Nanotechnology For Regenerative Medicine Biology Essay


Nanotechnology for Regenerative Medicine

Nanotechnology is a fast developing field with a wide range of potential applications. Nanotechnology involves understanding and controlling material at the nanometer scale (1). Improving lifestyles, an aging population, and over-increasing expectations for a better quality of life demand better, more proficient and inexpensive health care (2; 3). Regenerative medicine is a multidisciplinary field (4). The use of nanotechnology in regenerative medicine can promise new treatment modalities when applied to major medical challenges (5; 4). The application of nanotechnology in regenerative medicine can drastically change the treatment of diseases in the future (1). Nanotechnology has a broad area of application in regenerative medicine, for example in increasing the contrast for imaging, in biosensors, in chemical sensors, in constructing scaffolds, and in drug delivery (6; 7). Use of polymers for controlled drug-delivery helps deliver a drug for a long period of time and also increases the effectiveness of the drug (1). Nanoparticles and Nanotubes can be used for drug delivery. Nanoparticles can circulate in the body; they can also be taken up by the cell through endocytosis. Nanoparticles can also be used for diagnostic purposes. There are some obstacles in transforming current drug delivery methods to therapies, for example targeting nanoparticles at a specific site without being captured by organs like liver and spleen (1).

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In the last few decades, nanomedicines have started coming onto the market; for example, superparamagnetic nanoparticles are used in the MRI for increasing contrast for imaging (8; 9; 10). The problem of the shortage of organs for organ transplantation can be solved by regenerative medicine (11; 12). Regenerative medicine can be used to repair, retain, or improve tissues and organ functions (13). With the help of regenerative medicine, tissue can be regenerated to replace or restore defective tissue or organs. (4; 14). Regenerative medicine assures rejuvenating damaged tissues and organs by provoking previously irrevocable organs to repair themselves (14). In the field of regenerative medicine tissue and organs can be grown in the lab and implanted when the body cannot repair itself (14). With the help of material science, engineering and biology, tissue engineering can be done. Using biomaterial and nanotechnology scaffolds can be created for tissue engineering (1).Using cells and scaffolds, regeneration of tissues can be achieved. Here the scaffold acts as a surface for cell proliferation (8; 4; 15). Cell-surface interactions is very important as it determines the success or failure of the implant. So it is essential that the scaffold used produces the right signal to guide cell growth suitably (4; 16).

In this study, we have focused on three different applications of regenerative medicine. Our first aim was to investigate an anodization technique to produce surface modified nanoporous titanium that can be used as a potential system for engineering a distinctive biomaterial. Our second aim was to fabricate a halloysite-PCL (polycaprolactone) scaffold and assess the cell supportive ability. The final aim was to examine the drug release from the drug loaded halloysites, the drug loaded PCL scaffolds and the drug loaded halloysite-PCL scaffolds.

Nanoparticles and Nanotubes for Regenerative Medicine

Nanotechnology is under going fast development, and new research is continuously published (1; 10). Examples of nanoparticles are buckyballs, liquid crystals, liposomes, nanoshells, quantum dots and supramegnetic nanoparticles (17). Carbon nanotubes and halloysite nanotubes are examples of nanotubes (18). A couple of examples of potential application of nanomaterials are biosensor and drug carriers (19). Nanomaterials can be used as intracellular drug carriers and as biosensors to monitor the real time level of biomolecules, enzymes, and cell differentiation (20). Present methods of judging cell treatment include invasive and/or destructive methods, for example, tissue biopsies. Thus, non-invasive methods are very advantageous. Examples of non-destructive nanoimaging probes include nanoshells and quantum dots (QD) (18; 21).


Quantum Dots

Quantum dots are colloidal nanocrystalline semiconductors (22; 23). Properties of quantum dots depend on their size. Quantum dots can be used as fluorescent probes for labeling (23). To examine the cell and the cellular process fluorescence labeling is used. Conventional fluorescence labeling has some limitations, for example sensitivity to thermal changes in local environment, blinking, photobleaching and lower contrast using quantum dots that are colloidal metal particles. However, we can overcome these limitations (23; 24). Gold nanaoparticles are examples of colloidal metal particles. Their use helps in overcoming drawbacks of blinking and photobleaching compared to fluorophores. Fluorescence wavelength of quantum dots depands on the size of quantum dots (24). The quantum dot can be observed with high resolution compared to fluorophores by electron microscopy (23).


