Nanotechnology in Tissue Engineering

C H A P T E R

7 Nanotechnology in Tissue Engineering Neha Maheshwari1, Muktika Tekade1, Yashu Chourasiya2, Mukesh Chandra Sharma1, Pran Kishore Deb3 and Rakesh K. Tekade4 1

School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshila Campus, Indore, India Department of Pharmacology, Shri Bherulal Pharmacy Institute, Indore, India 3Faculty of Pharmacy, Philadelphia University, Amman, Jordan 4National Institute of Pharmaceutical Education and Research (NIPER)—Ahmedabad, Gandhinagar, India

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O U T L I N E 7.1 Tissue Engineering: An Overview 7.2 Nanotechnology in Tissue Engineering 7.3 Strategies Related to the Formation of Scaffolds 7.3.1 Photolithography 7.3.2 Templating 7.3.3 Ionic Self-Complementary Peptide 7.3.4 Bionanotubes/Lipid Tubules 7.3.5 Miscellaneous 7.4 Natural Materials Based Tissue Engineering Nanoscaffold 7.4.1 The Chitosan-Based Tissue Engineering Scaffold 7.4.2 The Albumin-Based Tissue Engineering Scaffold

Biomaterials and Bionanotechnology DOI: https://doi.org/10.1016/B978-0-12-814427-5.00007-X

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7.4.3 The Alginate-Based Tissue Engineering Scaffold 7.4.4 The Silica-Based Tissue Engineering Scaffold

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7.5 Synthetic Materials Based Tissue Engineering Nanoscaffolds 238 7.5.1 The Dendrimer-Based Tissue Engineering Scaffold 238 7.5.2 Poly(Lactic Acid-co-Glycolic Acid)-Based Tissue Engineering Scaffold 240 7.5.3 Polylactic Acid Based Tissue Engineering Scaffold 240 7.5.4 The Polyethylene Glycol Based Tissue Engineering Scaffold 242 7.6 Applications 7.6.1 Nanotechnology in Cell Tissue Engineering

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© 2019 Elsevier Inc. All rights reserved.

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7.6.2 Nanotechnology-Based Tissue Engineering for Cell Labeling, Purification, Detection, and Suicide Bombing

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7.7 Recent Patents Overview 252 7.7.1 Magnetic Pole Matrices 253 7.7.2 Differentiable Human Mesenchymal Stem Cells 253 7.7.3 Degradable Polyurethane Foams 253 7.7.4 Multilayer Polymer Scaffolds 254

7.8 Clinical Trial Status

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7.9 Conclusion

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Acknowledgments

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Abbreviations

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References

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Further reading

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7.1 TISSUE ENGINEERING: AN OVERVIEW The reliability and body function of the injured cells, tissues, and organs are preserved by the most vital and critical contrivance of the body, i.e., the self-repair mechanism. This mechanism slows down and recovery takes a long time with growing age, injury, or prevailing disease, and this process is limited to small localized tissue. Many times this natural mechanism is ineffective and therefore, more effective organ transplantation can be used in which the damaged organ is replaced via a healthier organ. Due to organ transplant, uncontrollable autoimmune suppression may occur, which increases the chances of graft rejection or often leads to the death of the patient. The development and progress of tissue engineering help to reconstitute artificial cells or tissues in place of damaged organs using cells of the recipient. Therefore the definition of tissue engineering can be as the application of principles and methods of engineering and life sciences for the development of biological substitutes, to refurbish, sustain, or recover tissue function (Vacanti and Langer, 1999). Instead of replacing the tissues or organs the aim of this process, viz., tissue engineering is to regenerate injured tissues by developing biological surrogates that restore, maintain, or improve tissue function (O’Brien, 2011). The 1988 National Science Foundation workshop officially coined the term tissue engineering to decrypt the simple understanding of the assembly purpose relationship in the development of biological substitutes and between normal and pathological mammalian tissue (O’Brien, 2011). However, the first pioneering work in tissue engineering dates back to the 16th century when professor of surgery and anatomy at the University of Bologna Gasparo Tagliacozzi (1546 99) described a nose replacement that he had constructed from a forearm flap in his work De Custorum Chirurigia per Insitionem (The Surgery of Defects by Implantation), which was published in 1597 (O’Brien, 2011). Howard Green and his coworkers had a crucial contribution to the field of tissue engineering. The first mass production of normal human diploid cells (keratinocytes) was carried out by them in 1981. This was the first commercial product made by tissue engineering a living autologous human skin epithelium, which was used for burn patients (Peck et al., 2011).

