Introduction

Tissue engineering is a rapidly growing area that aims to create, repair and/or replace tissues and organs by using combinations of cells, biomaterials, and/or biologically active molecules. Tissue engineering strategies promise to revolutionize current therapies for irreversible myocardial damage, heart failure, and significantly improve the quality of life for millions of patients. The most challenging goal in the field of cardiovascular tissue engineering is the creation of an engineered heart muscle. Today, unlike heart valves or blood vessels, heart muscle has no replacement alternatives. Recent advances in methods of stem cell isolation and culture in bioreactors and the synthesis of bioactive materials show promise to contribute to creation of engineered cardiac tissue in vitro [1-3].

The biomaterial scaffold plays a key role in most tissue engineering strategies. To guide the organization, growth, and differentiation of cells in tissue engineered constructs, the biomaterial scaffold should be able to provide not only a physical support for the cells but also the chemical and biological cues needed in forming functional tissues [4].

Poly Hydroxy  Butyrate Valerate  (PHBV)  is  a  biomaterial  that  is  used  in  a  variety  of  applications  including  surgical  sutures,  wound  dressing,  drug  delivery  and  tissue engineering.  This  is  due  to  its  specific  properties  such  as  good biocompatibility,biodegradablity,  non  toxicity  as  well  as  its  piezoelectricity  features.  However, this material is a hydrophobic polyester which should be modified with other materials until it improves its cell adhesion and hydrophilicity properties [5-9]. It has been generally accepted that extracellular matrix mimics may improve the attachment, proliferation, and the viability of the cultured cells [10]. Electro-spinning has been rapidly developed  into a technique to prepare nanofibers with the diameter ranging from tens of nanometers  to several microns [11-13].The electro-spun  fibrous mats also show extremely high  surface  area  and  large  porosity. Besides, the fibrous structure of the electro-spun mats may mimic the extracellular matrix. It is well-known that the gelatin is a natural Biomaterial derived from the collagen inside animals skin and bones [13].Therefore; during the last few years, many works considering the tissue engineering of electro-spun nanofibers have been reported. Most recently, electro-spun nanofibers were prepared by Yang et al and were  applied  in  neural  tissue  engineering  [14]. Although  the  presented  nanofibers may mimic the morphologies of extracellular matrix to some extent, some modifications are still required  to create a  friendly environment  for  the cells attachment, proliferation, and  functions  such  as  communications.  Some  natural  materials  such  as  collagen, fibronectin  and  some  peptides  have  been  reported  as  scaffold  modifiers  [15,16]. Controlling  surface  properties  is  very  important  for  the  high  performance  of  adhesion. Biomaterials wettability is an important factor in the surface modification of materials. Surface modification  of  hydrophobic  polymer  surfaces  can  be  achieved  by  wet  (acid, alkali),  dry  (plasma)  and  radiation  treatments  (ultraviolet  radiation  and  laser)  [17-20].

In this study, the coated gelatin PHBV nanofibers were obtained through the chemical method. The samples were evaluated by ATR-FTIR, SEM and also the cell culture with Cardiac Muscle cells.

Materials and Methods

Nanofiber Preparation

A  poly  (3-hydroxybutyrate-co-3-hydroxyvalerate)  PHBV  containing  5  mol%  of  3-hydroxyvalerate with 680,000 molecular weight was purchased from Sigma Chemical Co. 2, 2, 2-trifluoroethanol (TFE) to prepare PHBV solution was also purchased from Sigma-Aldrich Chemicals and was used as  received without  further purification. An electro-spinning  apparatus  used  in  this  study  was  prepared  from  the  Asia Nanomeghyas Company (Iran). The PHBV was dissolved at determined concentration in TFE. The PHBV solution  (2%w)  in a glass syringe was controlled by  the syringe pump. A  positive  high  voltage  source  through  a wire was  applied  at  the  tip  of  the syringe  needle.  In  this  situation,  a  strong  electric  field  was  generated  between  the PHBV solution and a collector. When  the electric  field  reached a critical value with increasing  voltage,  the mutual  charge  repulsion overcame  the  surface  tension of  the polymer solution and the electrically charged jet was ejected from the tip of a conical shape as the Taylor cone. Ultrafine fibers were formed by the narrowing of the ejected jet fluid as it underwent increasing surface charge density due to the evaporation of the solvent. The  electro-spun  PHBV  nanofibrous  mat  was  carefully  detached  from  the collector  and  was  dried  in  vacuo  for  2  days  at  room  temperature  to  remove  solvent molecules completely. The used parameters for this nanofibers preparation can be seen in Table1.

