NANOPOLYMERS *
CHAPTER 6 CHAPTER 6
6.4 BIOMEDICAL TECHNOLOGY
6.4.2 BIOMATERIALS
Biomaterials are materials that are implanted as prostheses in the body. Prostheses have been used for decades. In the 1920s and 1930s, materials such as vanadium steels, stainless steel, cobalt, titanium, and gold have been used. These materials lacked the surface properties that would make them biologi- cally compatible.
Although strength in the material can be given by the bulk material, the surface properties have an important role since they need to directly interact with the body. For obvious reasons, the surface mate- rial must not be toxic or carcinogenic in any way. It must also be biocompatible, being recognized as non-foreign material. If the material is recognized as a foreign material, the body will not accept it and proper healing will not occur, as described in the wound healing section discussed previously. Table 6.2 displays common materials used in medicine currently.
Biocompatibility is a difficult term to clearly define, but it is often defined in terms of if the material fulfils its intended purpose. This may have different characteristics depending on what type of applica- tion it is used for. Some applications require adhesion to cells. Biocompatible also does not mean being without side effects. There may be minimal side effects even though the material may be performing its duty well.
Polymeric nanofibers have the application of creating an extracellular matrix (ECM) that is often needed in the wound healing process. This has the potential to speed the healing process if the material is biocompatible. Natural polymers have also been integrated in the nanofiber. Lecithin and collagen have been known to increase the biocompatibility and even allow for the cells to proliferate easier on the ECM. This is due to the presence of natural proteins in the nanofiber, leading to a higher cytocom- patibility. The presence of a foreign body can lead to the host cells rejecting the implant in a process called foreign body reaction.
Table 6.2 Applications of Synthetic and Modified Biological Materials
Material Application Tissue Response
Titanium and alloys Joint prostheses, oral implants, fixation plates, pacemakers, heart valves
Inert CaP ceramic Joint prostheses, oral implants, bone replacement, middle ear
replacement
Bioactive
Alumina Joint prostheses, oral implants Inert
Carbon Heart valves Inert
Poly(tetrafluoroethylene) Joint prostheses, tendon and ligament replacement, artificial blood vessels, heart valves
Inert
Poly(methylmethacrylate) Eyes lenses, bone cement Tolerant
Poly(dimethylsiloxane) Breast prostheses, catheters, facial reconstruction, tympanic tubes
Unknown Poly(urethane) Breast prostheses, artificial blood vessels, skin replacements Inert
Poly(lactic) acid Bone fixation plates, bone screws Inert
Poly(glycolic) acid Sutures, tissue membranes Inert
From Malsch, N. H. (2005). Biomedical nanotechnology. Boca Raton: Taylor & Francis Group.
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6.4.2.1 Foreign body reaction
There are four potential results of a foreign body being introduced in a biological system: (1) inte- gration, (2) extrusion, (3) resorption, or (4) encapsulation. Integration is obviously the most desired outcome of a biomaterial implantation, since it will interact with the biological system as if it were a part of the system. The number of cases of true biointegration achieved has been limited, most frequently they have been cases of bone tissue implants with titanium coated with hydroxyapatite.
Most often soft tissue, such as skin, implantation results in the other three outcomes. Extrusion occurs when the implanted device is in contact with epithelial cells and the epithelium will form a pocket around the implantation. If it is close to the surface of the epithelium, it will be gradually pushed out of the host. Resorption occurs when the implant is made of degradable material and the implant degrades, resulting in a collapsed scar at the implantation site. Encapsulation of soft tissue is the most frequent foreign body reaction. The capsule consists of a membrane of a high amount of collagen with a layer of myofibroblasts outside the membrane. The implant is then isolated from the body (Malsch, 2005).
Nirmala et al. (2010) attempted to create a blend of lecithin and polyamide-6 nanofibers that would allow osteoblast cell cultures to attach to and grow on the nanofiber blend. Polyamide-6 with 0, 1, 3, 5 wt% lecithin solution was used to prepare the nanofiber composite. Different instruments were used to characterize the nanofibers including average pore diameter, pore volume, pore area, total porosity, structure by X-ray diffraction, and bonding configurations with Fourier-transform infrared. Osteoblast cell culture was introduced after carefully preparing and incubating the cultures (with fetal calf serum).
The osteoblast cells were grown on the polymer scaffolds and cultured.
All nanofibers were comprised of randomly oriented fibers with smooth surfaces and uniform diam- eters. The polyamide-6 nanofibers created 150–200 nm structures whereas the mesh-like nanofiber structure resulted in one magnitude less than that (10–30 nm). The porosity is important to provide framework for the seeded cells. The 5% lecithin blend provided higher porosity and diameter with much lower average pore volume than polyamide-6 unblended. There is improved water wettability for the higher wt% lecithin than pristine polyamide-6. Wettability is advantageous since osteoblastic culture can spread more easily on hydrophilic surfaces, rather than hydrophobic ones. The lecithin blends showed a melting peak at 220–215°C and the crystallinities of the nanofibers were ~185–180°C.
Fig. 6.9 shows the cell culture after 3 days on the various nanofiber blends after adhesion and pro- liferation were examined. The cells are clearly growing attached to the matrix. The cytotoxic effect of the nanofiber was measured by measuring levels of pyruvic acid with a spectrophotometer. The lactate dehydrogenase activity in the medium was examined. There was a slight increase in toxicity as the lecithin was incorporated into the cells.
