Letter pubs.acs.org/NanoLett
Gold Nanoparticle-Decellularized Matrix Hybrids for Cardiac Tissue Engineering Michal Shevach,†,‡ Sharon Fleischer,†,‡ Assaf Shapira,† and Tal Dvir*,†,‡,§ †
The Laboratory for Tissue Engineering and Regenerative Medicine, Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Science, ‡The Center for Nanoscience and Nanotechnology, and §Department of Materials Science and Engineering, Tel Aviv University, Tel Aviv 69978, Israel S Supporting Information *
ABSTRACT: Decellularized matrices are valuable scaffolds for engineering functional cardiac patches for treating myocardial infarction. However, the lack of quick and efficient electrical coupling between adjacent cells may jeopardize the success of the treatment. To address this issue, we have deposited gold nanoparticles on fibrous decellularized omental matrices and investigated their morphology, conductivity, and degradation. We have shown that cardiac cells engineered within the hybrid scaffolds exhibited elongated and aligned morphology, massive striation, and organized connexin 43 electrical coupling proteins. Finally, we have shown that the hybrid patches demonstrated superior function as compared to pristine patches, including a stronger contraction force, lower excitation threshold, and faster calcium transients. KEYWORDS: Cardiac tissue engineering, decellularized matrix, gold nanoparticles, omentum, scaffold
C
become rounded, and their gap junction proteins such as connexin 43 are internalized or lost, disrupting proper anisotropic transfer of the electrical signal.9 To address this challenge, we and others have integrated nanoscale conductive materials with synthetic scaffolds to serve as electrical couplers between cells.9−14 For example, gold nanowires were embedded within the pore walls of 3D macroporous alginate scaffolds during the fabrication process.9 When cardiomyocytes were grown within the scaffolds, the cells interacted with the wires, and the engineered tissue exhibited significantly faster, synchronized electrical signal propagation, as compared to the signal produced by tissues grown in pristine scaffolds.9 In a different study, gold nanoparticles (AuNPs) conjugated to the backbone of synthetic hydrogels were able to enhance connexin 43 expression in cultured cardiomyocytes.10 Other studies have demonstrated that cardiomyocytes cultured on carbon nanotubes or in carbon nanotube supplemented hydrogels have formed tight junctions with the tubes and exhibited advanced electrophysiological functions, such as enhanced cell contraction, cell−cell coupling, and spontaneous electrical activity.11,15 In recent studies, we have shown that gold can be deposited on synthetic electrospun fiber scaffolds in a quick and facile manner.13,14 Here, we report on the development of a novel
ardiovascular diseases, such as myocardial infarction (MI), remain the most common cause of morbidity and mortality in developed countries.1 The aim of cardiac tissue engineering is to develop functional 3-dimensional (3D) tissue patches in vitro, later to be delivered to the damaged area in the myocardium to promote regeneration.2,3 However, in order to induce proper cardiac tissue assembly into a functioning tissue, it is essential to create a micro inductive environment that can mimic the native cardiac extracellular matrix (ECM).2 In vivo, this unique microenvironment composed of nanoscale to microscale fiber architecture fosters cell−cell coupling, leading to a rapid transfer of the electrical wave.2,4,5 One of the remaining key challenges in cardiac tissue engineering is fabricating a nonimmunogenic scaffold that would not provoke an adverse immune response after transplantation. Such response may hamper the therapeutic potential of the transplanted patch and can lead to graft rejection and adversely affect the health of the patient.6,7 To address this, we have recently suggested that nonimmunogenic scaffolds can be fabricated from the patient’s own biomaterials. We have shown that parts of the omentum can be easily, quickly, and safely harvested from the patient, and after efficient cell removal and quick processing, a porous 3D scaffold can be obtained.8 Another challenge, which may jeopardize the potential of cardiac patches to regenerate the infarcted heart is the lack of quick and efficient electrical coupling between adjacent cells.9 After cell isolation and seeding within the scaffolds, the cells © XXXX American Chemical Society
Received: July 15, 2014 Revised: August 12, 2014
A
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
Figure 1. Schematic overview of the concept. Omental tissue is isolated from the patient and undergoes a quick decellularization process to produce the 3D scaffold biomaterial. The decellularized scaffold is decorated with conductive motifs using an e-beam evaporator to produce a hybrid scaffold. Cells are isolated from the same patient, cultured in vitro, and seeded on the hybrid scaffold to produce a personalized cardiac patch. The engineered patch is transplanted on the infarcted heart.
