. 2010, 64(10), 1444. [5] C. Chung, Y. K. Kim, D. Shin, S. R. Ryoo, B. H. Hong, D. H. Min, Acc. Chem. Res. 2013, 46(10), 2211. [6] W. C. Lee, C. H. Lim, H. Shi, L. A. Tang, Y. Wang, C. T. Lim, K. P. Loh, ACS Nano 2011, 5(9), 7334. [7] S. W. Crowder, D. Prasai, R. Rath, D. A. Balikov, H. Bae, K. I. Bolotin, H. J. Sung, Nanoscale 2013, 5(10), 4171. [8] J. Kim, K. S. Choi, Y. Kim, K. T. Lim, H. Seonwoo, Y. Park, D. H. Kim, P. H. Choung, C. S. Cho, S. Y. Kim, Y. H. Choung, J. H. Chung, J. Biomed. Mater. Res. A 2013, 101(12), 3520. [9] S. H. Ku, C. B. Park, Biomaterials 2013, 34(8), 2017. [10] Y. Wang, W. C. Lee, K. K. Manga, P. K. Ang, J. Lu, Y. P. Liu, C. T. Lim, K. P. Loh, Adv. Mater. 2012, 24(31), 4285. [11] F. H. Chen, K. T. Rousche, R. S. Tuan, Nat. Clin. Pract. Rheumatol. 2006, 2(7), 373. [12] F. Ruggiero, B. Petit, M. C. Ronziere, J. Farjanel, D. J. Hartmann, D. Herbage, J. Histochem. Cytochem. 1993, 41(6), 867. [13] Z. Zhang, J. M. McCaffery, R. G. Spencer, C. A. Francomano, J. Anat. 2004, 205(3), 229. [14] M. R. Pagnotto, Z. Wang, J. C. Karpie, M. Ferretti, X. Xiao, C. R. Chu, Gene Ther. 2007, 14(10), 804. [15] M. W. Wong, L. Qin, J. K. Tai, S. K. Lee, K. S. Leung, K. M. Chan, J. Biomed. Mater. Res. B 2004, 70(2), 362. [16] J. M. Coburn, M. Gibson, S. Monagle, Z. Patterson, J. H. Elisseeff, Proc. Natl. Acad. Sci. USA 2012, 109(25), 10012. [17] N. Li, Q. Zhang, S. Gao, Q. Song, R. Huang, L. Wang, L. Liu, J. Dai, M. Tang, G. Cheng, Sci. Rep. 2012, 3, 1604. [18] W. S. Hummers, R. E. Offeman, J. Am. Chem. Soc. 1958, 80, 1339. [19] C. Su, M. Acik, K. Takai, J. Lu, S. J. Hao, Y. Zheng, P. Wu, Q. Bao, T. Enoki, Y. J. Chabal, K. P. Loh, Nat. Commun. 2012, 3, 1298. [20] J. Wang, K. K. Manga, Q. Bao, K. P. Loh, J. Am. Chem. Soc. 2011, 133(23), 8888. [21] N. S. Hwang, S. Varghese, H. Li, J. Elisseeff, Cell Tissue Res. 2011, 344(3), 499. [22] D. A. Wang, C. G. Williams, F. Yang, N. Cher, H. Lee, J. H. Elisseeff, Tissue Eng. 2005, 11(1–2), 201. [23] C. G. Williams, T. K. Kim, A. Taboas, A. Malik, P. Manson, J. Elisseeff, Tissue Eng. 2003, 9(4), 679. [24] P. Otte, Z. Rheumatol. 1991, 50(5), 304. [25] A. M. Mackay, S. C. Beck, J. M. Murphy, F. P. Barry, C. O. Chichester, M. F. Pittenger, Tissue Eng. 1998, 4(4), 415.
