DuPont® was one of the first companies to attempt to produce silk fibroins on an industrial scale70, but perfecting the large-scale production of spider silks has proved arduous and challenging relative to some other materials. DuPont® manufactures ap­proximately 2 million tons of Kevlar each year, requiring 15,800,000 to 18,750,000 pounds of sulfuric acid.71 The process also requires petroleum products, substantial pressure and temperatures that approach 1,400 degrees Fahrenheit. The U. S. Army uses about 10,000 pounds of Kevlar for composite materials. Manufacturing costs could be lowered by reducing the amount of hazardous waste material generated during the production of Kevlar. Additionally, over the past 70 years, DuPont® has also been manufacturing nylon. This material initially nicknamed the “miracle fi­ber.” One of first applications for nylon was socks, but other uses have expanded into clothing, carpeting, ropes, and the automobile industry. Despite the benefits of nylon’s use for a wide range of different applications, its production has a history of environmental concerns that include the reliance of large quantities of crude oil, adipic acid, and production of nitrous oxide, a greenhouse gas.

The onset and peak temperatures of the exothermic curve on the first heating DSC thermogram for the SPE/TPG compound with a standard epoxy/hydroxy ratio of 1/1 were 142.3 and 192.9 °C, respectively. Based on the DSC data, the curing tem­perature of the SPE/TPG(1/1) was changed between 150 and 190 °C. The tan 5 peak temperature (43.0, 43.5 and 53.5 °C) measured by DMA increased with an increase of curing temperature (150, 170 and 190 °C). Also, the 5% weight loss temperature (344.3, 344.8 and 361.1 °C) increased with an increase of curing temperature. When the mixture was cured at a temperature higher than 190 °C, the cured material con­siderably colorized. The curing temperature was fixed to 190 °C, considering the stability of SPE/TPG and wood flour which is subsequently added.

Figure 4.31 shows DMA curves of the SPE/TPG(1/1)/WF biocomposites cured at 190 °C. The E’ at the rubbery plateau region over 50 °C for the composites was much higher than that of SPE/TPG, suggesting a superior reinforcement effect due to the wood fibers. The tan 5 peak temperature related to T for the composites (WF40:45.6 °C; WF50:45.7 °C; WF60:44.5 °C) was a little lower than that of the corresponding neat resins (53.5 °C). The reason is not clear, but it is thought that hydroxy groups of WF reacted with epoxy groups of SPE and the stoichiometry of epoxy and hydroxy is deviated. A similar decline of Tg by the addition of WF was also observed for the GPE/TA/WF biocomposites.22 Figure 4.32 shows TGA curves of WF, SPE/TPG(1/1) and SPE/TPG(1/1)/WF composites. Since the thermal de­composition temperature of WF was lower than that of SPE/PGT, the SPE/PGT/WF composite exhibited two-step thermo-degradation, and the 5% weight loss tempera­ture decreased with increasing WF content (0 wt.%: 361.1 °C, 40 wt.%: 294.7 °C, 50 wt.%: 286.3 °C, 60%: 279.6 °C).

Figure 4.33 shows the tensile properties for SPE/TPG(1/1)/WF composites. The tensile modulus of SPE/TPG(1/1)/WF increased with increasing WF content in the range of 0-50 wt.%. However, the tensile modulus of SPE/TPG(1/1)/WF60 was lower than that of SPE/TPG(1/1)/WF50 in agreement with the influence of WF content on the E’ measured by DMA. Also, the tensile strength of the composites with WF content 40-50 wt.% was a little higher than the corresponding neat resin (SPE/TPG(1/1)). The fact that the improvement of tensile strength is not so high as that of tensile modulus is related to the decease of elongation at break for the WF biocomposites. In the previous our study on GPE/TA/WF and SPE/QC(1/1.2)/ WF biocomposites, the tensile strength considerably decreased by the addition of WF.22,25 When TPG was used as an epoxy-hardener, the tensile strength of the WF composite did not decrease.