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Further, the work of Green was followed by Eugene Bell, who added a dermal component, finally developing “artificial skin,” and this led to the foundation of Organogenesis Inc. in 1986. This was the first FDA approved plant for the massproduced tissue-engineered product (Vacanti, 2006). Tissue and whole organ grafting are frequently used to replace the damaged/diseased tissues or organs of the body. Earlier this process solely depended on the transplanting of donated organs and decellularized tissues. However, graft rejection and less availability or unavailability of organ donors were the major problems that have been faced frequently. Tissue engineering techniques are emerging technologies and more robust and cheaper alternatives to such earlier techniques and deals with the improvement of the regenerative capability of tissues, and facilitate the development of new tissues or complete organ and replacement of damaged/diseased tissues. Tissue engineering is different from biomimetic materials as biomimetic materials are substances that elicit one or more responses similar to that of the extracellular natural matrix (ECM) around the cells in tissue. Now they are extensively employed in the field of tissue engineering. Tissue engineering is at the frontier of material science, bioengineering, chemistry, biology, and medicine, controlled to meet the unmet clinical needs through the development of new technologies and enhancement of existing ones (Atala et al., 2012). The major advantage of tissue engineering is that it mimics the functional and anatomical features of innate tissues, so it can maintain the working and renovate the normal functioning of the injured tissues (Lee et al., 2016). The strategies of tissue engineering generally involve a combination of biomaterials, cells, and biologically active factors to effect tissue formation (Atala et al., 2012). Finally, tissue engineering leads to the foundation of organs that provide external support and nonimplantable structures in the situation where the convenience of a compatible donor is not possible (Martin et al., 2004).

7.2 NANOTECHNOLOGY IN TISSUE ENGINEERING The branch of science that deals with the intent of making, classifying, and applying materials and devices at the molecular level with dimensions less than 100 nm is called nanotechnology (Lalu et al., 2017; Maheshwari et al., 2015a; Sharma et al., 2015; Tekade et al., 2017a). In medicine, the application of these materials can be programmed to interact with cells and tissues at a receptor level with the highest scale of specificity thus allowing a greater degree of incorporation of technology with biological systems not previously possible (Kuche et al., 2018; Tekade et al., 2017b). The question then arises: what is the importance of nanotechnology? The answer is that it provides an uncomplicated binding site and increases tissue production nanotechnology to help in developing cell bonding and vascularization. Also, nanotechnology ensures the controlled release (Danie Kingsley et al., 2013). The ideal range of nanoparticles is 10 100 nm. (Walmsley et al., 2015). Nanotechnology ensures the formation of nanospheres, which imitate subjected body tissues like bone and cardiac tissues. After significant randomization difference in cell attachment, production

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and separation were observed (Lamers et al., 2010). Nanosized scaffold also ensures good binding of a fibrin clot, which improves the movement of osteogenic cells to the engineered surface (Mager et al., 2011). They are also widely used as controlled drug release because of their small size, and thus high surface area; as a result, drug loading efficiency also increases (Maheshwari et al., 2012, 2015b). The approaches for nanotechnology-based tissue engineering involved transplantation of harvested cells/tissues at the damaged site in the body; introduction of substances at the damaged site, which aids the cells to grow and repair; and implanting support matrices carrying cell seeds (e.g., stem cells, osteoblasts) (Langer and Vacanti, 1993). Tissue engineering trained in nanotechnology includes three components, namely scaffolds, cells, and growth factors (Krishnan and Sethuraman, 2013). Earlier efforts were made to choose the type of material that can be used in preparing the scaffold, but now attention has been paid in geometry and surface functionality of the scaffold chosen that mimics the topography of the extracellular matrix. Nanotechnology-enabled the integration of the nanostructures in the scaffold that showed better results than the two-dimensional cultures. Growth factors are the crucial part of the tissue engineering as the cells must be provided with appropriate chemical factors at an appropriate time interval to acquire the native physiological, chemical, and the mechanical characters of the cell. When these growth factors were integrated as the nanocarriers the cell formed showed more resemblance in functional and physiological aspect as well. Cells that can be used are the stem cells, and the type of stem cell that should be employed depends upon the type of the cell that is needed to be engineered. The stem cells that can be employed for the tissue engineering of hepatocytes are human bone marrow stem cells (Raftery et al., 2016). The appropriate method for engineering the scaffold and the time interval used for the incorporation of the growth factors directly affects the function of the cell that has been engineered, so one must pay attention in this concern while employing nanotechnology in cellbased therapy. The triad of signal, scaffolds, and cells, which acts as a stencil for tissue engineering, is represented in Fig. 7.1.

FIGURE 7.1 Triad of signal, scaffolds, and cells that act as a stencil for tissue engineering.