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Cell-Assembled Graphene Biocomposite for Enhanced Chondrogenic Differentiation Wong Cheng Lee, Candy Haley Lim, Kenry , Chenliang Su, Kian Ping Loh,* and Chwee Teck Lim*
Graphene-based
nanomaterials are increasingly being explored for use as biomaterials for drug delivery and tissue engineering applications due to their exceptional physicochemical and mechanical properties. However, the twodimensional nature of graphene makes it difficult to extend its applications beyond planar tissue culture. Here, graphene–cell biocomposites are used to preconcentrate growth factors for chondrogenic differentiation. Bone marrow-derived mesenchymal stem cells (MSCs) are assembled with graphene flakes in the solution to form graphene-cell biocomposites. Increasing concentrations of graphene (G) and porous graphene oxide (pGO) are found to correlate positively with the extent of differentiation. However, beyond a certain concentration, especially in the case of graphene oxide, it will lead to decreased chondrogenesis due to increased diffusional barrier and cytotoxic effects. Nevertheless, these findings indicate that both G and pGO could serve as effective pre-concentration platforms for the construction of tissue-engineered cartilage and suspension-based cultures in vitro.
1. Introduction Dr. W. C. Lee, Kenry, Prof. C. T. Lim Department of Biomedical Engineering and Department of Mechanical Engineering National University of Singapore Singapore 117575, Singapore E-mail: [email protected] Dr. C. H. Lim, Dr. C. Su, Prof. K. P. Loh Department of Chemistry National University of Singapore Singapore 117543, Singapore E-mail: [email protected] Prof. C. T. Lim Mechanobiology Institute National University of Singapore Singapore 117411, Singapore Dr. C. H. Lim, Kenry, Dr. C. Su, Prof. K. P. Loh, Prof. C. T. Lim Graphene Research Centre National University of Singapore Singapore 117546, Singapore Dr. W. C. Lee, Prof. K. P. Loh, Prof. C. T. Lim NUS Graduate School of Integrative Sciences and Engineering National University of Singapore Singapore 117456, Singapore DOI: 10.1002/smll.201401635 small 2014, DOI: 10.1002/smll.201401635
Human bone marrow derived mesenchymal stem cells (hMSCs) provide a promising cell source for engineering cartilage tissues or for cell-based treatments of osteochondral defects owing to their multipotency, ease of availability and immunosuppressive properties.[1] Articular cartilage is a highly organized avascular tissue composed of chondrocytes embedded within an extracellular matrix of collagen, proteoglycans and noncollagenous proteins.[2] Cartilage damage and associated joint dysfunction affects a growing number of both young and elderly individuals. Due to the low mitotic activity of chondrocytes and the avascular nature of cartilage tissues, chondral defects are difficult to heal.[3] Thus, surgical interventions such as microfracture technique and autologous chondrocyte transplantations are frequently required to repair cartilage defects and restore joint function.[4] However, these techniques are limited by their clinical efficacy and the lack of supply of chondrocytes for clinical use has led to a call for new regenerative tissue engineering approaches to facilitate the differentiation of hMSCs into mature chondrocytes to augment the repair process. Using graphene (G) as a supporting scaffold for stem cell differentiation have received significant attention in recent years. Owing to their excellent mechanical stability and
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physicochemical properties,[5] graphene based tissue engineering approaches offered new possibilities for the restoration of damaged or lost tissues. To enhance stem cell growth and differentiation, graphene has been used as a planar culture platform for various types of stem cells and differentiation lineages. Graphene and related materials have been investigated for osteogenic,[6,7] adipogenic,[8] myogenic[9] and neural differentiation.[10] Our previous work indicated that osteogenic differentiation is enhanced on G due to its ability to pre-concentrate growth factors and chemical inducers via electron clouds π–π bonding.[6] However, this approach is limited to planar cell growth and is difficult to translate to applications that require suspension cultures for three-dimensional tissue formation. Specifically, during carti- Figure 1. Schematic of experimental design. (A) Serum proteins and differentiation factors lage formation, hMSCs are usually grown were pre-incubated with graphene solutions (G, GO or pGO) overnight before MSCs at in a pellet or on a micromass[11] due to passage 5 were added into the mixture. hMSCs fold and self-assemble into a bio-composite the phenotypic instability of monolayer pellet after 24 hours of incubation (B) 4 h, (C) 8 h, (D) 12 h, (E) 20 h and (F) 24 h. Pellets were subsequently differentiated in chondrogenic induction medium for 4 weeks. culture for chondrocyte maturation.