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Posts Tagged ‘nanostructure’


Introduction to Tissue Engineering; Nanotechnology applications

Author, Editor and Curator:  Tilda Barliya, PhD

 

Tissue Engineering is an emerging multidisciplinary field involving biology, medicine, and engineering that is likely to revolutionize the ways we improve the health and quality of life for millions of people by restoring, maintaining, or enhancing tissue and organ function. Tissue engineering emerged as organ transplantation is limited by the number of  available donors and high cost process, leaving thousands of people each year on the transplant waiting lists in the United States alone. Many die before an organ donor becomes available. Dr. Tal Dvir from the Langer’s lab at MIT have summarized this topic in his review (2. http://nextbigfuture.com/2011/01/nanotechnology-strategies-for-tissue.html)

Tissue engineering aims at developing functional substitutes for damaged tissues and organs, a process that involves the use of a combination of cells, engineering and material methods, including suitable biochemical and chemical factors to improve or replace biological functions. Rather than simply introducing cells into a diseased area to repopulate a defect and/or restore function, in tissue engineering the cells are often seeded in or onto biomaterials (scaffolds) before transplantation.

These biomaterial scaffold allows cells to attach and reorganize to form functional tissue by proliferating, synthesizing extracellular matrix, and migrating along the implant path (1,2,3) Figure 1.

Until recently, it was believed that the macroporous features of scaffolds used in tissue engineering mimicked the dimension scale of the extracellular matrix (ECM), and that the matrix itself (natural or artificial) only served as a support for the cells; morphogenesis was controlled passively by defining tissue boundaries. Emphasis was placed on critical engineering and material issues, such as improving mass transfer into the core of the cell constructs and designing biocompatible and biodegradable scaffolds with mechanical properties suitable for engineering various tissues. As the field evolved, attention focused on the biology of the scaffolds and how they affect various cell types.

Tissue engineers had recognized that some of the widely used scaffolds do not fairly recapitulate the cell microenvironment and that the ECM is a dynamic and hierarchically organized nanocomposite that regulates essential cellular functions such as:

  • morphogenesis,
  • differentiation
  • proliferation
  • adhesion
  • migration

Nanotechnological tools for tissue engineering may help design advanced nanocomposite scaffolds that can better mimic the ECM and eventually assemble more complex and larger functional tissues. In order to generate a functional tissue, effective organization of cells in the tissue is required with similar morphology and physiology of the parental tissue.

Morphogenesis in the three-dimensional (3D) scaffold should occur in a similar way to natural organ development. The cells reorganize owing to interaction with the ECM on the basis of:

  • topography,
  • mechanical properties (such as matrix stiffness, elasticity and viscosity)
  • concentration gradients of immobilized growth factors
  • ECM molecules.

Recently, Ott and co-workers (4) reported a study emphasizing the importance of the ECM structure in guiding the seeded cells and promoting morphogenesis. Rat hearts were decellularized by perfusion of detergents to preserve the underlying ECM and then reseeded with cardiac and endothelial cells (4). The cells migrated and self-organized in their natural location in the matrix and by day 8, under physiological load and electrical stimulation, the constructs were able to generate pump function (4). The importance of the ECM was shown for:

  • Heart
  • Lung
  • Arteries
  • Liver
  • Bone
  • Nerve

So why is the Extracellular Martix (ECM) so important?

The ECM is composed of an intricate interweaving of protein fibres such as fibrillar collagens and elastins, ranging from 10 to several hundreds of nanometres. The mesh is covered with nanoscale adhesive proteins such as laminin and fibronectin that provide specific binding sites for cell adhesion (interacting with integrins, cadherins and so forth) and have been shown to regulate important cell behaviours such as growth, shape, migration and differentiation. Polysaccharides such as hyaluronic acid and heparan sulphate fill the interstitial space between the fibres and act as a compression buffer against the stress placed on the ECM or serve as a growth factor depot (Figure 2).

Scaffold design considerations

The ECMs of various tissues in the body differ in the composition and spatial organization of the collagens, elastins, proteoglycans and adhesion molecules, to maintain specific tissue morphologies and organ specific shape and function, and to supply specific instructive cues. Therefore, the design considerations for scaffolds should vary according to the desired engineered tissue. For example, the biochemical, electrical and mechanical functions of the heart are uniquely dependent on their biological nanostructures. The heart’s 3D ECM network is composed of an intricate, micro- and nanoscale interweaving pattern of fibrillar collagen and elastin bundles that form a dense, elastic network with proteoglycans and with adhesive and non-adhesive molecules. In this defined mesh, the cardiomyocytes are forced to couple mechanically to each other, to form elongated and aligned cell bundles that interact with each other or with neighbouring capillaries and nerves.

