Nanotechnology for Regenerative Medicine
The improving lifestyle of society, an ageing population and the sky-scraping expectations for a better quality of life urge for better, more proficient and inexpensive health care (1). Use of nanotechnology in regenerative medicine can promise new treatment modalities, when applied to major medical challenges (2). Regenerative medicine is the technique of generating tissues to restore or replace tissue or organ function lost due to birth defects, disease, damage or age(3). This branch assure for rejuvenating damaged tissues and organs by thought-provoking previously irrevocable organs to repair themselves (4). Regenerative medicine also allows growing tissues and organs in the lab to securely implant them when the body cannot repair itself (4). Prominently, regenerative medicine has the capability to solve the problem of the shortage of organs for organ transplantation (5; 6).
Regenerative medicine is a multidisciplinary field (7). Application of nanotechnology in regenerative medicine can drastically change the approach diseases are treated in the future. In the last few decades nanomedicines have started coming onto the market (8). Regenerative medicine can be used to repair, retain or improve tissues and organ functions. Regeneration of tissues can be accomplished by using scaffolds, which support cell proliferation, and cells, which will provide biological functionality (8; 7; 9). In vivo cells respond to the signals they receive from the surrounding environment. These signals are controlled by nanometer-scaled components, so it is very important that the material used produces the correct signal to guide cell growth appropriately (10). Nanotechnology has wide area of application in regenerative medicine (11). Nanotechnology is an excellent tool for producing scaffolds that mimic the biological structures. This technology also offers efficient drug delivery system.
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In this study we are focusing on three different applications of regenerative medicine. Our first aim is 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 is to fabricate halloysite-PCL (poly-Æ-caprolactone) scaffold and assess the cell supportive ability. The final aim is to examine the effect of different drug loaded halloysite-PCL scaffold as potential antibacterial material.
Nanoparticles and Nanotubes for Regenerative Medicine
NanotechnologyÂ is under fast development and new research additions continuously appearing. The recent research in nanomaterials, increasing awareness of material science and tissue engineering. Examples of nanoparticles are buckyballs, liquid crystals, liposomes, nanoshells, quantum dots and supramegnetic nanoparticles. Carbon nanotubes and halloysite nanotubes are example of nanotubes. Potential application of nanoparticles includes biosensor and drug carriers. Nanoparticles can be used as intracellular drug carriers and as biosensor to monitor in real time level of biomolecules, enzymes , cell differentiation. Present methods of judging cell treatment includes invasive and/or destructive methods, for example, tissue biopsies. Thus non-invasive methods are very advantageous. List of conventional non-invasive techniques includes: fluoresce microscopy, positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound. Examples of nanoimaging probes include nanoshells and quantum dots (QD).
Quantum dots are colloidal nanocrystalline semiconductor. Properties of quantum dots depend on their size. Quantum dots can be used as fluorescent probe for labeling. To examine cell and cellular process fluorescence labeling is used. Conventional fluorescence labeling have some limitations, for example, sensitive to thermal changes to local environment, blinking, photobleaching and lower contract using quantum dot that is colloidal metal particles we can overcome these limitation. 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. Quantum dot can be observed with high resolution compared to fluorophores by electron microscopy.
Liquid crystals (LCs) present a photoswitch material. It has ability to flow while displaying anisotropic properties. Liquid crystals are sensitive to temperature, magnetic and electric field. They are used in liquid crystal displays (LCDs). They can also be used in targeted drug delivery.
Liposomal drug delivery is lipid-based drug delivery for treatment cancer, inflammation and pain relief. With the help of liposome drugs can be released all over the body. There are some limitation with liposome one of them is as lipids are natural products, immune system target for destruction due to this drug inside the liposome are released too early.
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The nanoshells are nanoparticles, consist of a dielectric core coated with an metallic layer. Nanoshells produced by layer-by-layer self-assembling and can be used in drug delivery.
