fabricating nano fiber constructs via centrifugal jet spinning for tissue engineering

Proceedings of the 7th Annual FEP Honors Research Symposium Copyright, 2015, Woods AW, Spears, SS. Please do not use the materials without expressed permission of the authors.

Fabricating Nano Fiber Constructs via Centrifugal Jet Spinning for Tissue Engineering

Anthony WoodsDepartment of Industrial Engineering

Sarah SpearsDepartment of Biomedical Engineering

Mentor: Kartik Balachandran Ph.D.Department of Biomedical Engineering

Abstract

Using the centrifugal jet spinning technique, we mixed PCL (polymer) and gelatin (hydrolyzed protein) at different ratios and spun them at different speeds to create varying nanofiber constructs. By analyzing scanning electron microscope images of the fibers with computer software, we hope to find the effect of rotational speed and solution concentration on fiber diameter and alignment. By developing trends between these parameters and the resulting fiber characteristics, we hope to make nanofiber constructs more biocompatible and help in their manufacturing process. We found that as rotational speed increased, fiber diameter decreased and alignment increased. Nanofibers are potentially useful in the medical field in the area of accelerated wound healing and scaffolding.

1. Background

Nanofiber production for biological applications is a relatively new field, in which nanofibers, or strands the size of 1 x10−9 meters, are spun in nanofiber production machines and collected. Nanofiber strands consist of polymers, proteins, or a combination of both. Preliminary nanofiber fabrication methods include the electrospinning technique, in which strands of a solution of a polymer in a solvent are pulled onto a rotating cylinder by placing the solution in an electric field with a high voltage. Researchers aim to optimize different surface properties such as “morphology, pore size, distribution, biocompatibility, and biodegradability” (Bonani 2010) to make them the most cohesive with human tissue and durable as possible. In the electrospinning process, changes in parameters including voltage, solution concentration, feed rate, and humidity are modified to see how fiber diameter, tensile strength, and fiber alignment change. The alignment of the nanofiber construct must be similar to a muscle on the nano level with the fibers being parallel to one another in order to be the most biocompatible. Changes in the fiber diameters are analyzed since the fiber diameter relates to the biodegradability rate of the nanofiber construct. In different injury applications, the optimal biodegradation rate of the construct will vary.

In double electrospinning, a second process for nanofiber production, two materials such as gelatin, or hydrolyzed protein, and PCL (poly-caprolactone) are morphed into one nanofiber strand, which pools the PCL’s structural prowess and the gelatin’s advantageous biological characteristics. Currently, in a process called centrifugal jet spinning, a hollowed out cylinder with micro orifices going from the inside to the outside of the cylinder is spun with solutions inside of it, producing a nanofiber formation on the outside of the cylinder due to the centrifugal force on the solutions. Researchers incrementally modify “nozzle geometry, rotation speed, and polymer solution properties” (Sant 2011) to mutate “fiber morphology, diameter, and web porosity” (Sant 2011) to yield the most biologically and mechanically cohesive fiber as possible. Testing these fibers consists

of analyzing scanning electron microscope (SEM) pictures, which are visually enhanced to the micro or nano level, using software to measure fiber diameter and alignment, performing Fourier Transform Infrared Spectroscopy tests on the constructs to test for the presence of amide bonds, which would indicate biocompatible characteristics, and testing their tensile strength using an Instron machine. Recently, researchers used collagen and gelatin combined with PCL to test and analyze how fiber diameter and alignment vary with the inclination of these components in the initial solutions. Researchers found that the gelatin-PCL combination has a greater fiber diameter, but lower “orientation order parameter” (Bodrossamay 2014) than the collagen-PCL combination. Researchers aim to see if using different ratios of gelatin to PCL of the same percentages in highly volatile solvents or changing the rotational speeds at which they are spun at elicit a more biomimetic fiber than currently discovered.

