developing field of nanotechnology for regenerative medicine

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Nanotechnology is fast developing field with wide range of potential applications. Nanotechnology involves understanding and controlling material at nanometer scale (1). Improving lifestyles, an aging population, and over-increasing expectations for a better quality of life demands 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). Application of nanotechnology in regenerative medicine can drastically change the way 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 polymer for controlled drug-delivery helps delivery drug for long period of time and also increase effectiveness of drug (1). Nanoparticles and Nanotubes can be used for drug delivery. Nanoparticles can circulate in body; it can also be taken up by cell through endocytosis. Nanoparticles can also be used for diagnostic purpose. There are some obstacles to transform current drug delivery to therapies for example targeting nanoparticles at specific site without captured organs like liver and spleen (1).

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 are 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 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 halloysite, the drug loaded PCL scaffold and the drug loaded halloysite-PCL scaffold.

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). Couple of examples of potential application of nanoparticles is biosensor and drug carriers (19). Nanoparticles 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 includes 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).

Nanoparticles

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 size of quantum dots (24). The quantum dot can be observed with high resolution compared to fluorophores by electron microscopy (23).

Nanoshells

The nanoshells are nanoparticles, which consist of a dielectric core coated with an metallic layer (25). Nanoshells are produced by layer-by-layer self-assembling and can be used in drug delivery (25).

Liquid Crystals

Liquid crystals (LCs) present a photoswitch material. They have the ability to flow while displaying anisotropic properties. Liquid crystals are sensitive to temperature, magnetic and electric fields (26). They are used in liquid crystal displays (LCDs). They can also be used in targeted drug delivery (27).

Liposomes

Liposomal drug delivery is lipid-based drug delivery for the treatment of cancer, inflammation and pain relief (28). 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, and the immune system targets them releasing the drugs too early (28).

Superparamagnetic Nanoparticles

Superparamagnetic nanoparticles are commonly used in the MRI for increasing contrast for imaging inflammation; tumors and degenerative diseases (29). They can also be used for targeted drug delivery and gene transfection. Iron oxides are most frequently used as core particles of superparamagnetic nanoparticles (29).

Nanotubes

Typically 1-100 nm in length. Nanotubes can be natural or made by generating small cylinders of silicon, gold or inorganic phosphate.

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) (30). Carbon nanotubes are made by chemical vapor deposition, laser ablation of carbon, and carbon-arc discharge (30). There are many applications of carbon nanotubes, for example, cells tracking, bio-sensing, drug delivery, chemical sensing and many more (30; 31; 32).

Halloysites

Halloysite nanotubes (HNTs) are natural nanotubes, very inexpensive and available in large quantities. They can be used for drug loading. Drugs of smaller molecular size will be trapted within the inner lumen of the halloysites, and drugs of larger molecular size attache to the outer surface of halloysites (33).

Nanomodified Surfaces

Cell-surface interactions are important in tissue engineering, as they impel protein adsorption and finally lead to cellular interaction (34; 35). Protein adsorption relies upon the structure of the protein and the surface property (34). An ideal scaffold used in tissue engineering should resemble natural, extra cellular matrices (ECM) to increase implant biocompatibility (36). Natural bone consists of organic compounds strengthened with inorganic compounds (37). 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 (37). An important surface property of scaffolds to consider includes surface roughness, chemistry, hydrophilicity, and hydrophobicity. 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 (38). Growth factors mediate the differentiation of osteoblasts from immature, non-calcium depositing cells to mature calcium depositing cells (39). 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. These are all examples of FDA approved bio-resorbable polymers and titanium is example of non bio-resorbable scaffold (36). 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. Application of these biohybrid nanosystems is extremely wide, for example, drug delivery, catalysis, and biosensors (40).

Titanium has good elastic modulus and mechanical properties similar to that of natural bone under a load bearing condition (37). Smooth titanium is 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, acid etching or plasma spraying techniques (41). These modification techniques help create micro-scale and/ or nano-scale surface this modification also helps in biological fixation to surrounding tissue through bone tissue. 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 (37; 41).

Tissue Engineering in Dental and Orthopedic

In the United States each year, over half a million people undergo total joint replacement (42). The average lifespan of a reconstructive joint implant is approximately 15 years (43). In all likelihood this means that each patient will have to undergo a second surgery to maintain functionality (43). 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 (37). Currently vanadium, cobalt, chromium and smooth titanium are used in dental and orthopedic implants (37). Out of all these metals, titanium is most frequently used due to its tensile strength and corrosion resistance (44; 45; 46). 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 (47). Natural bone is nanoporous at the surface (37). So if we modify the surface of titanium such that it becomes nanoporus, this may help in increasing the life span of the implant. So the first objective is to produce nanoporus titanium by the process of anodization.

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 (48). Tissue engineering builds on the interface between materials science and biocompatibility, and integrates cells, natural or synthetic scaffolds, and helps to create new tissues (49). This field is increasingly being viewed as having enormous clinical potential. At present, the replacement of lost or deficient tissues involves prosthetic materials, drug therapies, and tissue and organ transplantation (48). Nevertheless, all of these treatments have limitations, including the inability of synthetic prostheses to replace any but the simplest structural functions of a tissue. Each year many patients are registered on transplantation waiting lists (50). These problems have motivated the development of tissue engineering.

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 (51). 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 (52). Use of nanotechnology in drug delivery methods may diminish current problems in drug delivery (52). Carbon nanotubes, halloysite nanotubes, superparamagnetic nanoparticles, liposomes and nanoshells can be used for targeted drug delivery (53). Drugs can be encapsulated in any of these nanoparticles and nanotubes, and used for drug delivery (54). This encapsulation protects drugs from metabolism or excretion (55). This system can also be use in internal and external signal triggered release. Examples of external signals include magnetic field, ultrasound, infrared light and radiofrequency. pH is example of internal signal (56).

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 produce drug loaded halloysite-PCL scaffolds and test their effectiveness on bacteria.

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 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 halloysite and document the effect of the drug released from the halloysite-PCL scaffold on bacteria.

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