Communication Journal of Biomedical Nanotechnology

Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America

Vol. 9, 1299–1305, 2013 www.aspbs.com/jbn

Development of Small Diameter Fibrous Vascular Grafts with Outer Wall Multiscale Architecture to Improve Cell Penetration K. T. Shalumon, K. P. Chennazhi, Shantikumar V. Nair∗ , and R. Jayakumar∗ Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi 682041, Kerala, India

This work explains about the development of a unique tubular scaffold for vascular tissue engineering. The inner layer/layers was made up of aligned poly (lactic acid) (PLA) nano fibers and outer layers were composed of random multiscale fibers of poly(caprolactone) (PCL)/PLA providing larger pores for Smooth Muscle Cell (SMC) penetration. The fabricated scaffolds were characterized by SEM. Cell attachment and infiltration studies using SMCs on the multiscale fibers were characterized by SEM and confocal microscopy. Blood compatibility of the scaffold was analysed by haemolysis-coagulation assays, platelet activation studies and the effect of material/fiber alignment on the morphological stability of Red Blood Cells (RBCs) were evaluated using SEM. Since this hierarchically designed tubular scaffold closely bycould Publishing Technology University mimics the morphology of nativeDelivered vessel, this be a better candidateto: forRice vascular tissue engineering.

IP: 50.46.234.208 On: Mon, 02 Nov 2015 03:59:46 Copyright: American Scientific Publishers KEYWORDS: Nanofiber, Multiscale, Vascular Graft, Scaffold, Electrospinning.

INTRODUCTION The most important factor in vascular tissue engineering is that, an ideal scaffold should have right properties in all aspects to regulate the material-cell interaction. Over the past two decades in tissue engineering, endless efforts have been made to develop a suitable blood vessel substitute which satisfies all the requirements of a scaffold.1 2 Among these, engineering of small diameter tubular scaffolds for arterial substitution was a major challengeable task.3 4 The design should be clearly based on the structural integrity and porosity to provide cyto-friendly atmosphere. Electrospun nanofiber has been widely used for the production of tissue engineering scaffolds.5–14 Even though nanofibers are suitable for tissue engineering, the thick fiber density and absence of porosity hinders the nanofiber-based scaffolds from the applicability of it as a three dimensional tissue engineering construct. For the simulation of the natural properties of a vascular graft, the internal geometry of the tubular construct should be ∗

Authors to whom correspondence should be addressed. Emails: [email protected], [email protected], [email protected] Received: 20 October 2012 Revised/Accepted: 1 February 2013 J. Biomed. Nanotechnol. 2013, Vol. 9, No. 7

in such a way that it should enhance the adhesion and proliferation of endothelial cells, but without inducing the blood coagulation, haemolysis or platelet activation. Our group reported that aligned fibers enhance the Endothelial Cell (EC) proliferation uniaxially with respect to the flow of blood.15 According to the reported literatures, compared to nanofibers, multiscale fibers gives more cell penetration16–18 followed by proliferation when cultured in-vitro. Recently, multiscale fibers got lot of importance in tissue engineering due to its unique properties. Basically, it is a hierarchical arrangement of nanofibers and microfibers and thus, it exactly mimics Extra Cellular Matrix (ECM). In tissue engineering using electro spun scaffolds, fiber diameter plays a critical role. In this work, by taking advantage of the penetration capability of cells in multiscale scaffolds and orientation ability of ECs on aligned fibers, we propose a unique vascular graft design with multiscale architecture on the outer layer of the scaffold and aligned fiber morphology in the inner layer. Compared to nanofiber architecture on the outer scale, the introduction of multiscale fibers are expected to enhance the cell proliferation faster. Another important factor to be projected out is the in-situ development of the design, instead of

1550-7033/2013/9/1299/007

doi:10.1166/jbn.2013.1630

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Development of Small Diameter Fibrous Vascular Grafts with Outer Wall Multiscale Architecture

sticking two parts of the scaffolds together to get a sandwiched shaped scaffold. The composition of the fibers with PCL and PLA is also expected to be helpful in tuning the degradability of the scaffold when it uses in real clinical conditions.