fibrous scaffold with fibers ranging from 100 nm to several micrometers in diameter (Figure 2D). The second step involved decoration of the decellularized scaffold with conductive motifs. AuNPs, with nominal thicknesses of 4 and 10 nm, were deposited on the scaffold’s fibers using an e-beam evaporator, contributing to a change in the underlying scaffold color (Figure 3A). AuNPs scaffolds were visualized using an environmental SEM (ESEM) without additional conductive coating. The AuNPs were homogeneously deposited on the fibers, creating AuNPs/ECM hybrids (Figure 3B−D). Energy-dispersive X-ray spectroscopy (EDX) was used to confirm that the NPs observed on the fibers were indeed Au. As shown in Figure 3E, energy peaks associated with Au were observed in both the 4 nm and to a greater extent in the 10 nm hybrid scaffolds. One of the limitations of naturally derived scaffolds for cardiac tissue engineering is their low electrical conductivity.9 Therefore, we next sought to evaluate the conductivity of the engineered scaffolds by performing current−voltage (IV) measurements. The IV curve of pristine omental scaffold exhibited, as expected, a typical curve of an isolating material, similar to other protein-based scaffolds. In contrast, the 4 nm Au scaffolds have shown lower electrical resistance (Figure 3F). The 10 nm Au scaffolds exhibited typical curves of conductive materials (Figure 3G). Resistance calculation derived from IV curves further demonstrated the conductive nature of the hybrid scaffolds, while the pristine scaffolds showed significantly higher resistance (Figure 3H; p = 0.01 and p = 0.001). Such an electrical property of the scaffolds was previously demonstrated to be inductive microenvironment for the assembly of electrogenic cells such as cardiomyocytes.9 Since AuNPs existence on the fibrous scaffolds is essential for continuous maturation and development of the cardiac patch throughout the in vitro culturing period, but not necessarily important for in vivo applications after maturation, we next examined the stability of the hybrid scaffold in vitro in cell culture conditions. Au-deposited samples were submerged in culture medium and subjected to 7 days of constant shaking, in a cell culture incubator. Next, samples were washed, air-dried, and subjected to elemental analysis. As shown in Figure 4A and B, EDX analysis was performed on several points over a straight
conductive platform, integrating AuNPs with decellularized matrix. Such approach may alter the electrical properties of an autologous scaffold, composed of the patient’s own biomaterials, to better suit the needs of an engineered cardiac tissue and lead to cell organization, tissue maturation, and enhanced mechano-electrical function. When seeded with the patient’s own cells, the patches may be implanted back without triggering an immune response (Figure 1). Gold nanoparticle-decellularized matrix hybrids (AuNP scaffolds) were prepared in two main steps. First, native omentum underwent a decellularization process to produce a 3D biomaterial scaffold (Figure 2A−C).8 DNA removal was validated using Hoechst 33258 staining of ECM and fresh native tissue sections. Nuclei were detected in fresh omentum but not in the decellularized matrix (Supporting Information, Supplemental Figure 1). Scanning electron microscopy (SEM) images further validated complete cell removal and revealed a
Figure 2. Decellularization process. (A) Native omentum prior to cell removal. (B) Omentum during the decellularization process. (C) After complete cell removal, a 3D biomaterial scaffold is produced. (D) SEM image revealed an a-cellular fiber scaffold. Bar: D = 40 μm. B
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
Figure 3. AuNP scaffolds. (A) AuNPs were deposited onto the scaffolds using an e-beam evaporator. The color of the scaffolds has changed due to the difference in NP dimensions. (B, C) ESEM images of Au-deposited scaffolds. Homogeneously deposited AuNPs were visualized on the 4 nm (B) and 10 nm (C). (D) Higher magnification of C. (E) Energy-dispersive X-ray spectroscopy of the fibers. (F−H) Conductivity of the scaffolds. Current−voltage curves of pristine and 4 nm (F) and pristine 4 and 10 nm scaffolds (G). (H) Resistance calculation derived from current−voltage curves. Bar: B, C = 500 nm, D = 100 nm.
line on the ECM fiber. As the mounted samples have not been additionally coated with gold prior to visualization, the points outside the fibers have not shown peaks associated with Au (Figure 4B, points 1 and 5). On the contrary, points 2−4 on the fibers have shown Au associated peaks (Figure 4B), indicating the preservation of the Au content on the fibers even after exposing the samples to excessive rinsing over a long period of time. We next sought to evaluate the fate of the AuNP coating in more relevant, physiological conditions taking place in vivo. We hypothesized that, since deposition of the AuNPs occurs mainly on the amine and sulfur groups in the ECM, degrading the ECM by a relevant enzyme will simulate AuNP disassembly in vivo. Therefore, the hybrid scaffolds were placed for 7 days in culture medium containing collagenase type II (95 U/mL), an enzyme that is extremely active after cardiac remodeling following acute MI.16 Transmitting electron microscopy (TEM) images of the degradation products of the 4 and 10 nm Au scaffolds (Figure 4C and D) revealed disassembly of the AuNPs into spherical particles with average diameters of 3.62 ± 0.12 nm and 4.736 ± 0.17, respectively (Figure 4E). These sizes of AuNPs have been shown to be cleared by immune cells17 and to be nontoxic in vivo.18 To investigate the effect of the AuNPs within the scaffolds on tissue organization and function, we engineered cardiac patches. Cardiac cells were isolated from neonatal rat hearts and seeded
by a single droplet on the scaffolds. To assess the engineered tissue assembly, cardiac patches were immunostained for α sarcomeric actinin, a protein associated with cell contraction, and connexin 43, associated with electrical coupling between adjacent cells. On day 5, cardiac cells formed elongated and aligned cell bundles with massive striation on the pristine, the 4 nm and the 10 nm AuNP scaffolds, as judged by the immense actinin staining (Figure 5A−C; pink staining). However, while connexin 43, electrical coupling protein was randomly distributed and hardly noticed between cells grown in the pristine scaffolds (Figure 5A; green staining), cells grown in the AuNP scaffolds exhibited pronounced and organized staining, aligned between adjacent cardiomyocytes (Figure 5B and C). Compared to randomly organized distribution of connexin 43 molecules, aligned localization of the molecules between cardiomyocytes may lead to a faster, anisotropic propagation of the electrical signal and eventually may induce better functioning engineered tissues.19 Another parameter that may affect the function of the engineered patch is the ratio between cardiomyocytes and fibroblasts. In the heart, the cardiomyocytes to fibroblasts ratio is tightly regulated and is maintained constant in healthy individuals.20 The isolated cell population is mostly composed of cardiomyocytes, contracting cells, which cannot divide, and fibroblasts, which are highly proliferative and cannot contract. Therefore, it is essential to prevent the fibroblasts from taking C
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
Figure 5. Cardiac cell organization and engineered tissue function on day 5. (A−C) Immunostaining of cardiac α sarcomeric actinin (pink), connexin 43 (green), and nuclei (blue) of cardiac cells within the pristine (A), 4 nm (B), and 10 nm (C) scaffolds. Lower panels are higher magnifications. White arrows indicate the location of connexin 43 molecules. (D) Spontaneous contraction amplitude. (E) Excitation threshold. (F) Velocity of calcium transients during spontaneous contractions. The velocity is normalized to tissues engineered in pristine scaffolds. Bar = 20 μm. Figure 4. Stability and degradation of the AuNP scaffolds. (A, B) Stability of the AuNP scaffolds in normal culture conditions. ESEM images (A) and energy-dispersive X-ray spectroscopy (B) were performed after 7 days of constant shaking in culture medium. Points 1 and 5 located outside the fibers have not shown the Au associated peaks, while points 2−4 located on the fiber showed preservation of the Au content. (C, D) Degradation products of the AuNP scaffolds. TEM images of AuNPs after enzymatic degradation of 4 nm (C) and 10 nm (D) hybrid scaffolds. (E) Size distribution quantification of the enzymatically degraded hybrid scaffolds. Bar: A = 250 nm, C, D = 100 nm.