Figure 4.34 shows SEM images of WF and the fractured surfaces of SPE/ TPG(1/1) and SPE/TPG(1/1)/WF composites. The micrograph of SPE/TPG(1/1) showed no phase separation, indicating that SPE is homogeneously cured with TPG. The micrograph of WF shows that the fiber length and width of WF are ca. 0.2-0.4 mm and 40-200 mm, respectively. All the micrographs of SPE/TPG(1/1)/ WF biocomposites show that WF is tightly incorporated into the crosslinked epoxy resin and their interfacial adhesion is good. The fact that tensile strength did not decrease by the addition of WF is related to the good affinity of SPE/TPG(1/1) and WF. The good affinity is inferred from what TO is widely used as a coating material for woody surface and the structure of pyrogallol moieties of TPG resembles that of lignin of WF.

The density of biocomposite plays a significant role in weight reduction. For en­ergy savings, designer are striving to manufacture light weight and high strength components. However it is to be noted that the density of the biocomposite have a significant influence on their noise reduction capabilities. Jute fibers are heavier than water and have a density in the range of 1200 to 1400 kg/m3.

The flax fabric (Fig. 7.4.a) used in this study, is a plain weave fabric with an areal weight of 280±19 g/m2, manufactured by GroupeDepestele (France)68. The fabric is not balanced, as the space between the weft tows (1.59± 0.09 mm) is different to the one between the warp tows (0.26± 0.03 mm). The width of the warp and the weft tows are, respectively 2.53±0.12 mm and 3.25±0.04 mm. As a consequence, there are 360 warp tows and 206 weft tows per meter of fabric. The linear mass of the warp and the weft tows is the same and is equal to 494±17 g/km. The tows are constituted by globally aligned groups of fibers. The length of these groups of fibers varies between 40 to 600 mm with a maximum occurrence-taking place at 80 mm. This fabric is constituted of continuous tows (Fig. 4.b). Generally, when natural fibers are considered, twisted yarns are elaborated to increase its tensile properties. Indeed, as discussed by Goutianos et al.69 sufficient tensile properties of the yarns are necessary for these ones to be considered for textile manufacturing or for pro­cesses such as pultrusion or filament winding. In this study, the flax tows used to elaborate the plain weave fabric are un-twisted and exhibit a rectangular shape. The fibers or groups of fibers are slightly entangled to provide a minimum rigidity to the tows. This geometry has been chosen as it generates low bending stiffness tows, therefore limiting the crimp effect in the fabric and therefore limiting empty zones between tows. It has also been chosen because fabric manufactured from highly twisted yarns exhibit low yarn permeability preventing or partially preventing the use of processes from the LCM (Liquid Composite Moulding) family. Un-twisted tows have also been chosen because manufactured composites display better me­chanical properties than composites made with twisted yarns70. However, this rein­forcement was originally developed to manufacture large panels with low curvature and was therefore not optimized for complex shape forming.

A second flax woven reinforcement has also been used. This reinforcement 2 is a 4/4 flax woven hopsack construction with an areal weight of 508 ± 11 g/m2 manu­factured by the Composites Evolution Company, UK71. The hopsack is presented in Fig. 7.5. The cylindrical yarns are manufactured from aligned fibers held together by a polyester yarn going in a spiral manner along the flax yarn. The linear density of the flax yarns is 250 ± 9 tex (g/km). The lineic mass of the yarn holding the flax fibers is 20 ± 3 tex. The reinforcement used in this work is not balanced. A differ­ence of 20% in the number of flax yarns has been measured between warp and weft directions.

7.2 RESULTS

Sugar palm fiber is black in color, with diameter up to 0.50 mm.70 According to Siregar,68 sugar palm fiber has heat resistant of up to 150°C and the flash point is around 200°C. It has been reported that the fiber length of sugar palm fiber is up to 1.19 m and density is 1.26 kg/m3.71,72 Traditionally, sugar palm fiber was used as ropes, filters, broom, roof and handicraft application such as for making ‘ko — piah.’72,73 Tomlinson74 reported that the ropes made from sugar palm fiber have bet­ter performance than the ropes made from rattan fiber (Calamus sp.). The main advantages of sugar palm fiber are durable and good resistant to seawater. It is also not affected by heat and moisture compared to coir fiber. Unlike other natural fibers, sugar palm fiber can directly be obtained from the trees which do not need second­ary processes to yield the fibers.3 Due to these advantages, sugar palm fiber should be a good material in the development of new ‘green’ materials.