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To form a microenvironment of the human body, an extracellular matrix (ECM) is embedded through support cells (mesenchymal cells) and functional cells (parenchymal cells); this forms the basis of every tissue or organ in the human body (Barnes et al., 2007). Mainly three approaches are used for designing of novel tissue: induced tissue, cell isolation and substitution, and cell matrices. Induced tissue approach requires large-scale production of applicable signal molecules and their purification. It also includes a method for delivering these molecules. Cell isolation and substitution can avoid complications involved in surgery as it permits manipulation of the cell before infusion and replacement of specific cells. Immunological rejection is the limiting factor of this approach. Thirdly, in the cell matrices approach, cells are placed within the matrices, forming a closed system that includes isolation of cells by a semipermeable membrane, which allows molecules of low molecular weight, i.e., nutrients, and avoids high molecular weight molecules, which may lead to transplant destruction. Cells attached to matrices are termed as an open system. These are directly implanted, and further, can be integrated into the body (Langer and Vacanti, 1993). Recent development has been seen in the approaches of tissue engineering that tend to be known as in situ tissue engineering, including assimilation of reformative stimuli into biomaterial scaffolds with the target of augmenting regenerative response of host cells. Along with a considerate approach toward cellular and molecular processes sustaining regenerative medicine, more radical methods of manipulating cells have emerged that can also allow RNA interference (RNAi) and a more integrated approach toward pharmacogenomics (Tekade et al., 2015; Maheshwari et al., 2017). The decisive aim of all these approaches is to empower the body to heal itself by familiarizing the body with tissue-engineered scaffolds and also to recognize them as an integral part of itself, as new innate tissues are regenerated (Barnes et al., 2007). The various advantages of using nanotechnology in tissue engineering are presented in Fig. 7.2. The second strategy is frequently accompanied by the third. To grow cells efficiently in three-dimensional (3D) region, support matrices (scaffolds) play a very crucial role (Ma, 2004). Tissue engineering has become an emerging field since the 1980s (Chan and Leong, 2008) and is considered to be a field merging different disciplines like biology, engineering, and material science (Vacanti, 2006). Fabrication of scaffolds with optimum attributes is a very important concern in the tissue engineering approach. Biomimetic materials are utilized in materialization of such scaffolds. Various types of biomimetic materials have been exploited and tailored for manufacturing of scaffolds, which may aid the tissue regeneration. Biomimetic materials to be used as scaffold should possess certain important properties to serve as temporary extracellular matrix (ECM) and to produce improved tissue growth compared with the natural EMC (Fig. 7.3). It should be compatible with the biological system and microenvironment to which it has to be subjected as a scaffold. It should not produce any immunogenic stimulation or toxicity. The material should be such that the scaffold possesses sufficient porosity to facilitate the recruitment of cells and cell attachment (Ma, 2008).

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FIGURE 7.2 Schematic representation of the advantages of nanotechnology in tissue engineering.

FIGURE 7.3 Properties of suitable scaffolds.

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7.3 STRATEGIES RELATED TO THE FORMATION OF SCAFFOLDS Scaffolds are structurally fabricated to be biodegradable and porous, which can be obtained either from natural material, e.g., collagen (George and Ravindran, 2010), fibrin, xanthan gum(Kumar et al., 2018), chitosan (CS), heparin (Rambhia and Ma, 2015), or synthetic polymers e.g., polyglycolide, polylactide, polylactide-co-glycolide (Kretlow and Mikos, 2008). By using new material processing technologies, assembly can be made in the form of sponges; sheets, a highly complex structure with intricate channels and pores; or gels. The most important feature of scaffolds is that they tend to degrade after implantation and eventually get replaced by newer tissues (Griffith and Naughton, 2002).

7.3.1 Photolithography Photolithography is the most prevailing technique of a top-down approach used by the semiconductor industry to generate the integrated circuits. When the lithographic technique of nanotechnology is used in cell biology, it is possible to set up cell cultures where neurons are put in one well and in another well there are astrocytes, and these two wells are conjoined by a channel that facilitates the diffusion of soluble factors (Takano et al., 2002).

7.3.2 Templating Templating is an interesting phenomenon during the normal hard tissue development organic phase, in which collagen fibers work as a template to guide inorganic phase formation in bone and teeth. Bones and teeth are biocomposites that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. He et al. used dentin matrix protein 1, an acidic protein, to nucleate the formation of hydroxyapatite (Hap) in vitro in a multistep process (Shakiba et al., 2018). The nucleated amorphous calcium phosphate precipitates ripen and nanocrystals form. Pins et al. used a self-assembly process to form collagen fibers, guide the natural hard tissue formation, and engineer the bone tissue through this biomimetic approach (Pins et al., 1997).

7.3.3 Ionic Self-Complementary Peptide Zhang et al. synthesized a 16-amino acid peptide, B5 nm in size, with an alternating polar and nonpolar pattern (Ghanghoria et al., 2016). The peptides can form a stable strand and sheet conformations, with side chains with one polar side and one nonpolar side, and then undergo self-assembly to form nanofibers. These nanofibers can form interwoven mats that form 3D hydrogels, with high water contents (.99.5%), which may be suitable for tissue engineering. The other feature of this peptide is that if the charge orientation is changed, entirely different molecules can be obtained.

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7.3.4 Bionanotubes/Lipid Tubules Rudolph et al. used amphiphilic molecules that contain distinct hydrophobic and hydrophilic segments, to form self-assembled lipid tubules (Pawar et al., 2016). These tubules can be used as microvials for long-term release of active agents. However, the lipid tubules are very weak regarding mechanical properties. Meanwhile, they have very low thermal stability, e.g., a few tens of degrees Celsius will destroy them. To overcome these limitations, Rudolph et al. used the lipid tubule as a mold, pattern, or scaffolding to convert them into stronger objects with the same geometry (Pawar et al., 2016). For example, lipid tubules were subsequently coated with metals or inorganic materials to form scaffolds. This concept is very important for using self-assembly structures for realistic tissue regenerating applications. Using this concept, lipid tubules can be coated using sol gels to fabricate ceramic rods or hollow cylinders with diameters down to 0.5 m, coated with silanes to change the chemical nature of the tubule’s surface, or coated with metals. Modified tubules as microbials can be used for controlled release applications as well.