Post-isolation cells lose their ultrastructural elongated morphology and their interaction with their surroundings, and they adopt a random distribution on the flat surface of the scaffold, which compromises many of their physiological properties. Therefore, the structure and support of the ECM is crucial. See Figure 2.

Limitations of the ECM:

  • Weak mechanical properties
  • Lack of electrical conductivity
  • Absence of adhesive and micoenvironment- defining moieties
  • Inability of cells to self-assemble to 3D tissue structure.

The rational behind incorporating nanostructures is to compensate for other scaffold limitations (Table 2) Ref.2

The Heart for example requires more than alignment and mechanical support (Boyang Zhang, Ref 5)

  1. cell responses to micro- and nanopatterned topographical cues
  2. cell responses to patterned biochemical cues
  3. controlled 3D scaffolds
  4. patterned tissue vascularization
  5. electromechanical regulation (conductivity). of tissue assembly and function

Nanostructures can be used to record the electronic signals that are transmitted through cells such as neurons and cardiomyocytes. One way to record these signals is by lithographically defining nanostructures as field-effect transistors, which are sensitive to local electric field changes. In particular, silicon nanowire transistors are useful for measuring extracellular signals because they exhibit particularly exquisite field-effect sensitivity compared with conventional, planar devices; they are just tens of nanometres in diameter and can therefore interface with cells and tissue at a subcellular level; and they show nanotopographic features that encourage tight interfaces with biological systems.

 

Summary:

This introduction reviewed some of the aspects required for tissue engineering  with the affiliation to nanotechnology. In the next post, we will dive deeper into a specific tissue organ, the bioengineering aspect and how nanotechnology strategies may improve the design and outcome.

 

Ref

1. http://www.nanotech-now.com/news.cgi?story_id=35168

2.  Dvir T.,  Timko BR., Kohane DS., and Langer R. Nanotechnological strategies for engineering complex tissues. Nature Nanotechnology 2010; 12():. http://nextbigfuture.com/2011/01/nanotechnology-strategies-for-tissue.html

3. http://www.nature.com/nnano/journal/v6/n1/abs/nnano.2010.246.html

4.  Ott, H. C. et al. Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartificial heart. Nature Med. 14, 213–221 (2008).

5. Boyang Zhang, Yun Xiao, Anne Hsieh, Nimalan Thavandiran and Milica Radisic. Micro- and nanotechnology in cardiovascular tissue engineering. Nanotechnology 2011; 22(49): 494003

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Author: Tilda Barliya PhD.

I recently read this beautiful paper by Fredrik Nederberg from the IBM Almaden Research Center  and A*STAR Institute of Bioengineeringtitled “Biodegradable nanostructures with selective lysis of microbial membranes” (http://www.nature.com/nchem/journal/v3/n5/full/nchem.1012.html)

This paper gained a lot of attention as it merged as an innovation in nanotechnology and antibacterial therapeutics and therefore I have decided to introduce it here to the audience.

“Bacteria are increasingly resistant to conventional antibiotics and, as a result, macromolecular peptide-based antimicrobial agents are now receiving a significant level of attention. Most conventional antibiotics (such as ciprofloxacin, doxycycline and ceftazidime) do not physically damage the cell wall, but penetrate into the target microorganism and act on specific targets (for example, causing the breakage of double-stranded DNA due to inhibition of DNA gyrase, blockage of cell division and triggering of intrinsic autolysins). Bacterial morphology is preserved and, as a consequence, the bacteria can easily develop resistance. In contrast, many cationic peptides (for example, magainins, alamethicin, protegrins and defensins) do not have a specific target in the microbes, and instead interact with the microbial membranes through an electrostatic interaction, causing damage to the membranes by forming pores in them3. It is the physical nature of this action that prevents the microbes from developing resistance to the peptides. Indeed, it has been proven that cationic antimicrobial peptides can overcome bacterial resistance”.

“Most antimicrobial peptides have cationic and amphiphilic features, and their antimicrobial activities largely depend on the formation of facially amphiphilic a-helical or b-sheet-like tubular structures when interacting with negatively charged cell walls, followed by diffusion through the cell walls and insertion into the lipophilic domain of the cell membrane after recruiting additional
peptide monomers. The disintegration of the cell membrane eventually leads to cell death. Over the last two decades, efforts have been made to design peptides with a variety of structures, but there has been limited success in clinical settings, and only a few cationic synthetic peptides have entered phase III clinical trials. This is largely due to the cytotoxicity (for example, haemolysis) resulting from their cationic nature, their short half-lives in vivo (they are labile to proteases) and their high manufacturing costs”

A number of cationic polymers thatmimic the facially amphiphilic structure and antimicrobial functionalities of peptides have been proposed as a better approach, because they can be prepared more easily and their synthesis can be more readily scaled up compared with peptides.