Sugar palm fiber locally known as ijuk is one of the most popular fiber among the researcher over the last decade. The fiber is originally wrapped along the sugar palm trunk.75 The tree can grow up to 12.3 m tall and has a thick, black/brown hairy fibrous trunk, with the dense crown of leaves, which are white on the outside. The tree begins to produce black sugar palm fiber after about 5 years, before flowering and the type of its fibers are depending to age and altitude of sugar palm tree.71 The fibers that are taken after flowering will produce fiber approximately 1.4 m long. It can yield about 15 kg for each tree and around 3 kg is very best and stiffest. In Malaysia, black sugar palm fiber started used since 1416 during Malacca Sultanate History. In 1800, the sugar palm tree was planted by British East India Company in Penang to yield its high durability of rope made from black sugar palm fiber.

Previously, the characterization (tensile and chemical properties) of single fibers from different morphological parts of sugar palm tree, that is, sugar palm frond (SPF), sugar palm bunch (SPB), ijuk and sugar palm trunk (SPT).76 From the in­vestigation, it was found that the tensile strength of ijuk was 276.64 MPa and the tensile modulus was5.86 GPa. The elongation at break of ijuk was 22.3% which was approximately the same with oil palm and coir fibers in term of physical and me­chanical properties because they were from same palmae family.77 For the chemical analysis, it was shown that ijuk has a high cellulose content which is 52.29%. This proved that mechanical properties of sugar palm fiber are strongly influenced by the cellulose content.78 Cellulose was the main structural component that provides strength and stability to the plant cell walls and the fibers.49Generally, ijuk can be used as reinforcement in composites due to higher tensile strength and cellulose content in comparison with other established natural fibers such as kenaf, pineapple leaf, coir and oil palm bunch.

DSC is widely used to characterize the thermal properties of WPCs. DSC can mea­sure important thermoplastic properties, including the melting temperature (Tm), heat of melting, degree of crystallinity x(%), crystallization, and presence of recy- clates, nucleating agents, plasticizers, and polymer blends (the presence, composi­tion, and compatibility). Thermal analysis of the WPC samples was carried out on a differential scanning calorimeter (Perkin Elmer Instruments, Pyris Diamond DSC,

Shelton, Connecticut) with the temperature calibrated with indium. All DSC mea­surements were performed with samples of about (9.5 ± 0.1) mg under a nitrogen atmosphere with a flow rate of 20 mL/min. Three replicates were run for each speci­men. All samples were subjected to the same thermal history with the following thermal protocol, which was slightly modified from the one reported by Valentini et al.45.

1. First, the samples were heated from 40 to 180 °C at a heating rate of 20 °C/ min to remove any previous thermal history.

2. Second, the samples were cooled from 180 to 40.00 °C at a cooling rate of 10 °C/min to detect the crystallization temperature (Tc).

3.

Finally, the samples were heated from 40 to 180 °C at a heating rate of 10 °C/min to determine Tm. Tm and the heat of fusion (днт) were calcu­lated from the thermograms obtained during the second heating. The heats of fusion were normalized on the basis of the weight fraction of PP present in the sample. The values of днт were used to estimate X, which was adjusted for each sample in XcOr (%) based on the percentage of polypro­pylene in the composite. Crystallinity (Xcor) was estimated according to the

ДНm and ДН0 are, respectively heats (J/g) of melting of composite and 100% crystalline PP, taken as 207.1 J/g,46 and Xpp is the PP fraction in the composite.