7.3.5 Miscellaneous Premade porous cell-seeding scaffold techniques are one of the most basic techniques used for scaffold synthesis. The method comprises using pyrogens in biomaterials or use of solid free form or prototyping or use of woven or nonwoven fibers. Decellularized ECM cell-seeding technique utilizes the use of allogenic or xenogenic tissues with the help of decellularization technique. Self-secreted cell sheets ECM technique can be done by thermoresponsive culturing cells of a polymer such as poly(N-isopropylacrylamide) coated culture dish until convergence. The hydrogel matrix self-assembled cell encapsulation technique uses biomaterials that can self-assemble into hydrogels and are fabricated by self-assembling by using parameters such as temperature and humidity (Chan and Leong, 2008).

7.4 NATURAL MATERIALS BASED TISSUE ENGINEERING NANOSCAFFOLD Different types of biomimetic materials are used as starting material in the fabrication of support matrices. Various substances obtained from natural sources are used in tissue engineering. The versatile chemical compound can be employed for scaffold fabrication. These can be classified as natural and synthetic as presented in subsequent sections. There are various proteins, polypeptides, and polysaccharides that have been explored to be good scaffolding material in tissue engineering. Being substances from a natural source, they are likely to possess better compatibility and less antigenicity in the biological sites. Natural substances are more complex in structure orientation, which causes difficulty in the manufacturing of the scaffold while their complexity may be advantageous to mimic the extracellular milieu (Connor and Tirrell, 2007).

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7.4.1 The Chitosan-Based Tissue Engineering Scaffold Recently, CS, being the most promising natural polymer, has been used in many areas of medical science. It is a highly versatile biomaterial obtained from crustaceans. The property of CS with a special interest in tissue engineering is a minimal foreign-body response and high efficacy for fibrous encapsulation. In comparison to most of the synthetic polymers, CS possesses a hydrophilic surface with many amine-terminated groups that promote cell binding and proliferation. Moreover, being a natural polymer, the degradation products of CS are nontoxic. Only a few materials can be classified as biologically active, biologically degradable, and osteoconductive. CS belongs in this category along with Hap and is most widely used for bone tissue engineering. Importantly, CS provides a platform for surface modification, which is also very useful for producing different 3D scaffolds. Moreover, being a biocompatible polymer, it can be combined with a variety of materials including ceramics and other polymers in conjugation to produce biocomposite materials with higher mechanical and physical characteristics. When we talk about the methods for CS-based scaffold fabrication, the most commonly used techniques for producing CS-based scaffolds are phase separation and lyophilization, particulate leaching, gas foaming, and freeze gelation technique as depicted schematically in Fig. 7.4A D (Levengood and Zhang, 2014).

7.4.2 The Albumin-Based Tissue Engineering Scaffold Albumin is a naturally available serum protein that is reported to affect the binding of cells to different scaffold material with equal or better efficiency from collagen and fibronectin after slight modification. It can serve as an interface between cells and scaffold, thereby mediating the integration of these two components. Lyu designed an investigation to study the potential of the albumin-based construct as tissue engineering nanoscaffolds for the regeneration of cartilaginous material (Guelcher et al., 2012). Briefly, authors seeded porcine knee chondrocytes followed by cultivation in a porous ternary matrix composed of different polymers with surface albumin. The outcomes found that the amount of albumin rarely influences the viability of porcine knee chondrocytes in the developed material. Although, a higher concentration of albumin preferentially improves the adhesion of porcine knee chondrocytes on the scaffolding pore surface. The staining image of the cultured scaffold (Fig. 7.5A and B) revealed that the purple dots were the stains of porcine knee chondrocytes. As indicated in Fig. 7.5C and D, the crimson stains denoted the secreted glycosaminoglycans. The red intensity in the albumin-grafted construct was stronger than that in the albumin-free construct, suggesting that albumin sustained a stable glycosaminoglycan secretion. As shown in Fig. 7.5E and F, the brown stains were the produced type II collagen. Therefore it can be concluded that due to its nontoxic, biocompatible, and water-soluble properties, albumin can be used in the applications of tissue engineering.

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FIGURE 7.4

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Schematic representation of four commonly used chitosan scaffold fabrication methods. (A) Phase separation and lyophilization technique where a chitosan solution is introduced into a mold, frozen to allow for phase separation of acetic acid solvent and chitosan acetate salt and then lyophilized. (B) Particulate leaching technique, which can be combined with phase separation to fabricate chitosan scaffolds. A porogen such as gelatin is mixed with chitosan solution prior to phase separation and lyophilization. The resultant scaffold is submerged in a solvent to allow for porogen leaching resulting in additional porosity. (C) Gas foaming technique where chitosan solution containing a cross-linker (glutaraldehyde) is supersaturated with carbon dioxide at high pressure while also undergoing cross-linking. When the system is depressurized, thermodynamic instability leads to nucleation and growth of gas bubbles. Gas bubbles grow and/or coalesce and escape the polymer solution thereby generating pores. (D) Freeze gelation technique, which initially involves phase separation due to freezing. The scaffold is placed in a gelation solution of sodium hydroxide and ethanol below the chitosan freezing temperature. Following gelation, the scaffold is air-dried to remove residual liquid. Adapted with permission from Levengood, S.K.L., Zhang, M., 2014. Chitosan-based scaffolds for bone tissue engineering. J. Mater. Chem. B 2(21), 3161 3184.