compared to the scaffolds modified with 10 nm AuNPs (Supplementary Figure 2E). Finally, to assess the effect of the hybrid scaffolds on the performance of the engineered tissues, a series of complementary functional assays was conducted. Since a strong contraction force is critical for generating effective cardiac patches,3,22 we initially assessed the spontaneous contraction amplitude. As shown in Figure 5D and Supplementary Movies 1−3, the presence of the AuNPs (4 and 10 nm) significantly increased the contraction amplitude of the patches (p = 0.044, p < 0.001, respectively). Since the pristine and the hybrid scaffolds had similar longitudinal elastic modulus (Supplementary Figure 3), it is acceptable to assume that the 10 nm AuNP patches have generated significantly higher forces as compared to the pristine and 4 nm patches. Another indication for the level of functional assembly of the tissue is its ability to generate synchronous contractions throughout the patch. To investigate this, cardiac constructs were subjected to an external electrical field, increasing in increments of 0.1 V. We defined excitation threshold as the minimum voltage needed to induce synchronous contractions of the entire patch at the same frequency (higher than the normal contraction rate). As shown in Figure 5E, the presence of both 4 and 10 nm AuNPs within the scaffolds increased the coupling between cell bundles, resulting in a significantly lower excitation threshold (p = 0.003, p < 0.001, respectively). Interestingly, no significant difference was observed between the 4 nm and the 10 nm scaffolds. Finally, an assessment of the electrophysiological effects of AuNPs was performed in cardiac cell constructs, with 4 and 10 nm particles or without NPs. The constructs were incubated with calcium-sensitive dye, and the velocity of calcium transients during spontaneous contractions was measured (n = 5 for each group). Although the 4 nm Au scaffolds still had
over the culture in vitro. A shift in the cell ratio in favor of the fibroblasts would impede patch contraction. Previously, it was shown that gold nanorods can restrain the infarcted heart matrix remodeling by affecting cardiac fibroblasts.21 More recently, we have shown that AuNPs can attenuate fibroblast proliferation.14 Here, we sought to evaluate whether AuNPs can have the same effect in the hybrid scaffolds, which may eventually lead to better maintenance of the initial cardiomyocyte to fibroblast ratio. Cardiac cell constructs modified with AuNPs (4 and 10 nm) and pristine scaffolds were stained on day 5 for troponin T and vimentin markers for cardiomyocytes and fibroblasts, respectively (Supplementary Figure 2A−C). Quantitative analysis of cell types in the scaffolds revealed significantly lower fibroblast fraction in the AuNP scaffolds (Supplementary Figure 2D), implying that the initial ratio of contracting to noncontracting cells was better preserved. Consequently, the cardiac patch may generate significantly stronger contraction force. To further investigate the effect of the AuNPs on fibroblast proliferation, metabolic activity of the isolated fibroblast population was measured at day 5 and normalized to day 0. As shown, a significantly higher proliferation rate was observed within the pristine scaffolds as D
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
(4) Dvir, T.; Timko, B. P.; Kohane, D. S.; Langer, R. Nat. Nanotechnol 2011, 6 (1), 13−22. (5) Parker, K. K.; Ingber, D. E. Philos. Trans. Roy. Soc. London, Ser. B, Biol. Sci. 2007, 362 (1484), 1267−79. (6) Jones, K. S. Semin. Immunol. 2008, 20 (2), 130−136. (7) Anderson, J. M.; Rodriguez, A.; Chang, D. T. Semin. Immunol. 2008, 20 (2), 86−100. (8) Shevach, M.; Soffer-Tsur, N.; Fleischer, S.; Shapira, A.; Dvir, T. Biofabrication 2014, 6 (2), 024101. (9) Dvir, T.; Timko, B. P.; Brigham, M. D.; Naik, S. R.; Karajanagi, S. S.; Levy, O.; Jin, H.; Parker, K. K.; Langer, R.; Kohane, D. S. Nat. Nanotechnol. 