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the amount of plasma haemoglobin was calculated in mg/dL. The haemolytic property in percentage haemolysis (% haemolysis) was calculated and the morphology of the scaffold treated Red Blood cells (RBCs) were observed through SEM. The effect of scaffolds on the plasma coagulation was determined using prothrombin time (PT) and activated partial thromboplastin time (APTT).20 The analysis was done using a coagulation analyzer and reagent kits CK prest and Fibriprest from Diagnostica stago (France). The effect of aligned fibers in the platelet activation was also evaluated using Anticoagulant citrate dextrose (ACD) containing 20 mM citric acid, 110 mM sodium citrate and 5 mM D-glucose. Qualitative evaluation of the difference in morphologies of platelets on positive controls and scaffold was done using SEM. After the incubation of scaffold in Platelet Rich Plasma (PRP), 100 l of treated PRP was again incubated with 20 l of FITC labelled CD42b and PerCP-Cy5 labelled CD62P (BD bioscience, India). After incubation with these antibodies for 20 minutes, samples were washed with PBS and re-suspended in 1 ml PBS and measured using FACS.

EXPERIMENTAL DETAILS

Aligned PLA nanofibers were prepared from 11.5 wt% of PLA solution, at a voltage of 15 kV, by spinning onto a rotating mandrel with high speed. After electrospinning, aligned nanofibrous mat was removed from the mandrel and fused into a tubular form as described in our previous study.15 After the preparation of the aligned fibrous inner layer of the tubular structure (step-(d) of Fig. 1), PCL/PLA solutions with 25 and 12 wt% concentrations were spun intermittently over the aligned tubular scaffold (step e of Fig. 1). The inner-outside and cross sectional morphology of the scaffold was analysed by SEM. Mechanical properties of the scaffold was done using ORIENTEC Universal testing machine STA-1150 RTC with a crosshead speed of 3 mm/min and compared with single layered and multilayered nanofibrous tubular scaffolds. SMCs were seeded onto the multiscale scaffolds with a seeding density 25 × 104 cells/cm2 and incubated. Each RESULTS AND DISCUSSION sample was taken out of the incubator after 24 and 96 hrs. Tubular scaffold with multiscale-outer layer structure was Morphology of the cells on the scaffold was analyzed Delivered by Publishing Technology to: Rice University fabricated. Figure 2 represents the photograph (A) and by SEM. Confocal and Fluorescent microscopic measureIP: 50.46.234.208 On: Mon, 02 Nov 2015of 03:59:46 SEM images the ments were done for the same time points after proper cell Scientific Publishers tubular scaffold clearly showing axiCopyright: American ally aligned inner layer (B) and randomly aligned multifixing procedures. scale outer layer (C). The intention behind the fabrication The haemolytic potential of aligned PLA fibrous scafof multiscale structure on the outer side of the scaffold fold was spectrophotometrically analyzed by soret band 19 was solely based on the cell penetration capability of the based absorption of free haemoglobin in blood plasma. scaffold. The inner alignment will provide the unidirecThe absorbance was measured at 380, 415 and 450 nm tional orientation guidance for the Human Umbilical Vein using a spectrophotometer (Shimadzu, UV-1700) and (a)

(b)

(c)

(d)

(e)

Figure 1.

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Schematic representation of development of multiscale tubular scaffold by electrospinning. J. Biomed. Nanotechnol. 9, 1299–1305, 2013

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Development of Small Diameter Fibrous Vascular Grafts with Outer Wall Multiscale Architecture

Figure 2. Photograph (A) and SEM images of the tubular scaffold clearly showing (B) axially aligned inner layer and (C) randomly aligned multiscale outer layer.

unidirectional morphology of aligned fibers inside the scafEndothelial Cells (HUVECs)15 where as outer multiscale fold. More detailed exploration of the scaffold architecture architecture could enhance the SMC infiltration to a higher is shown in Figure 3. Both horizontal and vertical imaging extend compared to the normal nanofiber outer-layered of the tube gives an overall outlook of the design of the scaffolds.15 16 The diameters of the aligned fibers were in Delivered by Publishing to: Rice University scaffold. (A) and (E) show the cross sectional view of the the range of 500–800 nm whereas nano and microfibers Technology 02 Nov scaffold in 2015 which03:59:46 multiscale fibers are perfectly wounded in the random multiscale scaffold IP: had50.46.234.208 a fiber diameterOn: of Mon, Copyright: American Scientific Publishers over the inner axial layers. (B), (C) and (F) are the top 250–400 nm and 10 m respectively. Arrows indicates the