It was concluded that the polyamide-6/lecithin composite nanofibers improve the cell growth behav- iors. If the mechanical strength of the scaffold can be increased, the nanofiber can be used practically for bone regeneration. It can be used in vitro for transplantation to the site.
By examining the biocompatibility of human umbilical vein endothelial cells (HUVECs) on a poly(l-lactide-co-ε-caprolactone) (PLCL)/fibrinogen nanofiber blend, a biodegradable ECM capable of supporting some types of tissues can be created. PLCL is a biodegradable polymer which shows promise in soft tissue engineering, and fibrinogen is a soluble plasma glycoprotein, used to enhance the adhesion of cells to PLCL. Using the random copolymer PLCL with 70 mol% l-lactide, an 8%
PLCL solution was prepared. Similarly, a fibrinogen solution was prepared and they were mixed at 4:1,
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2:1, 1:1, 1:2, pure PLCL, fibrinogen, and tissue culture polystyrene as a control. This was put through an electrospinning process. The different composites were placed in culture plates with HUVEC.
Different tests were performed including cell proliferation tests, cell adhesion tests, and morphological characteristic tests.
It was found that the higher the fibrinogen content in the composite, the smaller the water contact and the smaller the fiber diameter of the nanofiber. It was found that initially there was no significant difference between the different composites, but after 3–5 days, the PLCL/fibrinogen blended scaffold- ing had increased the cell number quickly especially compared to the pure PLCL nanofiber. After day 7, it was found that the proliferation rates of the PLCL/fibrinogen nanofibers were 3.23, 4.35, 3.95, and 3.39 for 4:1, 2:1, 1:1, and 1:2 ratios, respectively. Fibrinogen had a much higher proliferation rate, but this had been previously known. Fig. 6.10 shows the proliferation of cells on the nanofiber scaffolds by way of metabolic activity of the cells. The PLCL/fibrinogen blend of 2:1 may be the most suitable culture. The live/dead assay demonstrated that very few cells were found dead possibly demonstrating that the nanofibers were relatively non-cytotoxic. This may be a good sign for future applications (Fang et al., 2010).
FIGURE 6.9
SEM image of cell growth on nanofiber with lecithin concentrations of (A) 0, (B) 1, (c) 3, and (D) 5 wt%.
From Nirmala, R., Park, H. M., Navamathavan, R., Kang, H. S., El-Newehy, M., & Kim, K. Y. (2010). Lecithin blended polyamide-6 high aspect ratio nanofiber scaffolds via electrospinning for human osteoblast cell culture. Material Science and Engineering C, 486–493.
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Nguyen, Kin, Song, and Lee (2010) examined the process of thermally treating Ag nanoparticles (NPs) within polyvinyl alcohol (PVA) nanofibers as a means to inhibit microbial growth for skin appli- cations. The silver interrupts microbial growth that can hinder the process of skin regeneration.
An aqueous solution of AgNO3 was added to distilled water and irradiated with microwaves, reduc- ing the Ag ion to have no charge. The PVA and Ag solution was then electrospun and heated from 0°C to 150°C at 5°C/min in a nitrogen atmosphere. A test of inhibition with Staphylococcus aureus and Escherichia coli, gram-positive and gram-negative bacteria, respectively, was carried out in an incuba- tor at 37°C for 18–24 h.
It was first found that the increased microwave irradiation time increased the diameter of the nanofibers, 100–200 nm and 100–500 nm for the 60 s and 90 s irradiation, respectively. This was due to the evaporation of water increasing the polymer concentration in solution. The samples vary in Ag concentration with sample A having a 9.93% Ag concentration and sample B with 5.73%. Sample A was irradiated for 60 s while sample B was irradiated for 90 s. Using UV-Vis absorption, it was con- firmed that Ag NPs were formed in the nanofibers. Fig. 6.11 shows the SEM imaging of the nanofibers loaded with Ag NPs. The electrospun mats were then heated for 24 h to 80°C, 120°C, and 150°C. The heat treatment at higher temperatures was shown to have Ag NPs that were larger in size on the surface of the nanofiber. It was found that the higher-temperature-treated nanofibers had a slightly increased antimicrobial property. The nanocomposites were much stronger and more brittle than pure PVA mat, with the heat treatment having an improvement on the strength of the nanofiber. The nanofibers showed antibacterial properties in a zone around the application area with the 60 s irritation sample at 150°C heat treatment having the best antimicrobial properties. The Ag NP loaded PVA shows great promise in the area of skin application to promote rapid healing (Nguyen et al., 2010).
FIGURE 6.10
Metabolic activity of cultured HUvEcs on PLcL/fibrinogen nanofiber scaffolds.
From Fang, Z., Fu, W., Dong, Z. D., Zhang, X., Gao, B., Guo, D., et al. (2010). Preparation and biocompatibility of electrospun poly (l-lactide-co- ε -caprolactone)/fibrinogen blended nanofibrous scaffolds. Applied Surface Science, 4133–4138.
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FIGURE 6.11
SEM micrograph of Ag NP loaded PvA nanofiber mats with 60 s irradiation and heat treatment at (A,B) 80°c, (c,D) 120°c, (E,F) 150°c and with 90 s irradiation and heat treatment at (G,H) 150°c.
From Nguyen, T.-H., Kin, Y.-H., Song, H.-Y., & Lee, B.-T. (2010). Nano Ag loaded PVA nano-fibrous mats for skin application. Journal of Biomedical Materials Research B, 225–233.
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