2011, 6 (11), 720−5. (10) You, J. O.; Rafat, M.; Ye, G. J.; Auguste, D. T. Nano Lett. 2011, 11 (9), 3643−8. (11) Shin, S. R.; Jung, S. M.; Zalabany, M.; Kim, K.; Zorlutuna, P.; Kim, S. B.; Nikkhah, M.; Khabiry, M.; Azize, M.; Kong, J.; Wan, K. T.; Palacios, T.; Dokmeci, M. R.; Bae, H.; Tang, X. S.; Khademhosseini, A. ACS Nano 2013, 7 (3), 2369−80. (12) Martinelli, V.; Cellot, G.; Toma, F. M.; Long, C. S.; Caldwell, J. H.; Zentilin, L.; Giacca, M.; Turco, A.; Prato, M.; Ballerini, L.; Mestroni, L. ACS Nano 2013, 7 (7), 5746−56. (13) Fleischer, S.; Shevach, M.; Feiner, R.; Dvir, T. Nanoscale 2014, 6, 9410−9414. (14) Shevach, M.; Maoz, B. M.; Feiner, R.; Shapira, A.; Dvir, T. J. Mater. Chem. B 2013, 1 (39), 5210−5217. (15) Martinelli, V.; Cellot, G.; Toma, F. M.; Long, C. S.; Caldwell, J. H.; Zentilin, L.; Giacca, M.; Turco, A.; Prato, M.; Ballerini, L.; Mestroni, L. Nano Lett. 2012, 12 (4), 1831−8. (16) Thompson, M. M.; Squire, I. B. Cardiovasc. Test. 2002, 54 (3), 495−8. (17) Bartneck, M.; Keul, H. A.; Zwadlo-Klarwasser, G.; Groll, J. Nano Lett. 2010, 10 (1), 59−63. (18) Chen, Y. S.; Hung, Y. C.; Liau, I.; Huang, G. S. Nanoscale Res. Lett. 2009, 4 (8), 858−864. (19) Bian, W.; Jackman, C. P.; Bursac, N. Biofabrication 2014, 6 (2), 024109. (20) Souders, C. A.; Bowers, S. L.; Baudino, T. A. Circ. Res. 2009, 105 (12), 1164−76. (21) Sisco, P. N.; Wilson, C. G.; Mironova, E.; Baxter, S. C.; Murphy, C. J.; Goldsmith, E. C. Nano Lett. 2008, 8 (10), 3409−12. (22) Fleischer, S.; Feiner, R.; Shapira, A.; Ji, J.; Sui, X.; Daniel Wagner, H.; Dvir, T. Biomaterials 2013, 34 (34), 8599−606.
relatively high resistance (Figure 3H), tissues engineered within both AuNP scaffolds (4 and 10 nm) transferred the electrical signal 2- and 3-fold faster than the tissues grown in the pristine scaffolds (p = 0.035, p = 0.032, respectively). After transplantation, faster transfer of the electrical signal may lead to better integration of the patch with the healthy part of the myocardium and thus improve the efficacy of the cardiac patch therapy in vivo. In summary, we have developed and characterized a hybrid material based on natural ECM scaffold and AuNPs for cardiac tissue engineering. We have demonstrated that the AuNPs were able to significantly increase the conductivity of the natural material without affecting the mechanical properties relevant for cardiac tissue engineering. Moreover, we have shown that, in normal cultivation conditions in vitro, the developed scaffold is stable, and in in vivo-simulating conditions the NPs may dissociate from the scaffold. Cardiac cells cultivated within the hybrid scaffolds exhibited elongated and aligned morphology, massive striation, and organized connexin 43 electrical coupling proteins. The presence of the AuNPs attenuated fibroblast proliferation and thus maintained a contracting to noncontracting cell ratio during cell culture. Finally, growing cardiac cells in AuNP scaffolds has led to the formation of cardiac patches generating stronger contraction forces. Our approach may be useful for engineering more homogeneous cardiac patches that could better integrate with the healthy part of the heart and improve heart function after MI. The technique could be also beneficial for culturing other electrogenic cells such as neurons and may assist to regenerate other tissues such as the injured spinal cord.
■
ASSOCIATED CONTENT
S Supporting Information *
Details of experimental procedures, supporting figures, and movies displaying cardiac patch contraction. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*T.D. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS T.D acknowledges support from the European Union FP7 program (Marie Curie, CIG), Alon Fellowship, Israel Science Foundation and the Nicholas and Elizabeth Slezak Super Center for Cardiac Research and Biomedical Engineering at Tel Aviv University. The work is part of the doctoral thesis of M.S. at Tel-Aviv University. We would like to thank Ekaterina Glukhikh for her assistance with the current−voltage measurements.