Figure 3. SEM images of cross sectional (A), (E), (H) surface (B), (C), (F), (G) and internal (D), (I) views of the tubular scaffold with multiscale architecture. J. Biomed. Nanotechnol. 9, 1299–1305, 2013

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view of the surface of the scaffold with multiscale archi(C). Long-term incubation (96 h) resulted in the maximum tecture. (D) Shows both outside and inside morphology of spreading and proliferation of cells in the entire multiscale the scaffold with random and aligned morphology respecsystem (D), (E) and (F). A cross confirmation of cell pentively. A closer view of the inter junction of aligned nanoetration was done using confocal microscopy. Figure 4(ii) fibers and multiscale fibers are represented in (H), where shows the maximum projection confocal images of cell (G) and (I) points to the existence of multiscale fibers in infiltration in multiscale outer layers of the tubular scafthe background of aligned nanofibers. fold at (A) 24 and (B) 96 h. (C) represents the Z-stacked Mechanical studies revealed the comparable elongation lateral view of cellular infiltration and (A), (B) and (C) strength of multilscale tubular scaffold with single layered represents the auto-fluorescent green colour of fibers. Red and multilayered architectures as reported earlier.15 From colour in (A1), (B1) and (C1) represents cells and (A2), our previous results, tubular scaffolds with nanofibrous (B2) and (C2) represents the combination of cells and single-layered and multi-layered outer layer architecture fibers. Figure 4(iii) is the DAPI stained image of the scafshow 1122 ± 041 and 1173 ± 037 N/mm2 respectively.15 fold with cells penetrated in to the interiors. In comparison, multiscale tubular scaffold having same When replacing the outer layer of the tubular scafinternal diameter as multi-layered nanofibrous scaffold fold from nanofibrous to multiscale structure, prevention shows a mechanical strength of 1169 ± 024 N/mm2 . This of SMC movement into the interiors of the scaffold is a clear evidence for the multiscale tubular scaffold for (for nanofibers) and poor cell adhesion behaviour (for its applicability in tissue engineering. microfibers) could be avoided. Hence, SMC can infilSMC adhesion studies were conducted to evaluate the trate till the top line of internal elastic lamina to form cell infiltration ability of the scaffolds to the interiors. a strong vascular tissue engineering construct in-vitro. Figure 4(i) represents the SEM images of SMC infiltration Multiscale scaffold mimics the ECM due to its unique in multiscale outer layers of the tubular scaffold at (A), characteristics. Here, nanofibers promote protein adsorp(B) and (C) 24 and (D), (E) and (F) 96 h. As expected, tion and thereby increase cell adhesion, differentiation cells were able to penetrate into the interiors very well and proliferation whereas microfibers provides mechanand started to spread over the available nanofibers in mulical support and porosity for cell infiltration.16–18 These tiscale fibers even at 24 h of incubation (A), (B) and distinctive properties of multiscale scaffolds are essential Delivered by Publishing Technology to: Rice University IP: 50.46.234.208 On: Mon, 02 Nov 2015 03:59:46 (i) (ii) Copyright: American Scientific Publishers

(iii)

Figure 4. (i) SEM images of SMC infiltration in multiscale outer layers of the tubular scaffold (A)–(C) at 24 and (D)–(F) at 96 h and (ii) is the maximum projection confocal images of cell infiltration in multiscale outer layers of the tubular scaffold at (A) 24 (B) 96 h and (C) represents the Z-stacked lateral view of cellular infiltration. (A)–(C) represents fibers, (A1)–(C1) are cells and (A2)–(C2) represents cells/fibers. (iii) is the DAPI stained image of the scaffold with cells penetrated in to the interiors.

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(i)

(ii)

Delivered by Publishing Technology to: Rice University IP: 50.46.234.208 On: Mon, 02 Nov 2015 03:59:46 Copyright: American Scientific Publishers

Figure 5. (i) (A) Effect of aligned PLA fibers in percentage haemolysis compared to positive control (PC) and negative control (NC) and (B) is the SEM image of morphology of red blood cells (RBC) on PLA fibers. (ii) (A) represents the morphology of activated platelet on a reactive surface and (B) shows the resting platelet morphology on aligned fibers. (C)–(E) is the FACS data showing the least platelet activation on PLA fibers.