Ethylene is a rather stable molecule that polymerizes only upon contact with cata­lysts. The conversion is highly exothermic, that is the process releases a lot of heat. Coordination polymerization is the most pervasive technology, which means that metal chlorides or metal oxides are used. The most common catalysts consist of titanium(III) chloride, the so-called Ziegler-Natta catalysts. Ethylene can be pro­duced through radical polymerization, but this route has only limited utility and typically requires high-pressure apparatus.

15.1.2.2. CLASSIFICATION

PE is classified into several different categories based mostly on its density and branching. Its mechanical properties depend significantly on variables such as the extent and type of branching, the crystal structure and the molecular weight. With regard to sold volumes, the most important polyethylene grades are HDPE, LLDPE and LDPE.

• Ultra-high-molecular-weight polyethylene (UHMWPE)

• High-density polyethylene (HDPE)

• Linear low-density polyethylene (LLDPE)

• Low-density polyethylene (LDPE)

(a) Ultra-high-molecular-weight polyethylene (UHMWPE)

These include can and bottle handling machine parts, moving parts on weaving machines, bearings, gears, artificial joints, edge protection on ice rinks and butch­ers’ chopping boards. It competes with aramid in bulletproof vests, under the trade names Spectra and Dyneema, and is commonly used for the construction of articular portions of implants used for hip and knee replacements.

(b) High-density polyethylene (HDPE)

HDPE is used in products and packaging such as milk jugs, detergent bottles, butter tubs, garbage containers and water pipes. One third of all toys are manufac­tured from HDPE. In 2007 the global HDPE consumption reached a volume of more than 30 million tons.

(c) Linear low-density polyethylene (LLDPE)

Lower thickness may be used compared to LDPE. Cable covering, toys, lids, buckets, containers and pipe. While other applications are available, LLDPE is used predominantly in film applications due to its toughness, flexibility and rela­tive transparency. Product examples range from agricultural films, saran wrap, and bubble wrap, to multilayer and composite films.

(d) Low-density polyethylene (LDPE)

LDPE is created by free radical polymerization. The high degree of branching with long chains gives molten LDPE unique and desirable flow properties. LDPE is used for both rigid containers and plastic film applications such as plastic bags and film wrap.

Starch is one of the most exciting and promising raw materials for the production of biodegradable products. It is the major polysaccharide reserve material of photosyn­
thetic tissues and of many types of plant storage organs such as seeds and swollen stems. The primary crops used for its production consist of potatoes, corn, wheat and rice. In all of these sources, starch is produced in the form of granules, which vary in size and somewhat in composition based on the resources. Starch granule is composed of two main polysaccharides, amylose and amylopectin with some minor components such as lipids and proteins. Amylose is linear polymer of (1^4)-linked a-D-glucopyranosyl units with some slight branches by (1^-6)-a-linkages (Fig. 17.1). Amylose can have a molecular weight between 104 and 106 g mol-1, but it is soluble in boiling water.40,41 Amylopectin is a highly branched mol­ecule composed of chains of a-D-glucopyranosyl residues linked together mainly by (1^4)-linkages but with (1^6)-linkages at the branched points. Amylopectin consists of hundreds of short chains of (1^4)-linked a-D — glucopyranosyl interlinked by (1^6)-a-linkages (Fig. 17.2). It is an ex­tremely large and highly branched molecule with a molecular weights rang­ing from 106 to 108 g mol-1. Therefore, it is insoluble in boiling water, but in their use in foods, both fractions are readily hydrolyzed at the acetal link by enzymes. Amylases attack the a-(1-4)-link of starch while the a-(1-6)-link in amylopectin is by glucosidases. The crystallinity of the starch granules is attributed mainly to the amylopectin and not to amylose, which although linear, presents a conformation that hinders its regular association with other chains.42,43

(Glucose-a(l -4 )-glucose)

Starch has received significant interest during the past two decades as a biode­gradable thermoplastic polymer. Starch offers an attractive and cheap alternative in developing degradable materials. Starch is not truly thermoplastic as most synthetic polymers. However, it can be melted and made to flow at high temperatures under pressure and shear. It has been widely used as a raw material in film production be­cause of increasing prices and decreasing availability of conventional film-forming resins based on petroleum resources. Starch films possess low permeability and are thus attractive materials for food packaging. Starch is also useful for making ag­ricultural mulch films because it degrades into harmless products when placed in contact with soil microorganisms.44,45