■
REFERENCES
(1) Lloyd-Jones, D.; Adams, R. J.; Brown, T. M.; Carnethon, M.; Dai, S.; De Simone, G.; Ferguson, T. B.; Ford, E.; Furie, K.; Gillespie, C.; Go, A.; Greenlund, K.; Haase, N.; et al. Circulation 2010, 23 (121 (7)), 948−54. (2) Fleischer, S.; Dvir, T. Curr. Opin. Biotechnol. 2013, 24 (4), 664− 71. (3) Iyer, R. K.; Chiu, L. L.; Reis, L. A.; Radisic, M. Curr. Opin. Biotechnol. 2011, 22 (5), 706−14. E
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Gold Nanoparticle-Decellularized Matrix Hybrids for Cardiac Tissue Engineering Michal Shevach,†,‡ Sharon Fleischer,†,‡ Assaf Shapira,† and Tal Dvir*,†,‡,§ †
The Laboratory for Tissue Engineering and Regenerative Medicine, Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Science, ‡The Center for Nanoscience and Nanotechnology, and §Department of Materials Science and Engineering, Tel Aviv University, Tel Aviv 69978, Israel S Supporting Information *
ABSTRACT: Decellularized matrices are valuable scaffolds for engineering functional cardiac patches for treating myocardial infarction. However, the lack of quick and efficient electrical coupling between adjacent cells may jeopardize the success of the treatment. To address this issue, we have deposited gold nanoparticles on fibrous decellularized omental matrices and investigated their morphology, conductivity, and degradation. We have shown that cardiac cells engineered within the hybrid scaffolds exhibited elongated and aligned morphology, massive striation, and organized connexin 43 electrical coupling proteins. Finally, we have shown that the hybrid patches demonstrated superior function as compared to pristine patches, including a stronger contraction force, lower excitation threshold, and faster calcium transients. KEYWORDS: Cardiac tissue engineering, decellularized matrix, gold nanoparticles, omentum, scaffold
C
become rounded, and their gap junction proteins such as connexin 43 are internalized or lost, disrupting proper anisotropic transfer of the electrical signal.9 To address this challenge, we and others have integrated nanoscale conductive materials with synthetic scaffolds to serve as electrical couplers between cells.9−14 For example, gold nanowires were embedded within the pore walls of 3D macroporous alginate scaffolds during the fabrication process.9 When cardiomyocytes were grown within the scaffolds, the cells interacted with the wires, and the engineered tissue exhibited significantly faster, synchronized electrical signal propagation, as compared to the signal produced by tissues grown in pristine scaffolds.9 In a different study, gold nanoparticles (AuNPs) conjugated to the backbone of synthetic hydrogels were able to enhance connexin 43 expression in cultured cardiomyocytes.10 Other studies have demonstrated that cardiomyocytes cultured on carbon nanotubes or in carbon nanotube supplemented hydrogels have formed tight junctions with the tubes and exhibited advanced electrophysiological functions, such as enhanced cell contraction, cell−cell coupling, and spontaneous electrical activity.11,15 In recent studies, we have shown that gold can be deposited on synthetic electrospun fiber scaffolds in a quick and facile manner.13,14 Here, we report on the development of a novel
ardiovascular diseases, such as myocardial infarction (MI), remain the most common cause of morbidity and mortality in developed countries.1 The aim of cardiac tissue engineering is to develop functional 3-dimensional (3D) tissue patches in vitro, later to be delivered to the damaged area in the myocardium to promote regeneration.2,3 However, in order to induce proper cardiac tissue assembly into a functioning tissue, it is essential to create a micro inductive environment that can mimic the native cardiac extracellular matrix (ECM).2 In vivo, this unique microenvironment composed of nanoscale to microscale fiber architecture fosters cell−cell coupling, leading to a rapid transfer of the electrical wave.2,4,5 One of the remaining key challenges in cardiac tissue engineering is fabricating a nonimmunogenic scaffold that would not provoke an adverse immune response after transplantation. Such response may hamper the therapeutic potential of the transplanted patch and can lead to graft rejection and adversely affect the health of the patient.6,7 To address this, we have recently suggested that nonimmunogenic scaffolds can be fabricated from the patient’s own biomaterials. We have shown that parts of the omentum can be easily, quickly, and safely harvested from the patient, and after efficient cell removal and quick processing, a porous 3D scaffold can be obtained.8 Another challenge, which may jeopardize the potential of cardiac patches to regenerate the infarcted heart is the lack of quick and efficient electrical coupling between adjacent cells.9 After cell isolation and seeding within the scaffolds, the cells © XXXX American Chemical Society
Received: July 15, 2014 Revised: August 12, 2014
A
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
Figure 1. Schematic overview of the concept. Omental tissue is isolated from the patient and undergoes a quick decellularization process to produce the 3D scaffold biomaterial. The decellularized scaffold is decorated with conductive motifs using an e-beam evaporator to produce a hybrid scaffold. Cells are isolated from the same patient, cultured in vitro, and seeded on the hybrid scaffold to produce a personalized cardiac patch. The engineered patch is transplanted on the infarcted heart.