for vascularization and thus for the development of a tissue construct.17 The mechanism of cell adhesion on nanofibers is through multiple focal points on the entire area of nanofibers.16–18 The combined entanglement of nano and micro fibers on the outer layer could give flexible nature to the scaffold and thus can function as an ideal vascular scaffold. Haemolysis was analyzed by soret band based absorption of free haemoglobin in blood plasma by measuring the absorption at 415 nm.19 In Figure 5(i), (A) represents the effect of aligned PLA fibers in percentage haemolysis and (B) SEM image of morphology of red blood cells (RBC) on PLA fibers. Percentage haemolysis of the sample was less than 0.5% compared to the positive control (PC) Triton X100. Triton treated samples show 95% haemolysis whereas negative control PBS (NC) shows no cytotoxicity. According to ISO/TR 7406 the critical safe limit of biomaterials is less than 5%. The morphology of plasma treated RBC shows uniform morphology of cell membrane J. Biomed. Nanotechnol. 9, 1299–1305, 2013

without disturbing the integrity of RBCs. The amount of plasma haemoglobin in mg/dL was calculated and from the results it was concluded that neither the material nor the alignment of the fiber induced haemolysis. Qualitative evaluation of the difference in morphologies of platelets on positive controls and scaffold was done using SEM and the percentage activation of platelet was quantified using flow cytometry. Figure 5(ii) represents (A) morphology of activated platelets on the surface of the coverslip (positive control) and (B) represents the non-activated morphology of platelets on aligned fibers. Positive control showed aggregates of platelets with numerous philopodial extensions while the PLA fibers showed a few platelets adhered on their surface which appeared rounded. In general, platelets have more affinity for activation on smooth surfaces compared to rough. Here, compared to platelets on cover slips, PLA fibers had rough surface morphology and hence not favourable for philopodial extensions to adhere. Moreover, pores 1303

Development of Small Diameter Fibrous Vascular Grafts with Outer Wall Multiscale Architecture Table I. PT and APTT values of PLA fibers treated with platelet poor plasma. Prothrombin time (Sec) (PT) Normal value 11–16

Activated partial thromboplastin time (Sec) (APTT)

Test value

Normal value

Test value

1556 ± 042

25–35

359 ± 036

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outer-multiscale part of the scaffold and it was further confirmed by the maximum projection and Z-stacking experiments by confocal microscope. Haemolysis assay for the aligned fibers showed negligible haemolysis compared to the positive control where as experiments on PT and APTT evaluation confirms that the blood coagulation values of the scaffold lies in the normal range. Platelet activation studies showed that neither the morphology nor the material property of aligned fibers induces platelet activation. Hence from all the results, we conclude that this unique design would be a suitable candidate for a small diameter vascular graft.