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FIGURE 7.5 Stained photographs of the cultured constructs. (A) H&E stain, albumin-free construct; (B) H&E stain, albumin-grafted construct; (C) safranin-O stain, albumin-free construct; (D) safranin-O stain, albumingrafted construct; (E) type II collagen stain, albumin-free construct; (F) type II collagen stain, albumin-grafted construct. Albumin-grafted constructs contained 150 g/mL of albumin. Adapted with permission from Guelcher, S.A., Patel, V., Hollinger, J.O., Didier, J., 2012. Degradable Polyurethane Foams, Google Patents.

7.4.3 The Alginate-Based Tissue Engineering Scaffold Apart from albumin, sodium alginate also provides high water solubility and is known to be used in different biomedical applications such as drug delivery and wound dressings, including tissue engineering. In tissue engineering, the cross-linking ability of alginate that can be modified by altering the composition and molecular weight of the alginate chains is of prime importance. Based on the fact that shorter degradation times may be more optimal for some tissue regeneration applications that aim to match tissue formation with polymer degradation rate, a group of investigators reported the alginate-based nanofibers made using two

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FIGURE 7.6 Scanning electron micrographs of nanofibers containing 37 kDa alginate/PEO/Pluronic F127 (8.0:1.6:2.0 wt.%) (A and B) prior to cross-linking. Nanofibers composed of 196 (C and D) and 37 kDa (E and F) alginate after cross-linking with calcium and soaking in water for 4 days to remove PEO. Adapted with permission from Dwivedi, N., Shah, J., Mishra, V., Mohd Amin, M.C.I., Iyer, A.K., Tekade, R.K., et al., 2016. Dendrimer-mediated approaches for the treatment of brain tumor. J. Biomater. Sci. Polym. Ed. 27(7), 557 580.

different molecular weights (37 and 196 kDa) for in vivo tissue scaffolds and investigated mechanical strength of the prepared scaffold (Dwivedi et al., 2016). Uniform nanofibers containing low molecular weight alginate, polyethylene oxide, and F127 surfactant were obtained (Fig. 7.6). It was the first study that reported alginate-based nanofibers from a low molecular weight alginate for scaffold application. The ability to turn the structure and characteristics like biocompatibility, higher Young’s modulus, cross-linking property, and cell affinity make the alginate valuable to be used in combination with other nanomaterials for tissue engineering applications.

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FIGURE 7.7 (A) Silica-based bone pellets. (B) Hemolysis of RBCs in the presence of silica-based developed formulation. 1 and 2 indicate distilled water and 0.9% saline solution as a positive and negative control, respectively. Adapted with permission from Werani, J., Gru¨nberg, M., Ober, C., Leuenberger, H., 2004. Semicontinuous granulation—the process of choice for the production of pharmaceutical granules?. Powder Technol., 140(3), 163 168.

7.4.4 The Silica-Based Tissue Engineering Scaffold Silica is a very favorable material from the tissue engineering point of view due to its porous nature. Apart from that, its surface modification ability also provides immense opportunities in tissue engineering applications. Calcium sulfate α-hemihydrate (CSH) is widely used as the material for bone cement. However, the initial burst release of the loaded drug in the first few days greatly hindered its application. Werani et al. developed a novel bone cement pellet with sustained release of vancomycin by mixing vancomycin loaded mesoporous silica nanoparticle and CSH together (Werani et al., 2004). The developed bone pellets (Fig. 7.7A) revealed nonsignificant pyrogenicity and posed no potential adverse effects upon hemolytic test (Fig. 7.7B). These results imply that the silica-based bone pellets are a suitable candidate to replace CSH bone cement in the treatment of open fractures.

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7.5 SYNTHETIC MATERIALS BASED TISSUE ENGINEERING NANOSCAFFOLDS Synthetic polymers are more frequently used with added advantages like ease for tailoring, improved mechanical attributes, low cost of production, and so on. These may be categorized as organic [polyesters like polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic acid-co-glycolic acid) (PLGA)] and inorganic. Synthetic materials are widely used for tissue regeneration in the field of regenerative medicine. Synthetic materials have less structural complexity than natural substances and can be readily modified to obtain desired attributes (Sreejalekshmi and Nair, 2011). They can be manufactured in the form of the scaffold in a more economical way and are less immunogenic than natural materials. Being a synthetic compound, they show diminished biological response compared with the natural extracellular matrix (O’Brien, 2011), but it is possible to tailor them easily, and the surface can be modified and attached with functional groups and biologically active moieties to impart such attribute into them to help enhance cell attachment and proliferation. The most commonly used synthetic polymers that can mimic the extracellular microenvironment and used in tissue engineering are polyesters, polyether esters, bioactive ceramics, etc. They are inorganic compounds that have been explored for fabrication of support matrices for tissue regeneration. Commonly used bioceramics for this purpose are Hap, bioactive glasses, calcium phosphates, calcium sulfates, etc. Due to their rigid nature earlier bioceramics were used for bioengineering of hard tissues but recently their application in the regeneration of soft tissues has also been explored (Baino et al., 2016). Bioceramics have higher biological responses as compared with other synthetic polymers. Calcium orthophosphate resembles the mineral component of bones and teeth and osteoconductivity, but brittleness limits its use in bone grafts, thus nanocrystallized form of calcium orthophosphates are used, which better mimic the component of mineralized tissues (Dorozhkin, 2010). Another commonly used ceramic material is Hap, which is an apatite of calcium phosphate. Hap is also naturally present in the bone and teeth. It is osteoconductive and highly biocompatible. Moreover, it is used as bone cement for filling of bone and teeth (Baino et al., 2015). However, its tensile strength is not sufficient to be used as a scaffold. They are generally used as a composite scaffold with other biomimetic materials when subjected to tissue regeneration application (Barrere et al., 2006). Polyesters are one amongst the most widely used synthetic polymers in tissue engineering. Linear aliphatic polyesters, PLA, PGA, and polycaprolactones are used either as single polymer or copolymers such as PLGA due to their tunable biodegradability and high biocompatibility (Chen and Ma, 2006).