[12] Although pellet cultures have been used clinically to construct hyaline cartilage and repair chondral applications in tissue engineering.[21–23] The schematic of defects,[13–15] this technique faces limitations such as lack of experimental design is shown in Figure 1A. Briefly, G based controlled release of cytokines for prolonged culture and solutions at different concentrations (10 µg/mL, 25 µg/mL poor process ability.[3] Therefore, a significant need exists for and 50 µg/mL) were incubated in 10% (v/v) fetal bovine a solution based culture technique that can efficiently entrap serum and chondrogenic chemical inducers overnight before cells and sufficiently provide the necessary mechanical and 200,000 hMSCs were added. After the cells were introduced, chemical cues to stimulate new cartilage tissue development. the cells and G (or pGO, G) folded and assembled together Current therapies aimed to regenerate articular cartilage into a composite pellet 24 h later as shown in Figures 1B-F. traumatic injuries are time-consuming and the quality of Cell pellets which were formed and differentiated in the repaired tissues needs improvement. Also, routine surgical absence of G (or pGO, G) (Figures A, B) were used as contechniques used to repair damaged cartilage tissues often trols and the extent of differentiation was assessed through result in significant fibrosis and delayed healing.[16] To date, histochemical staining for alcian blue (Figure 2C) and immuthere has been minimal progress made in tissue engineering nostaining for type II collagen production (Figures 2D-F). techniques to augment surgical strategies for cartilage regenMacroscopic images of the cells were used to assess comeration. Recently, the use of GO film for stem cell differen- posite formation and morphology (Figure 3). After selftiation towards cartilage tissues has been explored but it was assembly, we observed that the microparticles of G and pGO reported that the extent of differentiation decreased with distributed homogeneously within the construct (Figures 3A-C time,[8] as cell-cell interaction was confined to within the 2D and Figure 3G-I). Microparticles of GO appeared larger than G plane. Owing to the exceptional physicochemical properties and pGO and showed a higher propensity to assemble around of G, recent approaches aimed to enhance 3D stem cell dif- the surface of the composite (Figures 3D-F). Similarly, composferentiation using G scaffolds are gaining traction.[7,17] How- ites of GO were significantly larger than those assembled with ever, research on using G and related materials as ‘soluble, G and pGO (Figure 4C). No significant differences in pellet 3-dimensional scaffolds’ are lacking. Therefore, our study aims size were observed between the G and pGO composites. The to develop a solution-supported graphene-based construct to segregation of GO on the outer surface of the composite versus support pellet formation and differentiation of human bone the inner distribution of pGO and G inside the composites may marrow derived hMSCs towards the chondrogenic lineage. be due to the greater hydrophilicity of GO, which favours its interaction with the aqueous-based extracellular environment. Compared to G and pGO, GO has a higher concentration of oxygen functional groups[19] and thus has a tendency to cross2. Results and Discussion link via hydrogen bonding on the hydrophilic cell surfaces to First, we sought to quantitatively evaluate the biocompat- form a thin layer around the exterior of the composite. ibility of G, GO and pGO in vitro. Bone marrow derived To assess the biocompatibility of the G-based micropartihMSCs were used for these experiments because they cles on chondrogenic tissue formation, cell viability was monhave been extensively used for evaluating osteo-chondral itored by measuring the metabolic rates of the composites
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Figure 2. Chondrogenesis of hMSCs. (A) hMSCs develop into a micromass 24 h after incubation in chondrogenic induction media. Inset shows monolayer of cells before assembly into pellet. (B) Stereoscopic image of chondrogenic cell pellet in the absence of GO, G or pGO solution. Differentiation toward chondrogenic lineage was assessed by (C) Alcian Blue staining for sulfated mucosubstances and (D-F) type II collagen immunostaining. (Scale bars, 200 µm).