Table 1: Used parameters for nano fibers preparation.

Gelatin Immobilization

Gelatin (Sigma) was immobilized onto the nanofiber surface based on the following protocol.  The nanofibrous mat was submerged into the Gelatin solution (10 mg/mL in distilled water solution) and was shaken gently for 2 h at 40 °C. The samples were exposured to glutaraldehyde 12% in hot water bath. The obtained samples were placed inside a vacuum oven so that they would fully lose humidity.

Fourier transmission infrared spectroscopy

The samples were examined by FTIR (Bruker-Equinox 55; Bruker Optics, Billerica, MA) before and after adjustment. The samples were scratched into powder and were produced as capsules using KBr, and then were investigated.

Scanning electron microscopy

The surface characteristics of various modified and unmodified films were studied by scanning electron microscopy (SEM; Cambridge Stereo-scan, model S-360; Cambridge Instruments, Wetzlar, Germany) to analyze the changes in the surface morphology. The films were first coated with a gold layer (Joel fine coat, ion sputter for 2 hours) to provide surface conduction before their scanning.

Cellular analysis

Primary cultures  can be initiated  from  cardiac  muscle  tissues   of  newborn   rats  as  described   below.

Digest minced cardiac muscle tissue with  1% trypsin   (gibco)  in Ca²⁺ and   Mg²⁺ free   PBS  for  20  min  at  37°C.  Satellite cells   can   also   be isolated   by digesting   minced adult muscle with 0.2% (w/v) collagenase (Roche) in HBSS followed   by 0.1% (w/v)    trypsin   in PBS. Inactivate   the trypsin with an equal volume of 0.1% soya bean  trypsin  inhibitor  (Sigma) in PBS. Cells enzymatically released   from muscle tissue usually contain a significant number of fibroblasts in addition  to  myogenic   cells.

Separate fibroblasts   from myogenic cells using differential adhesion: incubate   cells for 30 min on non-coated plastic culture dishes, and collect the non-adherent muscle  Repeat   this differential cell adhesion step.

Plate myoblasts on to gelatin-coated dishes   in Eagle’s minimal   essential medium   (MEM) (Life Technologies) supplemented with 10% horse serum (Hyclone) and 2% embryo extract   (Difco).  It is advisable to add ascorbic   acid (Sigma) to 100 Il-M to enhance collagen production.

Myoblasts  enter the   post-mitotic G0 phase and   myoblast fusion (fusion- burst)   becomes evident   within 48 h after plating.  Around the time of fusion-burst,   transcription of   muscle-specific   genes   is up-regulated. The activation of muscle-specific genes is also observed in fusion-arrested myoblasts in low calcium   medium. Myofibrillogenesis   takes  place in  multinucleated  myotubes  and  spontaneous  twitching   can  be  observed   within  7 days of  plating. Aliquots of cell suspension in RPMI medium including 300,000 cardiomyocyte cells were seeded on a 6-multiwell cell culture plate (Orange County Industrial Plastics) which was precoated with samples. The film was put in an incubator (37°C, CO₂) over three hours for cell attachment, followed by rinsing of the loosely attached cells with phosphate buffer solution, and adding 2 mL of fresh medium to the cell culture in the incubator for seven days. Proliferation of cells was determined from measure-ment of viable cell numbers by MTT assay. The MTT tetrazolium compound was reduced by living cells into a colored formazan product that was soluble in tissue culture medium. The quantity of formazan product was directly proportional to the number of viable cells in the culture. The assays were performed by adding 1 mL of MTT solu-tion (Sigma-Aldrich) and 9 mL of fresh medium to each well after aspirating the spent medium, and incubating at 37°C for four hours with protection from light. The colorimetric measurement of formazan dyeing was performed at a wavelength of 570 nm using a microplate reader (Rayto,Shenzhen, People’s Republic of China).