7.5.1 The Dendrimer-Based Tissue Engineering Scaffold The word dendrimer is obtained from the Greek word dendron, which means “tree,” indicating that dendrimers are a tree-like structure (branched). Dendrimers are synthetic, highly branched, spherical, and monodisperse macromolecules with 3D nanometric structure.

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In literature, they are also called cascade molecules, arborals, dendritic molecules, or because of their nanoscopic size and monodispersity they are often referred to as nanoscopic compounds (Sva¨rd et al., 2013). Currently, dendrimers are proving their versatility in a number of fields, including biomedical sciences. The dendritic architecture is perhaps one of the most pervasive topologies observed. The main objective in developing dendrimer-based tissue engineering scaffolds includes providing nontoxic constructs in the 3D scaffold surface where cells can grow in the provided native ECM with the necessary hormones, signaling molecules, and other growth factors. The benefit of Poly(amidoamine) (PAMAM) dendrimers in scaffold engineering was investigated by two different research groups who studied the incorporation of dendrimers in tissue engineering. These authors employed an enhanced amount of alkaline phosphatase in, and mineralization characteristics of, the modified cellular microenvironment employing a dexamethasone carboxymethyl CS/PAMAM dendrimer. The study revealed enhancement in the ectopic early osteogenic differentiation of rat bone marrow stromal cells in the scaffold (Oliveira et al., 2009). Calcium deposition leading to higher mineralization was reported to be greater in the scaffold constructs exposed to a dexamethasone carboxymethyl CS/PAMAM dendrimer environment (Fig. 7.8).

FIGURE 7.8 Microscopic images of dendrimer-based tissue engineering scaffolds. Optical microscopy and scanning electron microscope images of HA (left) and SPCL (right) scaffolds seeded with RBMSCs, stained with Alizarin red (mineralization) after culturing in different culture media for 14 days: (A D) controls (scaffolds without RBMSCs); (E H) complete Eagle’s minimum essential medium; (I L) MEM medium with Dex-loaded CMCht/PAMAM dendrimer nanoparticles. HA, Hydroxyapatite; RBMSCs, rat bone marrow stromal cells; SPCL, starch polycaprolactone. Adapted with permission from Oliveira, J.M., Sousa, R.A., Kotobuki, N., Tadokoro, M., Hirose, M., Mano, J.F., et al., 2009. The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasoneloaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles. Biomaterials 30(5), 804 813.

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7.5.2 Poly(Lactic Acid-co-Glycolic Acid)-Based Tissue Engineering Scaffold PLGA PGA is a polymer having rapid degradation that is suitable for fabrication of scaffold used for the short-term graft. However, it also has poor tensile strength. Thus it cannot be used for bioengineering of hard tissues like bones, tendon, and ligament (Bogan et al., 2016). Recently, Kwak et al. (2016) developed micro/nanomultilayered scaffolds of PLGA and collagen by alternately electrospinning for bone tissue engineering. To fabricate the multilayered scaffolds having a sequential arrangement of microfibrous PLGA meshes and micro/nanomixed fibrous meshes of PLGA and collagen, the dual extrusion electrospinning technique was used (Fig. 7.9A). The simultaneous electrospinning parameters for PLGA and collagen solutions were optimized by individual electrospinning of PLGA and collagen solutions. The morphology and composition of mixed fibrous meshes in the multilayered scaffolds were controlled by varying electrospinning parameters of individual fibers (Fig. 7.9B). The results of this experiment showed that the dual extrusion electrospinning technique can be used further for designing 3D scaffolds with different topologies and compositions for drug delivery and bone tissue engineering in our ongoing programs for the applications of biomaterials in the fields of biomedical research.