By itself, starch is a poor alternative for any commodity plastic because, it is mostly water soluble, difficult to process, and brittle. Therefore, research on starch includes exploration of its water adsorptive capacity, the chemical modification of the molecule, its behavior under agitation and high temperature, and its resistance to thermo mechanical shear. Although starch is a polymer, its stability under stress is not high. At temperatures higher than 150 °C, the glucoside links start to break, and above 250 °C the starch grain endothermally collapses. At low temperatures, a phe­nomenon known as retrogradation is observed. This is a reorganization of the hydro­gen bonds and an aligning of the molecular chains during cooling. In extreme cases under 10° C, precipitation is observed. Thus, though starch can be dispersed into hot water and cast as films, the above phenomenon causes brittleness in the film.46

Plasticized starch is essentially starch that has been modified by the addition of plasticizers to enable processing. Thermoplastic starch is plasticized to com­pletely destroy the crystalline structure of starch to form an amorphous thermoplas­tic starch. Thermoplastic starch processing involves an irreversible order-disorder transition termed gelatinization. Starch gelatinization is the disruption of molecu­lar organization within the starch macromolecules and this process is affected by starch-water interactions. Most starch processing involves heating in the presence of water and some other additives like sugar and salt to control the gelatinization in the food industry, or glycerol as a plasticizer for biodegradable plastics applications. Most of the commercial research on thermoplastic starches has involved modified starches and or blends with additives and other appropriate polymers for its applica­tion as biodegradable plastics.47 The starch molecule has two important functional groups, the — OH group that is susceptible to substitution reactions and the C-O-C bond that is susceptible to chain breakage. The hydroxyl group of glucose has a nucleophilic character. To obtain various properties starch can be modified through its — OH group. One example is the reaction with silane to improve its dispersion in polyethylene.48 Crosslinking or bridging of the — OH groups changes the structure into a network while increasing the viscosity, reducing water retention and increas­ing its resistance to thermo mechanical shear.

One of the approaches to modify this starch is by acetylation to from starch acetate. Acetylated starch does have several advantages as a structural fiber or film­forming polymer as compared to native starch. The acetylation of starch is a well — known reaction and is a relatively easy to synthesize. Starch acetate is considerably more hydrophobic than starch and has been shown to have better retention of tensile properties in aqueous environments. Another advantage is that starch acetate has an improved solubility compared to starch and is easily cast into films from simple solvents. The degree of acetylation is easily controlled by trans esterification, al­lowing polymers to be produced with a range of hydrophobicities. Starch has been acetylated [with a high content (70%) of linear amylose] and its enzymatic degrada­tion has been studied. Apart from acetylation and esterification, some other modifi­cation of starch such as carbonilation of starch with phenyl isocyanates, addition of inorganic esters to starch to produce phosphate or nitrate starch esters, production of starch ethers, and hydroxypropylation of starches via propylene oxide modifica­tion has been performed. Generally all these modifications involve hydroxyl group substitution on the starch that will lower gelatinization temperatures, reduce retro — degradation and improve flexibility of final product.42

Starch has been used for many years as an additive to plastic for various pur­poses. Starch was added as a filler49 to various resin systems to make films that were impermeable to water but permeable to water vapor. The use of starch as a biode­gradable filler in LDPE was reported.50 A starch-filled polyethylene film was pre­pared which became porous after the extraction of the starch. This porous film could be readily invaded by microorganisms and rapidly saturated with oxygen, thereby increasing polymer degradation by biological and oxidative pathways.51 Otey et al. in a study on starch-based films, found that a starch — polyvinyl alcohol film could be coated with a thin layer of water-resistant polymer to form a degradable agricultural mulching film.47 Starch-based polyethylene films were formulated and containing up to 40% starch, urea, ammonia and various portions of low density polyethylene (LDPE) and poly(ethylene-co-acrylic acid) (EAA). The EAA acted as a compatibil — izer, forming a complex between the starch and the PE in the presence of ammonia. The resulting blend could be cast or blown into films, and had physical properties approaching to those of LDPE.52,53