on the surface of fibers and in between fibers provided a non-continuous surface architecture, which might be a non favourable atmosphere for platelet adhesion. In Figure 5(ii) (B), the coin shaped cell morphology is of Acknowledgment: The authors are thankful to the live RBCs, located along with non-adhered platelets points Nanomission, Department of Science and Technology towards the poor platelet adhesion and good RBC attach(DST), Government of India for supporting this work ment on PLA scaffolds. FACS was used to evaluate the under “Thematic Unit of Excellence” grant. The author surface marker expression of platelet activation on interK. T. Shalumon is grateful to Council of Scientific and action with PLA fibers. Two surface marker CD42b (Q4) Industrial Research (CSIR) for awarding Senior Research and CD62p (Q2) expression in platelets were used for the Fellowship (SRF). platelet activation study. The resting platelets show the expression of CD42b while the activated platelets express both markers CD42b and CD62p. ADP (50 uM) was used REFERENCES as positive control for the activation of platelets, which 1. C. Y. Xu, R. Inai, M. Kotaki, and S. Ramakrishna, Aligned resulted in 70% activation (C). PLA fiber treated platelets biodegradable nanofibrous structure: A potential scaffold for blood vessel engineering. Biomaterials 25, 877 (2004). showed an activation of 14% (D) which is almost compa2. M. R. Hoenig, G. R. Campbell, B. E. Rolfe, and J. H. Campbell, rable to the PBS treated negative control (showed a basal Tissue-engineered blood vessels-Alternative to autologous grafts. activation of 13.1%) (E). Hence the quantitative and qualiDelivered by Publishing Technology to: Rice University J. Americ. Heart. Assos. 25, 1128 (2005). IP:fact 50.46.234.208 Mon, 02 2015 tative results are conclusive of the that, the PLAOn: fibers 3. Nov W. Yang, J. F.03:59:46 Wang, T. Wang, H. Wang, S. Jin, and N. He, Study Copyright: American Scientific Publishers caused no significant activation of platelets. on chitosan/poly(caprolactone) blending vascular scaffolds by electrospinning. J. Biomed. Nanotech. 6, 254 (2010). The purpose behind the plasma coagulation assay is to 4. L. Soletti, A. Nieponice, Y. Hong, S. H. Ye, J. J. Stankus, W. R. analyze the behaviour of material for coagulation factors Wagner, and D. A. Vorp, In vivo performance of a phospholipidwhen it is in contact with blood plasma. PT gives us a meacoated bioerodable elastomeric graft for small-diameter vascular surement of the extrinsic pathway and APTT represents applications. J. Biomed. Mater. Res. A 96, 436 (2011). that of the intrinsic pathway of coagulation. The sample in 5. C. P. Barnes, S. A. Sell, E. D. Boland, D. G. Simpson, and G. L. Bowlin, Nanofiber technology: Designing the next generation of tisspecified dimension was treated with PPP for the schedsue engineering scaffolds. Adv. Drug Deli. Rev. 59, 1413 (2007). uled time interval and the effect was monitored by measur6. L. Buttafoco, N. G. Kolkman, B. P. Engbers, A. A. Poot, P. J. Dijking the PT and APTT. Table I represents the PT and APTT stra, I. Vermes, and J. Feijen, Electrospinning of collagen and elastin values of test sample and normal values of the correspondfor tissue engineering applications. Biomaterials 27, 724 (2006). 7. K. T. Shalumon, K. H. Anulekha, K. P. Chennazhi, H. Tamura, S. V. ing tests. The measured value of PT was 1556 ± 042, Nair, and R. Jayakumar, Fabrication of chitosan/poly(caprolactone) which includes in the normal range of PT 11–16 seconds. nanofibrous scaffold for bone and skin tissue engineering. Int. J. Similarly for APTT, the test and normal values obtained Biol. Macromol. 48, 571 (2011). were 359 ± 036 and 25–35 seconds respectively. From 8. K. T. Shalumon, D. Sathish, S. V. Nair, K. P. Chennazhi, H. Tamura, these results it was clear that the PLA samples are not and R. Jayakumar, Fabrication of aligned poly(lactic acid)-chitosan nanofibers by novel parallel blade collector method for skin tissue inducing any coagulation for blood plasma when comes in engineering. J. Biomed. Nanotech. 8, 405 (2012). contact with blood. Hence the results confirm that none of 9. Q. Wang, N. Zhang, X. Hu, J. Yang, and Y. Du, Chithe coagulation pathways are getting affected on contact tosan/polyethylene glycol blend fibers and their properties for drug with the PLA fibers. controlled release. J. Biomed. Mater. Res. 85A, 881 (2008).

CONCLUSIONS A new tubular scaffold design with inside aligned PLA nanofiber morphology and outside PCL/PLA multiscale morphology was developed by electrospinning technique and characterized by SEM and mechanical measurements. Cell adhesion studies by SEM with SMCs shows that cells were able to penetrate into the interiors of the 1304

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17. S. Sreenivasan, R. Jayakumar, K. P. Chennazhi, E. J. Levorson, A. G. Mikos, and S. V. Nair, Multiscale fibrous scaffolds in regenerative medicine. Adv. Polym. Sci. 246, 1 (2012). 18. Q. P. Pham, U. Sharma, and A. G. Mikos, Electrospun poly(caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: Characterization of scaffolds and measurement of cellular infiltration. Biomacromolecules 7, 2796 (2006). 19. V. B. Morris and C. P. Sharma, Folate mediated in vitro targeting of depolymerised trimethylated chitosan having arginine functionality. J. Coll. Interface Sci. 348, 360 (2010). 20. A. Anitha, K. P. Chennazhi, S. V. Nair, and R. Jayakumar, 5-flourouracil loaded N, O-carboxymethyl chitosan nanoparticles as an anticancer nanomedicine for breast cancer. J. Biomed. Nanotech. 8, 1 (2012).

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J. Biomed. Nanotechnol. 9, 1299–1305, 2013

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Development of small diameter fibrous vascular grafts with outer wall multiscale architecture to improve cell penetration.

This work explains about the development of a unique tubular scaffold for vascular tissue engineering. The inner layer/layers was made up of aligned p...
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