normalized to those formed in the absence of G-based microparticles (Figures 4A, B) at day 24 h and 72 h after composite formation. At day 1 post assembly, the metabolic rates of the composites remained high with increasing concentrations of G, GO and pGO (Figure 4A). However, at day 3, the viability of the cells decreased with increasing concentrations of G and GO despite their low initial cytotoxicity (Figure 4B). In contrast, the viability
of pGO remained high with increasing pGO concentration. Serum proteins and growth factors are essential for cell survival and proliferation. In order to determine whether the observed cell viability differences is due to the capacity of the G-based materials to pre-load growth factors, the protein loading capacity of G and its derivatives was quantified via spectrophotometric measurements. We found that the protein loading capacities of GO and pGO were significantly larger than that of G (Figure 4D). However, this trend did not correlate with the long-term (72 h) viability of the cells since the viability of those assembled with both G and GO decreased while those assembled with pGO remained high (Figure 4B). Therefore, the decrease in the viability of G and GO composites at day 3 and could be attributed to the increased diffusional barrier for the cells embedded in the inner layers of the composite. This effect is more pronounced in GO composites because of the tendency of the GO microparticles to assemble closer to the surface of the construct; thereby creating a thicker diffusion barrier. The higher viability of pGO is related to its porosity, which allows exchange of substances between the cells and its surrounding. After 4 weeks of culture and differentiation, Alcian blue staining was used to assess histological changes and sGAG synthesis. The extent of Alcian blue staining can be correlated to the amount of sGAG Figure 3. Chondrogenesis of hMSCs incubated with different concentrations of (A-C) produced. Composites formed with 10 µg graphene, (D-F) graphene oxide, (G-I) porous graphene oxide. hMSCs self assembled into of G, GO and pGO had uniform staining a graphene-cell biocomposite 24 h after incubation. Inset shows monolayer of cells after graphene solution is introduced. (Scale bars, 200 µm). G and pGO are more homogenously for Alcian blue, which was the characdispersed and distributed within the cell pellet than GO. A thicker GO shell can be seen in the teristic of cartilage matrix. This means that the composites could differentiate case of hMSCs incubated with GO. small 2014, DOI: 10.1002/smll.201401635
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Figure 4. Viability of cells incubated with different graphene solutions was assessed using Alamar Blue metabolic assay at (A) 24 h and (B) 72 h normalized to controls. Cell pellets formed in the absence of G or its derivatives were used as controls. Pellet diameter (C) was measured at 24 h (n = 3). (D) Protein loading capacity of serum proteins on G, GO and pGO was assessed 24h after incubation. After 74 h of culture, cell viability decreased (B) with increasing concentration of GO due to increased diffusional barrier while cell viability remained high for those incubated with pGO. (*p < 0.05; n = 3 for each group).
into chondrocytes and synthesize a cartilaginous matrix (Figures 5A, D and G). For both G and pGO, the extent of staining increased with increasing concentrations of the microparticle during composite formation. Spectrophotometric quantification of the amount of Alcian blue staining revealed that the amount of sGAG produced was highest for 25 µg of G, while sGAG synthesis decreased with increasing concentrations of GO (Figure 6A). To confirm observations from Alcian blue staining and quantification, immunohistochemistry staining of collagen II was carried out. Compared to weakly staining of GO composites when the concentration is increased (Figures 7E, F), staining of collagen II was enhanced when the concentration of pGO is increased (Figures 7G-I). Conversely, intense staining of collagen II was detected in composites with 25µg of G while the production of
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collagen II decreased when the concentration is increased to 50 µg, suggesting that the optimum cells to G ratio for chondrogenic differentiation is 8000 cells/µg of G. We next investigated changes in tissuespecific gene expression to evaluate lineage specificity of the differentiating stem cells in the various composites. Cell pellets formed in the absence of G or its derivatives were used as controls. Gene expression was evaluated after 4 weeks of culture. Cartilage specific markers- type II collagen, Sox9 and aggrecan were up-regulated in both pGO and GO composites compared with bare pellet cultures (Figures 6B-D), indicative of enhanced chondrogenesis. Specifically, collagen II gene was dramatically up-regulated at 25 µg of G but declined when the concentration was increased further. It appears that optimal spatial configuration and cell-cell contacts are provided by the G microparticles at this concentration. However, when the concentration of G was increased further, the extent of chondrogenic differentiation became limited by the decreased cell-cell communication and exchange of oxygen and other nutrients that regulate matrix production and chondrocyte phenotype. Decreased availability of glucose is known to alter the metabolism of cells in a pellet and their susceptibility to apoptosis.[24,25] Moreover, the cartilage markers remained relatively low in all GO composites as the GO shell increased the compactness of the composite which restricted hMSC proliferation and differentiation. In contrast, pGO further enhanced chondrogenic differentiation when the concentration of the microparticles is increased because the porous nature of pGO provides routes for diffusional exchange and sustains the metabolism of the cells in the pellet.