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The nanoshells are nanoparticles, which consist of a dielectric core coated with a metallic layer (25). Nanoshells are produced by layer-by-layer self-assembling and can be used in drug delivery (25). Nanoshells can be manufactured by a one-step or two-step approach (26). This shell can be made up of oxides, metals etc depending on use (27). As nanoshells are porous, drug loaded nanoshells can be used for the delivery of specific drug at a targeted site. This can be achieved by attaching antibodies to the outer surface so that the nanoshell-antibody complex can bind to the specific antigen in the body. (26). Nanoshells can be used for cancer therapy (26).

Liquid Crystals

Liquid crystals (LCs) present a photoswitch material. They have the ability to flow while displaying anisotropic properties. They have solid like and liquid like properties (28). Liquid crystals are sensitive to temperature, magnetic and electric fields (29). They can be used in targeted drug delivery and topical applications (30). In topical application drug diffusion depends on amount of free water, length of diffusion pathway and arrangement of structural elements (28).


Liposomal drug delivery is a lipid-based drug delivery for the treatment of cancer, inflammation and pain relief (31). It helps to reducing the dose dependent toxicity (32). With the help of liposomes, drugs can be released all over the body. There are some limitations with liposomes, one of them is that since lipids are natural products, the immune system targets them, releasing the drugs too early (31).

Superparamagnetic Nanoparticles

Superparamagnetic nanoparticles are commonly used in the MRI for increasing contrast for imaging inflammation, tumors and degenerative diseases (33). They can also be used for targeted drug delivery and gene transfection. Iron oxides are most frequently used as core particles of superparamagnetic nanoparticles (33). Endorem is an example of a superparamagnetic nanoparticle available in the market as a contrast agent for spleen and liver disease detection. Lumirem is an example of a superparamagnetic nanoparticle available in the market for detection of gastro-intestinal tract imagining (34).


Carbon Nanotubes

Carbon nanotubes are made from a graphite sheet. There are two types of carbon nanotubes, single wall nanotubes (SWNTs) and multiwall nanotubes (MWNTs) (35). Carbon nanotubes are made by chemical vapor deposition, laser ablation of carbon, and carbon-arc discharge (35). There are many applications of carbon nanotubes, for example cells tracking, bio-sensing, drug delivery, chemical sensing, and many more (35; 36; 37). The price of carbon nanotubes depends on the quality and method of production. The price of single wall carbon nanotubes is 50 to 100 times more than gold (38).


Halloysite nanotubes (HNTs) are natural nanotubes, very inexpensive and available in large quantities. Carbon nanotubes cost approximately $500 per gram while halloysite cost approximately $500 per ton (39). As halloysites have larger surface area, they can be used for drug loading. Drugs of smaller molecular size will be trapped within the inner lumen of the halloysites, and drugs of larger molecular size attache to the outer surface of halloysites (40). Other potential applications of halloysites include antifouling paint, fragrance, slow-release perfume, antiscalants, pesticides, pest repellents, slow release of cosmetics, in electronic industry and radiofrequency shielding (39). Thus, large surface area and low cost make halloysites superior compared to all other nanoparticles and nanotubes.

Nanomodified Surfaces

Surface properties of the scaffolds are very important as cell-surface interactions are dependent on the surface property of the scaffold. Cell-surface interactions impel protein adsorption and finally lead to cellular interaction (41; 42). Protein adsorption relies upon the structure of the protein and the surface property (41). Scaffolds used in tissue engineering should resemble natural, extra cellular matrices (ECM) to increase implant biocompatibility (43). An important surface property of scaffolds to consider includes surface roughness, chemistry, hydrophilicity, and hydrophobicity. Natural bone consists of organic compounds strengthened with inorganic compounds (44). Collagen fibers are the major structure seen at nano-scale in the bone. 90% of organic compound is collagen and the rest is noncollageneous protein and ground substances (44). So if the scaffold has nanopores or nanofibers at the surface, it will resemble natural extra cellular matrix. According to some studies, adsorption of particular proteins increases subsequent cell adhesion to the material surface, for example fibronectin. Calcium phosphate crystals (CaP) are the key feature determining Osseo-integration to surrounding bone tissue (45). Growth factors mediate the differentiation of osteoblasts from immature, non-calcium depositing cells to mature calcium depositing cells (46).