Superparamagnetic nanoparticles are commonly used in MRI for increasing contrast for imaging inflammation; tumors and degenerative diseases. It can also be used for targeted drug delivery and gene transfection. Iron oxides are most frequently used as core particle.
Typically 1-100 nm in length. Nanotubes can be natural or made by generating small cylinders of silicon, gold or inorganic phosphate.
Carbon nanotubes are made from graphite sheet. There are two types of carbon nanotubes, single wall nanotubes (SWNTs) and multiwall nanotubes (MWNTs). Carbon nanotubes are made by chemical vapor deposition, laser ablation of carbon and carbon-arc discharge. There are many applications of carbon nanotubes for example, utilized for cells tracking, biosensor, drug delivery, chemical sensor and many more.
Halloysite nanotubes (HNTs) are natural nanotubes, available in large quantities and very inexpensive. It can be used for drug loading. Drug of smaller molecular size entrapment within the inner lumen of the halloysites and drug of larger molecular size attaches to the outer surface of halloysite.
Cell-surface interaction is important in tissue engineering, as it impel protein adsorption and finally leads to cellular interaction. Protein adsorption relies upon on structure of the protein and thesurface property. An ideal scaffold used in tissue engineering should have resemblance to natural extra cellular matrices (ECM) to increase implant biocompatibility. Important surface property to consider includes surface roughness, chemistry, hydrophilicity and hydrophobicity. According to some studies adsorption of particular proteins increases subsequent cell adhesion on material surface, for example, fibronectin. Calcium phosphate crystals (CaP) are key feature for determine oseeo-integration to surrounding bone tissue. Growth factors mediate the differentiation of osteoblasts from immature, non-calcium depositing cells to mature calcium depositing cells. They are many types of scaffolds. They can be mainly subdivided in two types' bioresrobable and non-bioresrobable, PCL, PLA, PLGA are example of FDA approved bioresorbable polymer and titanium is example of non-bioresrobable polymer. Electrospinning technique is used for fabrication of polymeric electrospun scaffold also allows the incorporation of nanoparticles, enzymes, protein and chromophores, directly during preparation process. Application of these biohybrid nanosystem is extremely wide for eample, drug delivery, catalysis and biosensors.
Titanium has good elastic modulus and mechanical properties similar to that of natural bone under a load bearing condition. 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. These modification techniques help create micro-scale and/ or nano-scale surface this modification 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. Theses coating have long term failures due to weak adhesion to the metal substrate and dissolution once implanted.
Tissue Engineering in Dental and Orthopedic
It is predicted that tissue engineering will have a considerableÂ effect on dental practice during the next 25 years. The greatestÂ effects will likely be related to the repair and replacementÂ of mineralized tissues, the promotion of oral wound healingÂ and the use of gene transfer adjunctively. Tissue engineering buildsÂ on the interface between materials science and biocompatibility,Â and integrates cells, natural or synthetic scaffolds, and specificÂ signals to create new tissues.Â This field is increasingly beingÂ viewed as having enormous clinical potential.
Clinical problems relating to the loss and/or failure of tissuesÂ extend beyond dentistry to all fields of medicine, and are estimatedÂ to account for approximately one-half of all medical-relatedÂ problems in the United States each year. Currently, the replacementÂ of lost or deficient tissues involves prosthetic materials,Â drug therapies, and tissue and organ transplantation. However,Â all of these have limitations, including the inability of syntheticÂ prostheses to replace any but the simplest structural functionsÂ of a tissue. An extreme shortage of organs and tissues for transplantationÂ exists. Fewer than 10,000 organs are available for transplantationÂ each year in the United States, while more than 50,000 patientsÂ are registered on transplantation waiting lists.Â Such problemsÂ have motivated the development of tissue engineering, whichÂ can be defined as a "combination of the principles and methodsÂ of the life sciences with those of engineering to develop materialsÂ and methods to repair damaged or diseased tissues, and to createÂ entire tissue replacements."