2. Motivation

Nanofibers have a myriad of medical applications including treatments of “tendons, cartilage, blood vessels, and bone muscles.” (Bonani 2010) Utilized in treating coronary vascular diseases, nanofibers make certain procedures possible, which help doctors save many lives and improve the quality of life for a multitude of individuals. Nanofibers play a major role in treatment of coronary vascular diseases in that they are “used to repair weakened vessels or bypass blockages primarily in the abdomen and lower extremities [and] have saved countless individuals from massive bleeding from ruptured degenerated aortas” (Ratner 2013). For example, in the case of an

2Woods, AW Spears SS

Figure 1: Motor and Nozzle Platform Top Left) Motor and Nozzle and base Top Right) aerial view of cylinder and double reservoir Bottom Left) Micro orifice Bottom Right) Side view of cylinder

aortic aneurysm, an implanted stent graft, which contains nanofiber constructs acts as the existing blood vessel. Currently, the constructs do not conform well biologically to human tissue due to a lack of biomaterial in the constructs. Therefore, younger patients must have the grafts replaced by stent grafts with a greater diameter within ten or so years. With more biologically compatible nanofiber constructs combined with a growth factor, a young patient is less likely to have to have their stents replaced since the constructs grow with the patient and adhere to their cells well. Aside from vascular applications, nanofiber constructs can also be used to support and increase healing rates in almost any injury area, from spinal cord, burn, bone, or muscle injuries. In the case of a spinal cord injury, an implemented nanofiber construct can physically support, deliver drugs such as antibiotics over time, and foster increased cell proliferation in the damaged area. The outcome in this case would be an increased healing rate and effectiveness of the spinal cord injury. A construct can bridge a gap of an injured spinal cord area, allowing the spinal cord to function more effectively and heal quicker.

3. Research Objectives

Our first research objective is to perform a detailed literature review involving centrifugal jet spinning nanofiber production. Internalizing trends on how certain characteristics change when certain parameters vary will allow us to develop relatively new hypotheses or test other hypotheses. Next, we will to learn safety and operational protocol for the centrifugal jet spinning machine. Then, we will spin 15 samples of nanofiber constructs. We will spin 5 different ratios of 7% solutions of PCL to PCL/gelatin in HFIP (hexafluro-2-propanol) and a control group of PCL to PCL at 20,000, 25,000, and 30,000 rpms. Our fourth goal is to collect data from our experiments using computer software to analyze the scanning electron microscope images to attain the constructs’ fiber diameter and alignment. After that, we will create graphs comparing relationships between changes in solution concentrations and rotational speeds with fiber diameter and alignment outcomes. Finally, we will develop conclusions about the trends between the effects that changing solution ratios of PCL to gelatin and rotational speeds have on fiber diameter and alignment.

4. Research activities and Results

We successfully achieved our first goal in that we internalized trends between varying rotational speeds and solution concentrations and the effects on the nanofiber constructs. Using the knowledge and results of credible research articles, we knew that spinning two gelatin in PCL solutions at once does not elicit an extractible fiber. We learned how to make 7% solutions of PCL in HFIP (in addition, 3:1, 2:1, 1:2, PCL:gelatin in HFIP). To make a 7% solution of PCL:gelatin in HFIP, we mixed 50 ml of HFIP and 1.75 grams of PCL and gelatin together. To fully combine, the solution must be stirred on am automatic stirring table for about two hours. We successfully learned the centrifugal jet spinning protocol, assembling the machine, wiring the machine to the Elvis, or circuit board, and running the programs on the computer. We learned how to precisely extract the nanofiber samples from the machine by using a thin knife to carefully scrape the sample off from the outside of the cylinder. After placing the extracted nanofiber samples, we placed them in petri dishes, cut small squares of each one off, and placed them on a preparation platform to be taken to the SEM to attain images of them on the micro level. Our research mentor, took ten images of each experimental group (ex. PCL with PCL:gelatin 3:1 at 20,000 rpms). By familiarizing ourselves with a MATLAB program that analyzes the overall orientation of the nanofibers in the SEM photos, we assessed the alignment of each experimental group. The MATLAB program assigns light and dark values to an image, and assigns vectors to the light values, which represent each nanofiber. Once we adjust the light and dark values to match the image accurately, we run a program that combines the value of all of the vectors in the image and gives a number between 0 and 1, 1 meaning that the


nanofibers are all parallel to each other. We will run through that process for each of the 10 pictures taken for each of the 18 groups (6 different solutions and 3 different solution concentrations). We will average the values of each of the ten pictures to get an average alignment value for the group. To calculate the fiber diameter, we analyzed the SEM photos using a computer application called Image J. By converting the scale of 20 micrometers to pixels using the program, we calculated the length in micrometers of 5 close individual fibers in each image by dragging the mouse across the span of a fiber. To calculate the overall fiber diameter for each picture, we averaged these 5 values. Finally, we created excel graphs comparing changes in parameters with changes in fiber diameter and alignment. For example, to analyze how solution concentration changes the diameter and alignment, we will graph the 20,000 rpm spun groups with varying solution concentrations verses their fiber diameter and alignment.