fibrous scaffold with fibers ranging from 100 nm to several micrometers in diameter (Figure 2D). The second step involved decoration of the decellularized scaffold with conductive motifs. AuNPs, with nominal thicknesses of 4 and 10 nm, were deposited on the scaffold’s fibers using an e-beam evaporator, contributing to a change in the underlying scaffold color (Figure 3A). AuNPs scaffolds were visualized using an environmental SEM (ESEM) without additional conductive coating. The AuNPs were homogeneously deposited on the fibers, creating AuNPs/ECM hybrids (Figure 3B−D). Energy-dispersive X-ray spectroscopy (EDX) was used to confirm that the NPs observed on the fibers were indeed Au. As shown in Figure 3E, energy peaks associated with Au were observed in both the 4 nm and to a greater extent in the 10 nm hybrid scaffolds. One of the limitations of naturally derived scaffolds for cardiac tissue engineering is their low electrical conductivity.9 Therefore, we next sought to evaluate the conductivity of the engineered scaffolds by performing current−voltage (IV) measurements. The IV curve of pristine omental scaffold exhibited, as expected, a typical curve of an isolating material, similar to other protein-based scaffolds. In contrast, the 4 nm Au scaffolds have shown lower electrical resistance (Figure 3F). The 10 nm Au scaffolds exhibited typical curves of conductive materials (Figure 3G). Resistance calculation derived from IV curves further demonstrated the conductive nature of the hybrid scaffolds, while the pristine scaffolds showed significantly higher resistance (Figure 3H; p = 0.01 and p = 0.001). Such an electrical property of the scaffolds was previously demonstrated to be inductive microenvironment for the assembly of electrogenic cells such as cardiomyocytes.9 Since AuNPs existence on the fibrous scaffolds is essential for continuous maturation and development of the cardiac patch throughout the in vitro culturing period, but not necessarily important for in vivo applications after maturation, we next examined the stability of the hybrid scaffold in vitro in cell culture conditions. Au-deposited samples were submerged in culture medium and subjected to 7 days of constant shaking, in a cell culture incubator. Next, samples were washed, air-dried, and subjected to elemental analysis. As shown in Figure 4A and B, EDX analysis was performed on several points over a straight
conductive platform, integrating AuNPs with decellularized matrix. Such approach may alter the electrical properties of an autologous scaffold, composed of the patient’s own biomaterials, to better suit the needs of an engineered cardiac tissue and lead to cell organization, tissue maturation, and enhanced mechano-electrical function. When seeded with the patient’s own cells, the patches may be implanted back without triggering an immune response (Figure 1). Gold nanoparticle-decellularized matrix hybrids (AuNP scaffolds) were prepared in two main steps. First, native omentum underwent a decellularization process to produce a 3D biomaterial scaffold (Figure 2A−C).8 DNA removal was validated using Hoechst 33258 staining of ECM and fresh native tissue sections. Nuclei were detected in fresh omentum but not in the decellularized matrix (Supporting Information, Supplemental Figure 1). Scanning electron microscopy (SEM) images further validated complete cell removal and revealed a
Figure 2. Decellularization process. (A) Native omentum prior to cell removal. (B) Omentum during the decellularization process. (C) After complete cell removal, a 3D biomaterial scaffold is produced. (D) SEM image revealed an a-cellular fiber scaffold. Bar: D = 40 μm. B
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
Figure 3. AuNP scaffolds. (A) AuNPs were deposited onto the scaffolds using an e-beam evaporator. The color of the scaffolds has changed due to the difference in NP dimensions. (B, C) ESEM images of Au-deposited scaffolds. Homogeneously deposited AuNPs were visualized on the 4 nm (B) and 10 nm (C). (D) Higher magnification of C. (E) Energy-dispersive X-ray spectroscopy of the fibers. (F−H) Conductivity of the scaffolds. Current−voltage curves of pristine and 4 nm (F) and pristine 4 and 10 nm scaffolds (G). (H) Resistance calculation derived from current−voltage curves. Bar: B, C = 500 nm, D = 100 nm.
line on the ECM fiber. As the mounted samples have not been additionally coated with gold prior to visualization, the points outside the fibers have not shown peaks associated with Au (Figure 4B, points 1 and 5). On the contrary, points 2−4 on the fibers have shown Au associated peaks (Figure 4B), indicating the preservation of the Au content on the fibers even after exposing the samples to excessive rinsing over a long period of time. We next sought to evaluate the fate of the AuNP coating in more relevant, physiological conditions taking place in vivo. We hypothesized that, since deposition of the AuNPs occurs mainly on the amine and sulfur groups in the ECM, degrading the ECM by a relevant enzyme will simulate AuNP disassembly in vivo. Therefore, the hybrid scaffolds were placed for 7 days in culture medium containing collagenase type II (95 U/mL), an enzyme that is extremely active after cardiac remodeling following acute MI.16 Transmitting electron microscopy (TEM) images of the degradation products of the 4 and 10 nm Au scaffolds (Figure 4C and D) revealed disassembly of the AuNPs into spherical particles with average diameters of 3.62 ± 0.12 nm and 4.736 ± 0.17, respectively (Figure 4E). These sizes of AuNPs have been shown to be cleared by immune cells17 and to be nontoxic in vivo.18 To investigate the effect of the AuNPs within the scaffolds on tissue organization and function, we engineered cardiac patches. Cardiac cells were isolated from neonatal rat hearts and seeded
by a single droplet on the scaffolds. To assess the engineered tissue assembly, cardiac patches were immunostained for α sarcomeric actinin, a protein associated with cell contraction, and connexin 43, associated with electrical coupling between adjacent cells. On day 5, cardiac cells formed elongated and aligned cell bundles with massive striation on the pristine, the 4 nm and the 10 nm AuNP scaffolds, as judged by the immense actinin staining (Figure 5A−C; pink staining). However, while connexin 43, electrical coupling protein was randomly distributed and hardly noticed between cells grown in the pristine scaffolds (Figure 5A; green staining), cells grown in the AuNP scaffolds exhibited pronounced and organized staining, aligned between adjacent cardiomyocytes (Figure 5B and C). Compared to randomly organized distribution of connexin 43 molecules, aligned localization of the molecules between cardiomyocytes may lead to a faster, anisotropic propagation of the electrical signal and eventually may induce better functioning engineered tissues.19 Another parameter that may affect the function of the engineered patch is the ratio between cardiomyocytes and fibroblasts. In the heart, the cardiomyocytes to fibroblasts ratio is tightly regulated and is maintained constant in healthy individuals.20 The isolated cell population is mostly composed of cardiomyocytes, contracting cells, which cannot divide, and fibroblasts, which are highly proliferative and cannot contract. Therefore, it is essential to prevent the fibroblasts from taking C
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
Figure 5. Cardiac cell organization and engineered tissue function on day 5. (A−C) Immunostaining of cardiac α sarcomeric actinin (pink), connexin 43 (green), and nuclei (blue) of cardiac cells within the pristine (A), 4 nm (B), and 10 nm (C) scaffolds. Lower panels are higher magnifications. White arrows indicate the location of connexin 43 molecules. (D) Spontaneous contraction amplitude. (E) Excitation threshold. (F) Velocity of calcium transients during spontaneous contractions. The velocity is normalized to tissues engineered in pristine scaffolds. Bar = 20 μm. Figure 4. Stability and degradation of the AuNP scaffolds. (A, B) Stability of the AuNP scaffolds in normal culture conditions. ESEM images (A) and energy-dispersive X-ray spectroscopy (B) were performed after 7 days of constant shaking in culture medium. Points 1 and 5 located outside the fibers have not shown the Au associated peaks, while points 2−4 located on the fiber showed preservation of the Au content. (C, D) Degradation products of the AuNP scaffolds. TEM images of AuNPs after enzymatic degradation of 4 nm (C) and 10 nm (D) hybrid scaffolds. (E) Size distribution quantification of the enzymatically degraded hybrid scaffolds. Bar: A = 250 nm, C, D = 100 nm.