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Many strategies have evolved to engineer new tissues and organs,Â but virtually all combine a material with either bioactive moleculesÂ that induce tissue formation or cells grown in the laboratory.Â The bioactive molecules are frequently growth factor proteinsÂ that are involved in natural tissue formation and remodeling.Â The basic hypothesis underlying this approach is that the localÂ delivery of an appropriate factor at a correct dose for a definedÂ period of time can lead to the recruitment, proliferation andÂ differentiation of a patient's cells from adjacent sites.Â These cells can then participate in tissue repair and/or regenerationÂ at the required anatomic locale.
The second general strategy uses cells grown in the laboratoryÂ and placed in a matrix at the site where new tissue or organÂ formation is desired. These transplanted cells usually are derivedÂ from a small tissue biopsy specimen and have been expanded inÂ the laboratory to allow a large organ or tissue mass to be engineered.Â Typically, the new tissue will be formed in part from theseÂ transplanted cells.
With both approaches, specific materials deliver the moleculesÂ or cells to the appropriate anatomic site and provide mechanicalÂ support to the forming tissue by acting as a scaffold to guideÂ new tissue formation.Â Currently, most tissue engineering effortsÂ use biomaterials already approved for medical indications byÂ the U.S. Food and Drug Administration, or FDA. The most widelyÂ used synthetic materials are polymers of lactide and glycolideÂ , since these are commonly used forÂ biodegradable sutures. Both polymers have a long track recordÂ for human use and are considered biocompatible, and their physicalÂ properties (for example, degradation rate, mechanical strength)Â can be readily manipulated. A natural polymer-type 1 collagen-isÂ often used because of its relative biocompatibility and abilityÂ to be remodeled by cells. Other polymers familiar to dentistry, including alginate, are also being used.
Bone and cartilage generation by autogenous cell/tissue transplantation is one of the most promising techniques in orthopedic surgery and biomedical engineering . Treatment concepts based on those techniques would eliminate problems of donor site scarcity, immune rejection and pathogen transfer . Osteoblasts, chondrocytes and mesenchymal stem cells obtained from the patient's hard and soft tissues can be expanded in culture and seeded onto a scaffold that will slowly degrade and resorb as the tissue structures grow in vitro and/or vivo . scaffold or three-dimensional (3-D) construct
provides the necessary support for cells to proliferate and maintain their di!erentiated function, and its architecture the ultimate shape of the new bone and cartilage. Several scaffold materials have been investigated for tissue engineering bone and cartilage including hydroxyapatite (HA), poly(a-hydroxyesters), and natural polymers such as collagen and chitin. Several reviews have been published on the general properties
and design features of biodegradable and bioresorbable polymers and scaffolds [4,12].
In the United States each year, over half a million people undergo total joint replacement (14). The average lifespan of a reconstructive joint implant is approximately 15 years. In all likelihood this means that each patient will have to undergo a second surgery to maintain functionality (15). There are many drawbacks with replacement surgeries such as inferior recovery compared to the initial surgery, postsurgical complications and pain (16). The most common explanation for implant failure is improper growth on the implant surface (17). Currently vanadium, cobalt, chromium and smooth titanium are used in dental and orthopedic implants. Out of all these metals, titanium is most frequently used due to its tensile strength and corrosion resistance (13; 18; 19). But the problem with titanium implants is that it does not mimic the natural bone structure. So there are higher chances of implant failure (20). Natural bone is nanoporous at the surface. 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.
Nanotechnology for Bioactive Molecule and 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. Controlled release of antibiotics and antiseptic drug from halloysite PCL scaffold 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 PCL-halloysite scaffold, find the best concentration and the exact location of halloysite in the PCL-halloysite scaffold by Fluorescein isothiocyanateÂ (FITC) labeling of halloysite and check its biocompatibility. The third objective of this project is to produce drug loaded halloysite-PCL scaffold and test it effectiveness on bacteria.
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 it is similar to natural bone surface.
2. To electro-spin halloysite-PCL scaffold and find the best concentration and the exact location of halloysite in the halloysite-PCL scaffold by Fluorescein isothiocyanateÂ (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.