20K 25K 30K

0.90

0.86

0.82

PCL/PCL:gel (2:1)

Rotational Speed (RPM)

Alig

nmen

t

20K 25K 30K

0.92

0.90 0.81

PCL:PCL


Alig

nmen

t

20K 25K 30K

0.90

0.86

0.82

PCL/PCL:gel (3:1)


Alig

nmen

t

20K 25K 30K

0.89

0.870.83

PCL/PCL:gel (1:1)


Alig

nmen

t


20K 25K 30K

0.930.86

0.83

PCL/PCL:gel (1:2)


Alig

nmen

t

Figure 2 (above): Graphs for each solution group showing that as rotational speed increases, alignment also increases

20k 25k 30k

735 679587

PCL:PCL

Rotational Speed (RPM)Fibe

r Dia

met

er (n

m)

20k 25k 30k

697 668479

PCL/PCL:gel (3:1)


r Dia

met

er (n

m)

20k 25k 30k

673

639

603

PCL/PCL:gel (2:1)


r Dia

met

er (n

m)

20k 25k 30k

708 675574

PCL/PCL:gel (1:1)


r Dia

met

er (n

m)


20k 25k 30k

457635 711

PCL/PCL:gel (1:2)


r Dia

met

er (n

m)

Figure 3 (above): Graphs showing fiber diameter of each of the solution groups that show that as the rotational speed increases, fiber diameter decreases.

PCL PCL 3:1 PCL 2:1 PCL 1:1 PCL 1:2

0.81

0.87

0.82 0.83 0.83

20k RPM

Solutions

Alig

nmen

t


0.90 0.91

0.86 0.87

0.83

25k RPM

Solutions

Alig

nmen

t


0.92 0.920.90

0.89

0.93

30k RPM

Solutions

Alig

nmen

t


735 697 673 708

457

20k RPM

SolutionsFibe

r Dia

met

er (n

m)



679668

639

675

635

25k RPM

SolutionsFibe

r Dia

met

er (n

m)


587479

603 574711

30k RPM

SolutionsFibe

r Dia

met

er (n

m)

Figure 4 (above): Graphs showing a weak correlation between solution concentration and fiber diameter and alignment

5. Conclusions and Future Work

Two strong trends developed from our research. As the rotational speed increased, the fiber diameter decreased and the fiber alignment increased. These trends are shown in figures 2 and 3. We found no correlation between solution concentration and fiber diameter or alignment. This can be seen in figure 4. The trends attained in this project will help in the goal of consistent fabrication of more biocompatible nanofibers than current processes allow. For example, by knowing what the fiber diameter of a fiber spun at a certain RPM will be and the biodegradability rate of a fiber of that diameter, one can accurately fabricate a nanofiber for a specific medical application. Some future research includes using spectroscopy to test for amide bonds on the nanofibers, indicating the presence of protein in the construct. This would ensure the presence of protein in the fibers that will make the fibers more biocompatible. Some further tests such as tensile strength tests can also be done on the fibers to ensure their structural integrity. By seeding animal or human cells to the fibers, allowing time for the interacting of the cells with the construct, and visualizing the interactions using a scanning electron microscope, researchers will be able to further analyze the biocompatibility of certain fiber constructs.


Works Cited

Badrossamay M, Balachandran K, Parker K, et al. Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning. Biomaterials [serial online]. March 2014;35(10):3188-3197. Available from: MEDLINE, Ipswich, MA. Accessed March 8, 2015.

Bonani W, Maniglio D, Motta A, Tan W, Migliaresi C. Biohybrid nanofiber constructs with

anisotropic biomechanical properties.Journal Of Biomedical Materials Research. Part B, Applied Biomaterials [serial online]. February 2011;96(2):276-286. Available from: MEDLINE, Ipswich, MA.

Ratner, Buddy D. Hoffman, Allan S. Schoen, Frederick J. Lemons, Jack E. (2013).

Biomaterials Science - An Introduction to Materials in Medicine (2nd Edition) - 7.3.3

Stents and Grafts (for Atherosclerotic Vascular Disease). (pp. 470). Elsevier.

Sant S, Hwang C, Lee S, Khademhosseini A. Hybrid PGS-PCL microfibrous scaffolds with improved mechanical and biological properties. Journal Of Tissue Engineering And Regenerative Medicine [serial online]. April 2011;5(4):283-291. Available from: MEDLINE, Ipswich, MA.


fabricating nano fiber constructs via centrifugal jet spinning for tissue engineering

Documents