The authors Yang, Hedrick and their co-workers have developed a polymer-based peptide alternative which avoids all of these problems. The polymer incorporates three key components: a non-polar hydrophobic head and tail, which drives the polymer to self-assemble into a nanoparticle; a positively charged block that selectively interacts with the bacterial cell membrane; and a carbonate backbone that slowly breaks down inside the cell, ensuring good biocompatibility. “The starting materials of our synthesis are inexpensive, and the synthesis of the antimicrobial nanoparticles is simple and can be scaled up easily for future clinical application.

“Polycarbonates are attractive biomaterials because of their biocompatibility, biodegradability, low inherent toxicity and tunable mechanical properties”

 

In general, Preclinical results confirm that the nanoparticles can efficiently kill fungi and multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), even at low concentrations. The nanoparticles also showed insignificant activity against red blood cells, and no significant toxicity was observed during the in vivo studies in mice, even at concentrations well above their effective dose.

In more specific, the authors evaluated the minimal inhibitory concentrations (MICs) of the polymers against Gram-positive bacteria such as Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and the fungus Cryptococcus neoformans. MIC is an important parameter commonly used to evaluate the activity of new antimicrobial agents, and is generally defined as the minimum concentration of an antimicrobial agent at which no visible growth of microbes is observed

Some of the MIC were evaluated against conventional antimicrobial agents that are used in clinical settings to treat infections caused by these microbes, such as vancomycin for S. aureus, MRSA and E. faecalis, and amphotericin B for C. neoformans.  When compared with these conventional antimicrobial agents, the polymers demonstrated comparable antimicrobial activities against all the microbes except for E. faecalis. This is important, because vancomycin-resistant E. faecalis, and S. aureus, as well as amphotericin B-resistant C. neoformans have been reported, and the resistant strains of these microbes are not susceptible to conventional antimicrobial agents. This suggests that there is an urgent need to develop safe and efficient macromolecular antimicrobial agents.

The hypothesis was that the cationic micelles can interact easily with the negatively charged cell wall by means of an electrostatic interaction, and the steric hindrance imposed by the mass of micelles in the cell wall and the hydrogen-binding/electrostatic interaction between the cationic micelle and the cell wall may inhibit cell wall synthesis and/or damage the cell wall, resulting in cell lysis. In addition, the micelles may easily permeate the cytoplasmic membrane of the organisms due to the relatively large volume of the micelle, destabilizing the membrane as a result of electroporation and/or the sinking raft model, leading to cell death.

Haemolysis is a major harmful side effect of many cationic antimicrobial peptides and polymers. The haemolysis of mouse red blood cells was evaluated after incubation with polymers 1 and 3 at various concentrations. Although the polymers disrupt microbial walls/membranes efficiently, they do not damage red blood cell membranes.

Toxicity tests in vivo showed that the micelles do not cause significant acute damage to liver and kidney functions, nor do they interfere with the electrolyte balance in the blood. Importantly, these parameters remain unchanged, even at 14 days post-injection.

In addition, no mouse treated with the polymer died, and no colour change was observed in the serum samples and urine of the mice treated with the polymer when compared with the control group. These findings demonstrate that the polymer did not induce significant toxicity to the mice during the period of testing. Nonetheless, preclinical studies should be conducted in the future to further evaluate potential long-term toxicity of the antimicrobial polymers before clinical applications.

In summary, the authors  have designed and synthesized novel biodegradable, cationic and amphiphilic polycarbonates that can easily self-assemble into cationic micellar nanoparticles by direct dissolution in water. The cationic nanoparticles formed from the polymers, with optimal compositions, can efficiently kill Gram positive bacteria, MRSA and fungi, even at low concentrations. Importantly, they have no significant haemolytic activity over a wide range of concentrations, and cause no obvious acute toxicity to the major organs and the electrolyte balance in the blood of mice at a concentration well above the MICs. These antimicrobial polycarbonate nanoparticles could be promising as antimicrobial drugs for the decolonization of MRSA and for the treatment of various infectious diseases, including MRSA associated infections.

The data presented by the authors is very promising and open a new door to antimicrobial therapy. Several questions and new avenues comes in minds:

  • Can these polymers be proven for there efficiency in specific disease animal models?
  • Can these NPs or similar approach can be applied to gram-negative bacteria?
  • Can these polycarbonate  affect massive biofilms?!

Looking forward to reading more news and results from this research group.

 

 

 

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