7.5.3 Polylactic Acid Based Tissue Engineering Scaffold PLA has a slower degradation rate and better mechanical strength as compared with PGA, thus PLA is suitable for scaffolding for regeneration of bone and ligament (Venugopalan and Rajendran, 2015). PLA-based scaffold for cardiac tissue is also explored due to its sufficient elastic property. Carbon nanotubes (CNTs) are used with PLA and other polymers to improve conductivity and to make them useful for regeneration of skeletal muscle, cardiac muscle, and neuronal tissues (Ahadian et al., 2017). PLA can also be used as particle-based scaffolding such as microspheres, which undergo self-assembly in vivo to form 3D scaffold (Liu et al., 2011). In a recent investigation, Wang et al. developed conductive nanofibrous scaffold by electrospinning considering its advantages for cardiomyocytes-based tissue engineering (Ghadiri et al., 2017). The investigators designed conductive nanofibrous sheets with electrical conductivity and nanofibrous structure made of poly(L-lactic acid) blending with polyaniline for cardiac tissue engineering and cardiomyocytes-based 3D actuators. The outcomes of CX43 immunostaining analysis show the reason of how the conductivity of nanofibrous sheets influence the beating frequency and displacement of 3D actuators (Fig. 7.10). In comparison, both tubular shaped and folding shaped actuators formed by PLA nanofibrous sheets showed much weaker staining for CX43 (Fig. 7.10A and C). Strong positive staining for CX43, a gap junction protein responsible for cell cell coupling and synchronous beating of cardiomyocytes (CMs), were exhibited on tubular and folding shaped actuators formed by PLA/PANI3 conductive nanofibrous sheets (Fig. 7.10B and D).

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FIGURE 7.9 (A) Schematic representation of dual electrospinning technique for fabrication of multilayered 3D scaffolds. (B) structure and composition of multilayered scaffolds having micro/nanomixed fibrous PLGA and Col-HA mesh arranged in alternate fashion with microfibrous PLGA mesh as fabricated with dual extrusion electrospinning technique. PLGA, Poly(lactic acid-co-glycolic acid). Adapted with permission from Kwak, S., Haider, A., Gupta, K.C., Kim, S., Kang, I.-K., 2016. Micro/nano multilayered scaffolds of PLGA and collagen by alternately electrospinning for bone tissue engineering. Nanoscale Res. Lett. 11(1), 323 with slight modification.

The developed PLA and polyaniline conductive nanofibrous sheets with conductivity and extracellular matrix-like nanostructure revealed promising potential in cardiac tissue engineering and cardiomyocytes-based 3D actuators.

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FIGURE 7.10 Representative fluorescence images of cardiomyocytes immunostained for CX43 (red) on tubular bioactuators formed by cardiomyocytes-laden PLA (A) and cardiomyocytes-laden PLA/PANI3 (B), and folding bioactuators formed by cardiomyocytes-laden PLA (C) and cardiomyocytes-laden poly(L-lactic acid)/ polyaniline (D) after culture for 12 days. PLA, Polylactic acid. Adapted with permission from Ghadiri, M., VasheghaniFarahani, E., Atyabi, F., Kobarfard, F., Mohamadyar-Toupkanlou, F., Hosseinkhani, H., 2017. Transferrin-conjugated magnetic dextran-spermine nanoparticles for targeted drug transport across blood-brain barrier. J. Biomed. Mater. Res. A 105(10), 2851 2864.

7.5.4 The Polyethylene Glycol Based Tissue Engineering Scaffold Polyether esters, which are generally used as scaffolding material, are polyethylene glycol (PEG) and polybutylene terephthalate (PBT). PEG is highly biocompatible and suitable for soft tissues (Elisseeff et al., 2002). However, it has a faster degradation rate. Degradation rate for PEG can be rendered to desirable extend by copolymerized with polyesters like PLA and PGA (Han and Hubbell, 1997). PEG is elastic in nature and PBT is stiff, their composite as PEG/PBT is generally used to combine their attributes, and their proportion can be tuned to obtain scaffold with the desired property. Shakir et al. (2015) developed an interesting nanocomposite material containing nanohydroxyapatite, CS, and PEG (n-HAP/CS/PEG) using the coprecipitation method. The objective behind the development of this novel nanocomposite is to find suitable analog that can potentiate the natural formation of bone and therefore be applied to bone tissue engineering. The developed formulation was compared with the formulation lacking PEG (n-HAP/CS). The results of X-ray diffraction and TEM suggested that the crystallinity and thermal stability of the n-HAP/CS/PEG scaffold have decreased and increased respectively, relative to n-HAP/CS scaffold. The comparison of SEM images of both the scaffolds

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FIGURE 7.11 SEM micrographs of (A) n-HAP/CS and n-HAP/CS/PEG nanocomposites and their simulated body fluid studies after (B) 15 days and (C) 30 days. CS, Chitosan; n-HAP, nanohydroxyapatite; PEG, polyethylene glycol. Adapted with permission from Shakir, M., Jolly, R., Khan, M.S., Iram, N.E., Sharma, T.K., Al-Resayes, S.I., 2015. Synthesis and characterization of a nano-hydroxyapatite/chitosan/polyethylene glycol nanocomposite for bone tissue engineering. Polym. Adv. Technol. 26(1), 41 48 with slight modifications.

indicated that the incorporation of PEG influenced the surface morphology while a better in vitro bioactivity has been observed in n-HAP/CS/PEG than in n-HAP/CS-based on simulated body fluid study, referring a greater possibility for making direct bond to living bone if implanted (Fig. 7.11).