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They are many types of scaffolds. They can be mainly subdivided in two types: bio-resorbable and non-bio-resorbable. Scaffolds such as Poly(e-caprolactone )(PCL), poly(d, l-lactide) PLA, Poly(lactide-co-glycolide) PLGA are all examples of FDA approved bio-resorbable polymers, and titanium is an example of a non bio-resorbable scaffold (43).

The electrospinning technique is used for fabrication of the polymeric electrospun scaffold. This technique also allows the incorporation of nanoparticles, enzymes, proteins and chromophores directly during the preparation process. The application of these biohybrid nanosystems is extremely wide, for example drug delivery, catalysis, and biosensors (47). Titanium has good elastic modulus and mechanical properties similar to that of natural bone under a load bearing condition (44; 48). Smooth titanium is a not sufficient bioactive to form a direct bonding with bone, which leads to implant failure. So surface modification should be done to create porous material by sand blasting or plasma spraying techniques (49). With the help of plasma spraying, hydroxypatite (HA) or other calcium phosphates can be used for coating titanium. These coatings have long term failures due to weak adhesion to the metal substrate and dissolution once implanted (44; 49). These problems can be overcome by the anodization process, where we can produce micro and/or nano pores on the surface of the titanium implant which helps in biological fixation to the surrounding tissue through bone tissue.

Tissue Engineering in Dental and Orthopedic

Tissue engineering is based on the materials science and biocompatibility. It incorporates cells, natural or synthetic scaffolds to create new tissues (50). This field is increasingly being viewed as having enormous clinical potential. At present, the treatment of lost or deficient tissues includes prosthetic materials, drug therapies, and tissue and organ transplantation (51). Nevertheless, all of these treatments have some type of limitations, for example failure of synthetic prostheses to replace any simplest structural functions of a tissue. Each year many patients are registered on transplantation waiting lists (52). These problems have motivated the development of tissue engineering. Tissue engineering will have a significant effect on dental and orthopedic practice in the future, for example, in the repair and replacement of mineralized tissues, support of oral wound healing and the use of gene transfer (51).

In the United States each year, over half a million people undergo total joint replacement (53). The average lifespan of a reconstructive joint implant is approximately 15 years (54). In all likelihood this means that each patient will have to undergo a second surgery to maintain functionality (54). There are many drawbacks with replacement surgeries, such as inferior recovery compared to the initial surgery, postsurgical complications and pain. The most common explanation for the implant failure is improper growth on the implant surface (44). Currently, vanadium, cobalt, chromium and smooth titanium are used in dental and orthopedic implants (44). Out of all these metals, titanium is most frequently used due to its tensile strength and corrosion resistance (55; 56; 57). But the problem with titanium implants is that it does not mimic the natural bone structure. So there is a higher chance of implant failure (58). Natural bone is nanoporous at the surface (44). So if we modify the surface of titanium such that it becomes nanoporous, this may help in increasing the life span of the implant. So one of the objectives is to produce nanoporus titanium by the process of anodization.

Nanotechnology in Drug Release

Controlled drug delivery is one of the most promising biomedical applications of nanotechnology. The use of nanomaterials as nanocarriers for improving delivery methods has shown to be advantageous technically and viable economically (59). The Present generation of drugs is given systemically.  Some of the harmful effects and problems of systemic drug delivery include drug solubility, metabolism and excretion of drugs, trouble in maintaining drug concentrations and toxicity to nontarget tissues (60). Use of nanotechnology in drug delivery methods may diminish current problems in drug delivery (60). Carbon nanotubes, halloysite nanotubes, superparamagnetic nanoparticles, liposomes and nanoshells can be used for targeted drug delivery (61). Drugs can be encapsulated in any of these nanoparticles and nanotubes, and used for drug delivery (62). This encapsulation protects drugs from metabolism or excretion (63). This system can also be use in an internal and external signal triggered release. Examples of external signals include magnetic field, ultrasound, infrared light and radiofrequency. pH is an example of an internal signal (64).