Results 

FTIR Results 

The  results,  from  the  ATR-FTIR  spectrum  of  the  uncoated  nanofiber  sample  and  the  nanofiber  sample  modified  with  Gelatin,  have  been  shown  in figure 1. In figure 1a, the strong band in 1722 cm^-1 has been shown to be related to the C = O group. The stretching band in 800-975 cm^-1 has been shown to be related to the- C-O-C- group and the stretching band in 2900-3000 cm^-1 has been shown to be related to the -CH₃ groups. Figure  1b  also  shows  the  strong  band  in  1722  cm^-1  as  related  to  the  C=O  group  ,  the stretching band in 800-975 cm^-1 as related to the -C-O-C- group and the stretching bands in 2800-3000 cm^-1 as related  to the -CH3 groups. The stretching band in 3400 cm^-1 and 3761 cm^-1 are related to the OH and NH groups due to the presence of gelatin.

Figure 1: FTIR analysis of uncoated nanofiber film (a) and coated nanofiber film (b). Click here to View figure

SEM Investigations

The figures 2 and 3 show the Electron Microscopy Images of the uncoated and the coated nanofibers with gelatin in different magnifications. Figure   shows  the nanofiber mat prepared  to  the  electro-spinning  method  of  different  magnifications  (2a ; 5000x –  2b; 20000x).

Figure 2: SEM images of PHBV nanofibers in different magnifications (2a ; 5000x – 2b; 20000x.) Click here to View figure

Figure 3: SEM images of coated gelatin PHBV nanofibers in different magnifications (3a; 5000x – 3b; 20000x). Click here to View figure

Figure  3  shows  the  coated nanofiber  mat  prepared  to  the  electro-spinning method of different magnifications (3a; 5000x – 3b; 20000x).

Image analysis of the electrospun normal nanofibers fabricated from 2wt% PHBV-TFE solution and coated nanofiber revealed an unimodal distribution of fiber diameters with an observed average diameter of 280 nm and 500 nm respectively (Figure 4).

Figure 4: Fiber diameter analysis of normal and coated PHBV nanofibers. The average fiber diameter were about 280 and 500 nm for normal (A) and coated (B) PHBV nanofibers respectively. Click here to View figure

Cellular Study

Figure 5 shows the MTT assay for TCPS (control), the uncoated nanofiber and the coated nanofiber samples. The results showed a high viability for the samples of the uncoated nanofiber and the coated nanofiber (110, 132 % respectively), but the coated nanofiber showed a better viability than the uncoated nanofiber. Also, these samples caused more cells to proliferate. Figure 6 showed images of the cell culture on the coated nanofiber and the control sample. The image a is related to the control sample and the image b is related to the the coated nanofiber. Cellular images showed well growth in the vicinity of the coated nanofiber.

Figure 5: MTT analysis of the samples.   Click here to View figure

Figure 6: Cardiac Muscle Cells growth on the samples. a: Control (TCPS) ,  b: Coated nanofiber . Click here to View figure

Discussion

In this study, the PHBV nanofibers with a size average about 280 nm were designed. Nanofibers were successfully coated with gelatin via the chemical methods shown in the analysis The smooth and homology modified nanofibers have been clearly  shown  in  the  figures. The size  average  was  obtained  for  the  modified  nanofibers  to  be  about  500  nm, whose increasing  is due  to gelatin coated on  the PHBV surfaces.  The  results  showed  a  high  viability  for  the  samples  of  the  gelatin coated nanofibers. Also, these samples caused more cells to proliferate. Cellular images showed well growth in the vicinity of  nanofibers especially the coated nanofiber. These gelatin coated nanofibers could be used well for heart tissue engineering