Additionally, crosslinked starch may be induced by the addition of organic/inor — ganic esters, hydroxyethers, aldehydes and irradiation. Kulicke et al. examined solu­tion phase crosslinking of starch with epichlorohydrin and trisodium trimetaphos- phate.54 Jane et al. examined the crosslinking of starch/zein cast films for improving water resistance.55 Iman et al. studied the crosslinking of starch/jute composite with glutaraldehyde to improve its performance characteristics such as mechanical properties, thermal properties, flame retardancy, etc.56 The possibility of chemically combining starch or starch-derived products with commercial resins in such a man­ner that the starch would serve as both filler and a crosslinking agent may provide a possible approach for incorporating starch into plastics.

Commercial starch polymer based products are provided in Table 17.1 given below:

One of the first starch-based products was developed probably by the National Starch in the brand name ECO-FOAMTM and used as packaging material. ECO — FOAMTM materials are derived from maize or tapioca starch and include modified starches. This relatively short-term, protected-environment packaging use is ideal for thermoplastic starch polymers. National Starch now has additional thermoplas­tic starch materials, blends and specialty hydrophobic thermoplastic starches for a range of applications including injection molded toys, extruded sheet and blown film applications [http://www. ecofoam. com/loosefill. asp]. Novament has been de­veloping thermoplastic starch based polymers since 1990. Mater-BiTM polymers are based on starch-blend technologies and product applications include biodegradable mulch films and bags, thermoformed packaging products, injection molded items, personal hygiene products and packaging foam [http://www. novament. com]. Simi­larly, Biotech GmbH produces Bioplast™ based on starch for a wide range of ap­plications including accessories for flower arrangements, bags, boxes, cups, cutlery, edge protectors, golf tees, horticultural films, mantling for candles, nets, packag­ing films, packaging materials for mailing, planters, planting pots, sacks, shopping bags, straws, strings, tableware, tapes, technical films, trays and wrap films [http:// www. biotech. de/engl/index_engl. htm]. Recently, Plantic Technologies Ltd. pro­duced soluble Plantic™ thermoformed trays for confectionery packaging.42

Vijay Kumar Thakur, PhD

Staff Scientist School of Mechanical and Materials Engineering, Washington State University, USA

Vijay Kumar Thakur, PhD, PDF, is a Staff Scientist in the School of Mechanical and Materials Engineering at Washington State University, Pullman, Washington, USA. He is an editorial board member of several international journals including Advanced Chemistry Letters, Lignocel — luloses, Drug Inventions Today International Journal of Energy Engineering and Journal of Textile Science and Engineering (USA), to name a few, and also member of scientific bodies around the world. His former appointments include Research Scientist in Temasek Laborato­ries at the Nanyang Technological University, Singapore; Visiting Research Fellow in the Department of Chemical and Materials Engineering at Languha University, Taiwan, and Post Doctorate in the Department of Materials Science and Engineering at Iowa State University, USA. In his academic career, he has published more than 100 research articles, patents and conference proceedings in the field of polymers and materials science. He has published 10 books and 25 books on the advanced state of the art of polymers/materials science with numerous publishers. He has extensive expertise in the synthesis of polymers (natural/synthetic), nano materials, nanocomposites, biocomposites, graft copolymers, high performance capacitors and electrochromic materials.