3. Conclusions We have demonstrated the ability of G, GO and pGO to support pellet formation and chondrogenic differentiation of hMSCs. These G microparticles serve as ‘growth factor factories’ for the formation and maturation of chondrogenic tissues. The loading amount of GO and its derivatives in the composite accelerates chondrogenic differentiation initially, but excessive amounts affect the viability of the cell composites. Our results show that engendering porosity in G allows a higher loading of GO and further enhances the differentiation rate of the cells. Overall, we envision that these novel synthetic cell-assembled composites can provide an easy and effective way to construct cartilage tissues for various regenerative applications.
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Cell-Assembled Graphene Biocomposite for Enhanced Chondrogenic Differentiation
Figure 5. Representative images of Alcian blue staining of the cross section of cells incubated with G (A-C), GO (D-F) and pGO (G-I) after 4 weeks of culture. Inset shows the respective biocomposite pellet. (Scale bars, 200 µm). The extent of Alcian blue staining increased when the concentration of G and pGO were increased while the extent of staining decreased when the concentration of GO was increased.
Figure 6. Biochemical and gene expression analysis of in vitro chondrogenesis. (A) Alcian blue dye was extracted from the biocomposites and its optical density measured and normalized to pellets in the absence of graphene solutions. Real-time PCR analysis of markers for cartilage normalized to cell pellets formed in the absence of G or its derivatives (B-D). Data are presented as average ± SD (n = 3). Gene expression analysis of in vitro chondrogenesis corroborates with histological and immunological staining. Greatest extent of chondrogenic differentiation was observed for cells incubated with 25 µg of G. Differentiation decreased when the concentration of GO is increased because the amount of chemical factors becomes limiting due to increased diffusional barrier. In contrast, differentiation increased with the concentration of pGO. This can be ascribed to the porous nature of pGO which facilitates diffusional exchange even when the concentration of pGO is increased. small 2014, DOI: 10.1002/smll.201401635
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Figure 7. Representative images of type II collagen immunostaining of the cross section of cells incubated with G (A-C), GO (D-F) and pGO (G-I) after 4 weeks of culture. Blue = DAPI, Green = type II collagen. (Scale bars, 200µm). Cells incubated with 25 µg of G produced the largest amount of type II collagen (B) while the amount of type II collagen produced decreased with increasing concentration of GO (D-F). In contrast, the extent of type II collagen produced increased with increasing concentration of pGO (G-I).
4. Experimental Section Cell Culture and Composite Formation: Bone marrow derived hMSCs were isolated from the mononuclear fraction of a bone marrow aspirate obtained from a commercial source (Lonza) based on their attachment to tissue culture plastics, and expanded in high glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% Pen-Strep. Passage four (P4) hMSCs were used in composite pellet formation. Briefly, 1 mL of the supplemented DMEM containing 100 nM dexamethasone, 50 µg/mL ascorbic acid-2-phosphate (Sigma), 10 ng/mL of transforming growth factor-beta 3 (TGF-β3) 100 µg/mL sodium pyruvate (Sigma), 40 µg/mL of proline (Sigma), 1% insulin, transferrin and sodium selenite (ITS-mix, Gibco) was incubated with 10 µg, 25 µg or 50 µg of G, GO or pGO overnight before 200,000 hMSCs were added. The mixture was centrifuged at 1000g for 10 min. Composite pellet spontaneously formed after 24 h of incubation and was subsequently cultured at 37 °C for 4 weeks in 5% carbon dioxide atmosphere and the medium was replaced every 3 days. Macroscopic images of pellets (n = 3) under each condition were used to measure the pellet diameters. Pellets formed in the absence of G or its derivatives were used as controls. Preparation of G, GO, and p-GO: GO was synthesized by the conventional Hummer's method.[18] Porous graphene oxide (pGO) was synthesized by subjecting the as-prepared GO to sequential base and acid treatment. Such treatments had been verified to produce nanoscale porosity on the GO sheets as verified by scanning tunnelling microscopy, as well as resulting in its chemical reduction to produce reduced GO.[19] G, which was made of few layer graphene flakes, was prepared from the electrochemical exfoliation of