Controlled release of antibiotics and antiseptic drugs from halloysite PCL scaffolds can be used for wound healing. The basic unit of healing in any tissue type (for example bone or skin) is the same. The second objective of this project is to electrospin the PCL-halloysite scaffold, to find the best concentration and the exact location of the halloysite in the PCL-halloysite scaffold by Fluorescein isothiocyanate (FITC) labeling of the halloysite and to check its biocompatibility. The third objective of this project is to check the drug released from the drug loaded halloysite, PCL scaffold and halloysite-PCL scaffolds.

Project Aim

1. To find out the best parameter of anodization to produce nanoporous titanium. Compare osteoblast cell proliferation and differentiation on smooth versus nanoporous titanium surfaces. Nanoporous surfaces should lead to better cell proliferation and differentiation leading to enhanced implant durability and osteointegration for patients with degenerative joint problems, as the surfaces are similar to natural bone surface.

2. To electro-spin the halloysite-PCL scaffold and find the best concentration and the exact location of the halloysite in the halloysite-PCL scaffold by Fluorescein isothiocyanate (FITC) labeling of the halloysite, compare osteoblast cell proliferation and differentiation on PCL and halloysite-PCL scaffolds.

3. To load halloysite nanotubes with drugs, for example antibiotics and antiseptic, measure the drug released from the halloysites, PCL scaffolds and halloysite-PCL scaffolds.


This chapter describes the major topics of this dissertation. Nanofabrication of scaffolds can be done with different techniques (65). These techniques can create unordered or ordered surface. Colloidal lithography, chemical etching, polymer demixing and electrospinning are example of nanofabrication technique that create unordered feature (65; 66). Photolithography and electron beam lithography are examples of nanofabrication technique that create ordered feature (65; 66). Nanomodified surfaces resemble the nanoscale features presented to cells by extracellular matrix proteins. Collagen is major extracellular matrix proteins; it has specific nanotopography (66).

Tissue engineering has emerged as superior therapeutic option currently available in regenerative medicine. Tissue engineering includes the use of cells and biomolecules in artificial implants, which can compensate for body functions that have been lost or damage due to disease or accidents (12; 8). Tissue engineering is based upon scaffold-guided tissue regeneration (67). This process includes the seeding scaffolds with cells, which then differentiate and produce tissue mimicking natural tissues (68). Once tissue engineered constructs are ready; they are implanted into the patient to replace unhealthy or injured tissues (68). As time passes, host tissues invade scaffold with blood vessels and nerves; at the end scaffolds are dispersed. Few examples of aapplications of tissue-engineering are engineering of cartilage, bone and skin for autologous implantation (4; 69). There are some implants which are not biodegradable for example titanium. Successful clinical use of resorbable, non-resorbable, bioinert and bioactive implants will significantly advance medical needs (70; 71; 72).

In this study we are using two different types of scaffolds: anodized titanium and halloysite-PCL scaffolds. Anodized titanium can be used in orthopaedic and dental implants. Halloysite-PCL scaffold can be used for drug delivery, wound healing and tissue engineering.

Titanium Implants

Titanium implants are used in dental and orthopedic application (73). It is very difficult to produce properties of bone in synthetic implants (74). These synthetic implants do not give satisfactory results; the average life of a knee, hip or ankle implant is 10 to 15 years (75). Accordingly, patients need to undergo repeated implant surgery. The reason for implant fail is, that implants are unable to produce a sufficiently strong cellular response and so integrate the implant into surrounding tissue (76). In this study, we examined if nanoporous titanium, manufactured by anodization, helps improve an implant's life span or not. Nano means one billionth (). Nano-titanium has a unique property due to nano pores on it; these provide improved magnetic, electric properties and better structural integrity leading to a better cellular response (76; 77; 78).

Anodization of Titanium

An oxide layer forms on the anode surface, when a constant voltage is applied between the anode and cathode (79). The chemical reactions for anodizing titanium are as below. Figure 2.1 shows anodization set up.