compared to the scaffolds modified with 10 nm AuNPs (Supplementary Figure 2E). Finally, to assess the effect of the hybrid scaffolds on the performance of the engineered tissues, a series of complementary functional assays was conducted. Since a strong contraction force is critical for generating effective cardiac patches,3,22 we initially assessed the spontaneous contraction amplitude. As shown in Figure 5D and Supplementary Movies 1−3, the presence of the AuNPs (4 and 10 nm) significantly increased the contraction amplitude of the patches (p = 0.044, p < 0.001, respectively). Since the pristine and the hybrid scaffolds had similar longitudinal elastic modulus (Supplementary Figure 3), it is acceptable to assume that the 10 nm AuNP patches have generated significantly higher forces as compared to the pristine and 4 nm patches. Another indication for the level of functional assembly of the tissue is its ability to generate synchronous contractions throughout the patch. To investigate this, cardiac constructs were subjected to an external electrical field, increasing in increments of 0.1 V. We defined excitation threshold as the minimum voltage needed to induce synchronous contractions of the entire patch at the same frequency (higher than the normal contraction rate). As shown in Figure 5E, the presence of both 4 and 10 nm AuNPs within the scaffolds increased the coupling between cell bundles, resulting in a significantly lower excitation threshold (p = 0.003, p < 0.001, respectively). Interestingly, no significant difference was observed between the 4 nm and the 10 nm scaffolds. Finally, an assessment of the electrophysiological effects of AuNPs was performed in cardiac cell constructs, with 4 and 10 nm particles or without NPs. The constructs were incubated with calcium-sensitive dye, and the velocity of calcium transients during spontaneous contractions was measured (n = 5 for each group). Although the 4 nm Au scaffolds still had
over the culture in vitro. A shift in the cell ratio in favor of the fibroblasts would impede patch contraction. Previously, it was shown that gold nanorods can restrain the infarcted heart matrix remodeling by affecting cardiac fibroblasts.21 More recently, we have shown that AuNPs can attenuate fibroblast proliferation.14 Here, we sought to evaluate whether AuNPs can have the same effect in the hybrid scaffolds, which may eventually lead to better maintenance of the initial cardiomyocyte to fibroblast ratio. Cardiac cell constructs modified with AuNPs (4 and 10 nm) and pristine scaffolds were stained on day 5 for troponin T and vimentin markers for cardiomyocytes and fibroblasts, respectively (Supplementary Figure 2A−C). Quantitative analysis of cell types in the scaffolds revealed significantly lower fibroblast fraction in the AuNP scaffolds (Supplementary Figure 2D), implying that the initial ratio of contracting to noncontracting cells was better preserved. Consequently, the cardiac patch may generate significantly stronger contraction force. To further investigate the effect of the AuNPs on fibroblast proliferation, metabolic activity of the isolated fibroblast population was measured at day 5 and normalized to day 0. As shown, a significantly higher proliferation rate was observed within the pristine scaffolds as D
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters
Letter
(4) Dvir, T.; Timko, B. P.; Kohane, D. S.; Langer, R. Nat. Nanotechnol 2011, 6 (1), 13−22. (5) Parker, K. K.; Ingber, D. E. Philos. Trans. Roy. Soc. London, Ser. B, Biol. Sci. 2007, 362 (1484), 1267−79. (6) Jones, K. S. Semin. Immunol. 2008, 20 (2), 130−136. (7) Anderson, J. M.; Rodriguez, A.; Chang, D. T. Semin. Immunol. 2008, 20 (2), 86−100. (8) Shevach, M.; Soffer-Tsur, N.; Fleischer, S.; Shapira, A.; Dvir, T. Biofabrication 2014, 6 (2), 024101. (9) Dvir, T.; Timko, B. P.; Brigham, M. D.; Naik, S. R.; Karajanagi, S. S.; Levy, O.; Jin, H.; Parker, K. K.; Langer, R.; Kohane, D. S. Nat. Nanotechnol. 