References 

1. Akhyari, P., Fedak, P. W., Weisel, R. D., Lee, T. Y., Verma, S., Mickle, D. A., et al: Mechanical stretch regimen enhances the formation of bioengineered autologous cardiac muscle grafts. Circulation 2002, 106: I137– I142.
2. Akins, R. E., Boyce, R. A., Madonna, M. L., Schroedl, N. A., Gonda, S. R., McLaughlin, T. A., et al: Cardiac organogenesis in vitro:reestablishment of three-dimensional tissue architecture by dissociated neonatal rat ventricular cells. Tissue Eng 1999,5, 103– 118.
3. Anversa, P., Sussman, M. A., & Bolli, R: Molecular genetic advances in cardiovascular medicine: focus on the myocyte. Circulation 2004, 109, 2832– 2838.
4. Langer, R., & Tirrell, D. A. (2004). Designing materials for biology and Nature 428, 487–492.
5. Williams SF, Martin DP, Horowitz  DM, Peoples OP: PHA applications: addressing the price performance issue: I. Tissue engineering Int. Biol. Macromol 1999,25:111
6. Liu J, Zhao B, Zhang Y, Lin Y, Hu P, Ye C: PHBV and predifferentiated human adipose-derived stem cells for cartilage tissue engineering. J Biomed Mater Res A 2010,94(2):603-610
7. Meng W, Kim SY, Yuan J, Kim JC, Kwon OH, Kawazoe N, Chen G, Ito Y, Kang IK: Electrospun PHBV/collagen composite nanofibrous scaffolds for tissue engineering. J Biomater Sci Polym Ed 2007,18(1):81-94
8. Ndreu A, Nikkola L, Ylikauppila H, Ashammakhi N, Hasirci V: Electrospun biodegradable nanofibrous mats for tissue engineering. Nanomedicine (Lond) 2008,3(1):45-60
9. Torun Köse G , et al:Tissue engineering of bone using collagen and PHBV matrices . Technology and Health Care 2002,10: 3-4
10. Gerard C, Catuogno C, Amargier-Huin C, Grossin L, Hubert P, Gillet P, Netter P, Dellacherie E, Payan E: The effect of alginate, hyaluronate and hyaluronate derivatives biomaterials on synthesis of non-articular chondrocyte extracellular matrix. J Mater Sci Mater Med 2005,16(6):541-551
11. Li D , Xia YN: Electrospinning of nanofibers: Reinventing the wheel? . Advanced Materials 2004,16: 1151-1170
12. Huang ZM, Zhang Y Z, Kotaki M and Ramakrishna S: A review on polymer nanofibers by electrospinning and their applications in nanocomposites . Sci. Technol 2003,63:2223
13. Smith LA and Ma PX: Nano-fibrous scaffolds for tissue engineering. Colloids Surf. B Biointerfaces 2004,39:125
14. Yang F, Murugan R, Wang S and Ramakrishna S: Electrospinning of Nano/micro Scale Poly(L-lactic acid) Aligned Fibers and their Potential in Neural Tissue Engineering.Biomaterials 2005, 26 : 2603
15. Ito T, Nakamura T, Suzuki K, Takagi T, Toba T, Hagiwara A, Kihara K, Miki T, Yamagishi H, Shimizu Y:Regeneration of hypogastric nerve using a polyglycolic acid (PGA)-collagen nerve conduit filled with collagen sponge proved electrophysiologically in a canine model. J. Artif. Organs 2003,26: 245
16. Kapur TA and Shoichet MS: Chemically bound nerve growth factor for neural tissue engineering applications. J Biomater Sci: Polymer Ed 2003,14:383–394
17. Lu HW, Lu QH, Chen WT, Xu HJ and Yin J : Cell culturing on nanogrooved polystyrene petri dish induced by ultraviolet laser irradiation . Materials Letters 2004, 58(1-2):29-32
18. Roucoules V, Gaillard F, Mathia TG and Lanteri P : Hydrophobic mechano chemical treatment of metallic surfaces.Wettability measurements as a means of assessing homogeneity.in Colloids And Interf. Sciences 2002,97(1-3):177-201
19. Hay KM, Dragila MI, Liburdy J: Theoretical model for the wetting of a rough surface . of Colloid Interf. Science 2008,325:472–477
20. Kogelschatz U: Dielectric – barrier discharges: their history , discharge physics and industrial applications. Plasma chem. Plasma 2003,23:1-46