Chemical reaction at Ti and Ti oxide interface

Ti ↔ Ti2+ +2e-

Chemical reaction at Ti oxide and electrolyte interface

2H2O ↔ 2O2-+ 4H+

Chemical reaction at both interfaces:

Ti2+ + 2O2- ↔ TiO2 + 2e-

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Figure 2. Anodization set up (80)

As titanium oxide have more resistance compared to the electrolyte and the metallic substrate, the voltage will drop over the titanium oxide film on the anode (79). Provided the voltage applied is sufficiently strong to drive the ion conduction through the oxide layer, the oxide film will keep on growing. Thus the final oxide film thickness is almost linearly dependent on the applied voltage (79).

Anodization of titanium can be done with different chemicals, for example, in H2SO4/HF solutions, phosphate electrolytes, chloride-ion-containing media, acid/ethanol mixture (81; 82; 83; 84). Titanium has corrosion resistant properties due to its titanium oxide layer (85). This layer is formed automatically when titanium is exposed to the air (86). Still, this layer is not reactive enough, so it is unable to form a direct bonding with the bone (87). Due to this insufficient osseointegration, the implant life is very short. So here we are trying to improve surface properties of implants by doing anodization to make nanoporous similar to natural bone (85).

Effects of processing parameters

After the anodization process, morphology, chemistry and roughness of oxide film formed differ depending on the processing parameter used. These processing parameters are current density, voltage, pH, electrolyte composition, and temperature (44). Different types of acids, alkaline solutions and neutral salts are used as electrolytes for the anodization process. Commonly, it was observed that compared to all the electrolytes the titanium oxide layer thickness in H2SO4 was the highest (44).

Anodized oxide film

Structure and properties of anodic oxide films depends on the processing parameters. Properties of anodic oxide films include surface roughness and porous texture with cracks on it. After anodization, thickness of the protective oxide layer increases and it could lead to less ion release in the human body. The oxide barrier layer is considered to contribute to the improvement of corrosion resistance.

Chemical properties

Currently, many different implant materials are under investigation, including titanium, ceramics, polymers, and biologically synthesized substances. Compared to all material, titanium is the best material because it is more biocompatible, durability and corrosion resistance. Still, according to previous studies, metal such as smooth titanium failed to produce proper osseointegration (78).

Need for long lasting and better quality implants

Current implants do not help in changing bone mass that occurred due to osteoporosis and fractures. The average life of these implants is 10 to 15 years. The life time of the implant is very short for babies and young people. Bone implants replace missing bone and provide surface where bone and a vascular network can be regenerated and better osseointegration can occur. Once the implant is implanted at the injured site, protein from blood, bone marrow and other tissues for example, fibronectin and vitronectine, starts adhering to the implant surface. These proteins then control subsequent cell adhering. Protein bound to the implant surface osteoblast cells then binds to the implants. Thus it very important to have a proper surface where initial protein adsorption occurs. Here chemical and physical properties of the implant's surface determine the initial adsorption of the protein. So the goal is to produce implants with such chemical and topographic properties, that can solve current problems in orthopedic and dental implants (76; 77; 78).

Osteoporosis and implants

Osteoporosis is a bone disorder resulting in reduced bone density and bone strength. Osteoporosis can be asymptomatic. Symptoms and problems of osteoporosis include fractures, pain, and deformity. Osteoporosis is usually found in the aged population, especially women, who are very prone to this disorder. Implants that provide better structural integrity by providing quicker and better cellular response are very useful for repairing a fracture in osteoporosis patients (76; 78). Anodized titanium may prove useful in generating faster and better cellular response in osteoporosis and aged people.

Electrospinning PCL-Halloysite Scaffolds

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Figure 2. Electrospinning set up (86)

Scaffolds of micro and nano scale polymer fibers can be fabricated by placing a polymer solution to an electric field. This technique is known as electrospinning. The electrospinning set up is shown in figure 2.2. The electrospining set consists of a syringe pump, a high voltage source and a collector. Surface tension holds a polymer solution at the tip of the needle. Higher voltage in the needle causes induction of charges in the polymer solution. The polymer jet is formed when the charge repulsion within the solution overcomes the surface tension (87; 88; 89). Fluid jet experience instability when jet leaves the tip of the needle. This results in the bending of the fluid jet due to applied electric field (90). During the polymer jet tavel path, the evaporation of the solvent occurs, and the fibers are collected on a collector plate. Solutions with higher conductivity have more tendencies for jet formation (91; 92; 93). In this study we are trying to electro spin halloysite-PCL scaffold and drug loaded halloysite-PCL scaffold.