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graphite powder in organic solvent (lithium perchlorate and propylene carbonate), as reported previously.[20] According to analysis by atomic force microscope, the typical flake sizes of GO and G are between 1–2 µm, while pGO is usually sub-micron in sizes. Cell Viability Test: Viability of composite pellets was determined based on the metabolic rate of the hMSCs 24 h and 72 h after composite formation. Cell pellets formed in the absence of G or its derivatives were used as controls. Briefly, pellets were washed twice with PBS and incubated in supplemented DMEM with 10% of Alamar Blue (Thermo Scientific) for 3 h. After incubation, the fluorescence of 100 µL of the solution was measured with excitation wavelength at 530 nm and emission wavelength at 590 nm. The percentage reduction of Alamar Blue reagent of each composite was calculated and normalized to the control. Histology and Immunostaining: After 4 weeks of culture, the composites were fixed overnight in 10% formalin solution and stored in 70% ethanol. The fixed composites were embedded in paraffin, sectioned and stained with Alcian Blue. For immunostaining, sections were permeabilized for 10 min in 0.1% Triton X in PBS, washed and blocked with 1% BSA for 30 min before incubating in rabbit polyclonal antibody against type II collagen (Abcam) using a 1:100 dilution factor overnight. After extensive washing, fluorescei isothiocyanate-conjugated anti-rabbit antibodies (Molecular Probes) were used for fluorescent detection of type II collagen. Finally, 4′,6-diamidino-2-phenylindole (DAPI) solution (0.1 mg/mL, Millipore) was applied to dye cell nucleus as counter staining. Confocal microscopy (LSM 510 Meta Confocal Microscope, Zeiss) was performed to collect images. Spectrophotometric Determination of Protein Loading Capacities: Protein loading capacities of G, GO and pGO were determined
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as previously described.[6] Briefly, G, GO and pGO at 1 mg/mL in PBS were sonicated for 1 h prior to incubation with 10% (v/v) of fetal bovine serum in DMEM to ensure the formation of a homogeneous dispersion. The mixtures were vortexed in a shaker for equilibration. After 24 h of incubation, the mixture was centrifuged at 14,000 rpm for 10 min and the supernatant collected for spectrophotometric measurement at 280 nm. The amount of FBS adsorbed was determined from the change in protein adsorption before and after the addition of G, GO and pGO using a UV-vis spectrophotometer (NanoDrop 2000, Thermo Scientific). Real-Time Quantitative RT-PCR Analysis: Total RNA was extracted from pellets using RNeasy Mini Kit (Qiagen) according to the vendor’s protocol. RNA concentration was determined by using a spectrophotometer (NanoDrop 2000, Thermo Scientific), and 200 ng of RNA was used to synthesize cDNA with an Iscropt cDNA synthesis kit (Biorad laboratories). Quantitative real-time PCR reactions were carried out and monitored with a Stratagene Mx3000P system. QuantiTect SYBR Green PCR kit (Qiagen) was used to quantify the transcription level of collagen II, aggrecan and sox 9 genes. Primers for GAPDH, collagen II were synthesized as reported.[6] One µL of cDNA from each sample was then mixed with 10 µL of QuantiTect SYBR Green PCR master mix, 0.25 µL of each primer, and 8.5 µL of RNase-free water. Real-time PCR reactions were performed at 95 °C for 15 s and an extension step at 60 °C for 1 min. The transcription levels were normalized to GAPDH and were calculated using the 2ΔCt formula with reference to the undifferentiated hMSCs. Statistical Analysis: All sample values were expressed as the mean ± standard deviation (SD), and the data was analyzed using Prism software. Statistically significant values were defined as p < 0.05 (95% confidence interval) based on one-way analysis of variance (ANOVA).
Acknowledgements K. P. Loh acknowledges NRF-CRP Grant NRF-CRP-9-2011-03 “Towards Commercialization of Graphene Technologies (R-143-000-546-281).”
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: June 6, 2014 Revised: September 2, 2014 Published online:
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