2011, 6 (11), 720−5. (10) You, J. O.; Rafat, M.; Ye, G. J.; Auguste, D. T. Nano Lett. 2011, 11 (9), 3643−8. (11) Shin, S. R.; Jung, S. M.; Zalabany, M.; Kim, K.; Zorlutuna, P.; Kim, S. B.; Nikkhah, M.; Khabiry, M.; Azize, M.; Kong, J.; Wan, K. T.; Palacios, T.; Dokmeci, M. R.; Bae, H.; Tang, X. S.; Khademhosseini, A. ACS Nano 2013, 7 (3), 2369−80. (12) Martinelli, V.; Cellot, G.; Toma, F. M.; Long, C. S.; Caldwell, J. H.; Zentilin, L.; Giacca, M.; Turco, A.; Prato, M.; Ballerini, L.; Mestroni, L. ACS Nano 2013, 7 (7), 5746−56. (13) Fleischer, S.; Shevach, M.; Feiner, R.; Dvir, T. Nanoscale 2014, 6, 9410−9414. (14) Shevach, M.; Maoz, B. M.; Feiner, R.; Shapira, A.; Dvir, T. J. Mater. Chem. B 2013, 1 (39), 5210−5217. (15) Martinelli, V.; Cellot, G.; Toma, F. M.; Long, C. S.; Caldwell, J. H.; Zentilin, L.; Giacca, M.; Turco, A.; Prato, M.; Ballerini, L.; Mestroni, L. Nano Lett. 2012, 12 (4), 1831−8. (16) Thompson, M. M.; Squire, I. B. Cardiovasc. Test. 2002, 54 (3), 495−8. (17) Bartneck, M.; Keul, H. A.; Zwadlo-Klarwasser, G.; Groll, J. Nano Lett. 2010, 10 (1), 59−63. (18) Chen, Y. S.; Hung, Y. C.; Liau, I.; Huang, G. S. Nanoscale Res. Lett. 2009, 4 (8), 858−864. (19) Bian, W.; Jackman, C. P.; Bursac, N. Biofabrication 2014, 6 (2), 024109. (20) Souders, C. A.; Bowers, S. L.; Baudino, T. A. Circ. Res. 2009, 105 (12), 1164−76. (21) Sisco, P. N.; Wilson, C. G.; Mironova, E.; Baxter, S. C.; Murphy, C. J.; Goldsmith, E. C. Nano Lett. 2008, 8 (10), 3409−12. (22) Fleischer, S.; Feiner, R.; Shapira, A.; Ji, J.; Sui, X.; Daniel Wagner, H.; Dvir, T. Biomaterials 2013, 34 (34), 8599−606.
relatively high resistance (Figure 3H), tissues engineered within both AuNP scaffolds (4 and 10 nm) transferred the electrical signal 2- and 3-fold faster than the tissues grown in the pristine scaffolds (p = 0.035, p = 0.032, respectively). After transplantation, faster transfer of the electrical signal may lead to better integration of the patch with the healthy part of the myocardium and thus improve the efficacy of the cardiac patch therapy in vivo. In summary, we have developed and characterized a hybrid material based on natural ECM scaffold and AuNPs for cardiac tissue engineering. We have demonstrated that the AuNPs were able to significantly increase the conductivity of the natural material without affecting the mechanical properties relevant for cardiac tissue engineering. Moreover, we have shown that, in normal cultivation conditions in vitro, the developed scaffold is stable, and in in vivo-simulating conditions the NPs may dissociate from the scaffold. Cardiac cells cultivated within the hybrid scaffolds exhibited elongated and aligned morphology, massive striation, and organized connexin 43 electrical coupling proteins. The presence of the AuNPs attenuated fibroblast proliferation and thus maintained a contracting to noncontracting cell ratio during cell culture. Finally, growing cardiac cells in AuNP scaffolds has led to the formation of cardiac patches generating stronger contraction forces. Our approach may be useful for engineering more homogeneous cardiac patches that could better integrate with the healthy part of the heart and improve heart function after MI. The technique could be also beneficial for culturing other electrogenic cells such as neurons and may assist to regenerate other tissues such as the injured spinal cord.
■
ASSOCIATED CONTENT
S Supporting Information *
Details of experimental procedures, supporting figures, and movies displaying cardiac patch contraction. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*T.D. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS T.D acknowledges support from the European Union FP7 program (Marie Curie, CIG), Alon Fellowship, Israel Science Foundation and the Nicholas and Elizabeth Slezak Super Center for Cardiac Research and Biomedical Engineering at Tel Aviv University. The work is part of the doctoral thesis of M.S. at Tel-Aviv University. We would like to thank Ekaterina Glukhikh for her assistance with the current−voltage measurements.
■
REFERENCES
(1) Lloyd-Jones, D.; Adams, R. J.; Brown, T. M.; Carnethon, M.; Dai, S.; De Simone, G.; Ferguson, T. B.; Ford, E.; Furie, K.; Gillespie, C.; Go, A.; Greenlund, K.; Haase, N.; et al. Circulation 2010, 23 (121 (7)), 948−54. (2) Fleischer, S.; Dvir, T. Curr. Opin. Biotechnol. 2013, 24 (4), 664− 71. (3) Iyer, R. K.; Chiu, L. L.; Reis, L. A.; Radisic, M. Curr. Opin. Biotechnol. 2011, 22 (5), 706−14. E
dx.doi.org/10.1021/nl502673m | Nano Lett. XXXX, XXX, XXX−XXX