Parameters affecting electrospinning

There are different parameter, which can affect the diameter of the electrospun fiber for example distance between needle tip and the collection screen, rate of syring pump, voltage applied and solution parameters. Solution parameters includes weight percent of polymer, weight percent of solvent, molecular weight of polymer, viscosity and surface tension. There are some other paramerts which do have some effect on fiber morphology, this includes air flow, humidity and temperature.

Drug delivery

Drug delivery, is one of the promises of nanotechnology. The use of nanomaterial's as nanocarriers for improving the delivery methods has been advantageous technically and viable economically. These nanocarriers, formed by the process of nanoencapsulation, are of two types: named as nanospheres and nanocapsules. Halloysite is an example of nanocapsules (94).


Halloysite is a nanotubular clay particle. It is mined from natural deposits in different countries. Utah has the largest deposit in the USA. Halloysite is a two-layered aluminosilicate. It has a hollow tubular structure in the submicron range. Halloysite is chemically similar to kaolinite (94; 95; 96; 97). It is formed from kaolinite over millions of years due to the action of weathering and hydrothermal processes. Layers are rolled into nanotubes due to the strain caused by lattice mismatch between adjacent silicone dioxide and aluminum oxide sheets (94; 95; 96).

Chemical Composition and Structure of halloysite

Halloysite occurs in nature as a hydrated mineral. It is also called halloysite, due to its thickness, which is close to 10 A. Heating halloysite (10A) can easily and irreversibly dehydrate to form halloysite (7A). The chemical formula of halloysite is Al2Si2O5(OH)4.2H20, which is similar to kaolinite, except for the presence of an additional water monolayer between the adjacent layers. Figure 2.3 shows the halloysite nanotubes. The outside diameters of the halloysite vary from 40 to 190 nm. The average outside diameter is 70 nm. The diameters of the internal lumen vary from 15 to 100 nm. The average inner diameter is 40 nm. The lengths of halloysite vary from 1 to 20 µm (96; 98; 99; 100). The morphology of halloysite nanotubes vary depending on their geological occurrences and crystallization conditions. Chemical compositions of different morphologies are also different (99).

Physical and Chemical Properties of halloysite

Halloysite minerals have relatively high specific surface areas. Surface areas vary from 50 to l40 m2g'1 with deposit type. Halloysites are abundant with narrow cylindrical pore. Due to this reason they can absorb a relatively big class of compounds. They include inorganic and organic salts as well as polymers and biologically active agents (96; 99; 101). Absorption of small organic molecules and salts primarily takes place by their intercalation into an interlayer space (101; 102; 103), while big polymer molecules like proteins and drugs are bound to its outer and inner faces. Intercalation does not occur with bigger molecules like proteins, polymers and other macromolecules due to their large molecular size (95; 99). The organic compounds that have small molecular sizes and hydrophilic functional groups such as OH and NH2 were observed to form an intercalation complex with the halloysite nanotube. This is due to the formation of hydrogen bonds between them and alumina or silica layers (97; 102).The adsorptive and ion exchange properties of halloysite nanotubes are greatly affected by their surface charges. Surface charges are pH-dependent. Halloysite has a higher negative surface charge compared with kaolinite showing the predominance of silica properties at the outer surface (94).

Capillary Force

The capillary force helps in better adsorption of several materials. Capillary force is calculated by using the following formula,

h = (2y cosθ) / (pgr)

where, y is the liquid-air surface tension (J/m2 or N/m), p is the density of liquid (kg/m3), g is acceleration due to gravity (m/s2), θ is the contact angle, r is radius of the tube (m) (104).

For a water-filled tube in air at sea level, Y is 0.0728 J/m2 at 20 0C, θ is 20' (0.35 rad), p is 1000 kg/m3 , g is 9.8 m/s2, therefore, the height of the water column is given by:

h = (1.4 x 10 -5)/r

For halloysite nanotubes of average inner radius of 7nm the capillary force in terms of the height of the water column is h - 2000 in. Hence it is understandable that this much higher capillary force helps the halloysite in the quick adsorption of several materials (104; 105).