Professor and Berry Family Director School of Mechanical and Materials Engineering, Washington State University, Pullman, USA

Professor Kessler is an expert in the mechanics, process­ing, and characterization of polymer matrix composites and nanocomposites. His research thrusts include the develop­ment of multifunctional materials (including the develop­ment of self-healing structural composites), polymer matrix composites for extreme environments, bio-renewable poly­mers and composites, and the evaluation of these materials

using experimental mechanics and thermal analysis. These broad-based topics span the fields of organic chemistry, applied mechanics, and processing science. He has extensive experience in processing and characterizing thermosets including those created through ring-opening metathesis polymerization (ROMP), such as poly di — cyclopentadiene, and the cyclotrimerization of cyanate ester resins. In addition to his responsibilities as professor of Mechanical and Materials Engineering at Washington State University, he serves as the Director of the school. He has developed an active research group with external funding of over 10 million dollars—including funding from the National Science Foundation, ACS Petroleum Research Fund, Strategic En­vironmental Research and Development Program (SERDP), Department of Defense, Department of Agriculture, and NASA. His honors include the Army Research Of­fice Young Investigator Award, the Air Force Office of Scientific Research Young Investigator Award, the NSF CAREER Award, and the Elsevier Young Composites Researcher Award from the American Society for Composites. He has published more than 110 journal papers and 3700 citations, holds 6 patents, edited a book on the char­acterization of composite materials, presented more than 200 talks at national and international meetings, and serves as a frequent reviewer and referee in his field.

MTS array as applying for mazan (CellTiter 96® Aqueous One Solution Cell Prolif­eration Assay, Promega, Madison, WI) used the novel tetrazolium compound (MTS) and the electron coupling reagent, phenazine metho sulfate (PMS). MTS is chemi­cally reduced by cells into formazan, which is soluble in tissue culture medium. The measurement of the absorbance of the formazan can be carried out using 96 well microplates at 492 nm. The assay measures dehydrogenase enzyme activity found in metabolically active cells.

The metabolic activity of cells was monitored using the MTS that is chemically reduced by metabolically active cells into formazan. The amount of formazan pro­duced is an indicator of the cell viability. The measurement of formazan absorbance was performed in 96 well plates after several days of incubation. Standard curves were prepared by diluting a series of cell suspensions from 15.7 cells/mL to 157,000 cells/ mL. An aliquot (1.0 mL) of each dilution was transferred to wells of a 24-well tissue culture plate in triplicate. Subsequently, 150 nL of the MTS solution was added to each suspension.

The plate was incubated at 37 °C in a humidified atmosphere containing 5% CO2 in the dark for 4 h, after which 1.0 mL of the kit’s solubilization/stop solution was added to each well. Plates was sealed and incubated overnight. Absorbance was read at 570 nm wavelength and also at 650 nm as a reference wavelength using the plate fluorimeter.

The scaffolds were sterilized by incubating with 70% ethanol for 30 min, seeded with human osteoblast-like cells (SaOS-2) and cultured for up to 14 days. The vi­ability of the osteoblasts seeded to the electrospun scaffolds was determined first by using the MTS assay (Fig. 2.13). The amount of formazan produced is proportional to number of living cells in culture since the chemical reduction of formazin to a colored product is dependent on the number of viable cells. The MTS viability assay demon­strated that the cells exposed to these scaffolds maintain the ability to proliferate for up to 14 days that the experiment lasted for.

To evaluate cell morphology on the scaffolds, samples were prepared for elec­tron microscopy (EM) staining. Samples were washed 2 times with PBS and fixed with 2.5% gluteraldeyde [Sigma] for 2 h then washed with PBS. The cell-scaffold constructs were then attached to aluminum stubs, sputter-coated with gold, and then examined under a LEO-Gemini Schottky FEG scanning electron microscope.

The cells exposed to the nano scaffolds interacted with multiple fibers. Anchoring sites for cell attachment to the fibers were visualized by SEM. The nanoclusters of HAp mineral were consistently located at the edge of cells, which provides additional evidence that they act as anchoring sites for cell attachment to the fibrous hybrids. This improvement in cell adhesion and growth are deemed to the biological role of HAp. Figure 2.14 shows the morphology of the cells cultured on the scaffolds for 1, 7 and 14 days. The size and number of the cells are both increasing with time.

Our studies suggest that the morphology and structure of the CA-HAp composite scaffolds play important roles in facilitating cell spreading and differentiation and en­hance apatite mineralization. Based on our observations, the electrospun CA scaffolds with nanosized HAp are considered as a promising candidate for bone tissue engineer­ing application.