7.6 APPLICATIONS Tissue engineering has a wide scope of applications and is a continuously developing field. It includes the development of artificial cartilage to the heart. The knowledge of tissue engineering is applied to develop soft tissue as well as hard tissue, helping to regenerate the damaged cells and increases the life of the patient. Recent advances in developing scaffolds using various biomaterials have opened the doors for a large number of semisynthetic and synthetic polymers. Organ damage is a severe issue nowadays, and a proper and healthy donor organ is difficult to obtain; in such cases tissue engineering shows a wide area for development. Many industries are focusing on this thrust area for pursuing their further research. Since it includes the

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intervention of humans and their cells, tissue engineering suffers many issues for regulatory approval. It is the most emerging field, and much development is accepted for this area. Further reading on the application of nanotechnology in tissue engineering is presented in following subsections.

7.6.1 Nanotechnology in Cell Tissue Engineering Cellular engineering is the biological process that involves the principle of molecular biology to generate the cell products for the repair of tissue or organ. In the nanotechnology-driven era of research, new sophisticated techniques implicated in cell biology and nanotechnology do not only place emphasis on more complex in vivo like extracellular environments but also consider the dynamic complex biological process at the receptor level. Eventually, the goal is to obtain a comprehensive knowledge of how the building blocks of humans, i.e., the cells, work at the molecular level (Mendes, 2013). Nanomaterials may be used as smart interfaces for additional understanding and controlling the complex relationship of actions and interactions that occur in the cell (Nikalje, 2015). 7.6.1.1 Nanotechnology in Bone Cells Tissue Engineering Bone tissue transplantation is widely used in tissue transplantation in the world. Every year around 22 lakhs bone grafting take place worldwide (Saiz et al., 2013). Tissue engineering is widely used to produce, restore, and fix cells, tissues, or organs by using body cells with some bioadditives that help to construct the tissues similar to the body tissues. Tissue engineering combines the practice of engineering material science, medicine, and biology (Danie Kingsley et al., 2013). For bone tissue engineering one must know bone functioning, rejuvenation, and healing. To create a tissue-engineered bone one must consider the following to get an effective outcome. Primarily, a bioactive matrix, i.e., scaffolds are required to maintain the interstitium of cellular integrity and tissue growth. Secondarily, the specialized phenotypic cells are then inculcated into the matrix and the matrix must have the ability to induce osteogenesis. Thirdly, the final product must match the basic functions of bone (Lyles et al., 2015). In the body, osteoblasts and osteoclasts have the function of maintaining homeostasis of bone development (Tautzenberger et al., 2012). Osteoblasts promote cell formation, whereas osteoclasts cause bone breakage. In BTE, the osteoblast is supported by drug and growth factors, and osteoclasts are inhibited by specific inhibitors (Walmsley et al., 2015). Therapeutic agent, growth factor, or genetic material is encapsulated in the biodegradable or nonbiodegradable scaffold of nanosize. Both biodegradable and nonbiodegradable nanospheres are used here, but the biodegradable are generally the most preferred. The examples of nonbiodegradable nanoparticles are gold, dendrimer, and silica whereas degradable nanoparticles are PLA and PLGA (Jensen et al., 2011). As mentioned earlier, the inner part is planned such that it improves cell binding and separation where the outer part promotes assimilation with adjacent tissue to prevent cell

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extrusion and movement. In a few studies, it has been reported to use metallic plates and screws also for improved attachment in animals (Reichert et al., 2011). However, it is not a practical approach for a prolonged duration. Then the inner face is taken to micro- and nanosize, which resolves the major limitation of fixation and nearer tissue attachment. The other advantage of micro- and nanolevel scaffold is that they imitate extracellular matrix arrangement (Saiz et al., 2013). Scaffold manufacturing techniques are solvent casting, particulate leaching, freezedrying, gas forming, solution casting, phase separation, etc. (Ronca et al., 2016) and the various techniques used for bone tissue engineering through micro- and nanotechnology are soft lithography, photolithography, microcontact printing, and electrospinning (Ber et al., 2005). Although, more work is still required to be carried out as it is not clear between a nano or microscale, which one is better (Saiz et al., 2013). BTE is a versatile approach to bone rejuvenation. However, still, the regeneration of large bones is a big challenge (Walmsley et al., 2015). A few new design concepts and techniques are required to be developed for a new bioactive scaffold (Saiz et al., 2013). The current strategy is based on the formation of 3D nanocomposite by the additive manufacturing method. The 3D technique has an advantage over the traditional method of scaffold formation in that it has replicable internal morphology, good structural management, and altered mechanical and mass transfer properties. Purposely, this method formulates the objects in layer-by-layer style (Ronca et al., 2016). This 3D-based nanocomposite scaffold has better bone rejuvenation in less time (Saiz et al., 2013). In an investigation, a novel hybrid strategy involving the combination of mechanically strong, porous scaffolds and nanofeatured self-assembling peptide hydrogels as an osteoinductive scaffold system was reported (Igwe et al., 2014). In this strategy, the mechanically strong scaffold component would allow for mechanical stability of the loadbearing defect site; whereas, the hydrogel phase will allow for efficient cell delivery into the defect implantation site, cell niche establishment, and promotion of mineralization (Fig. 7.12). Growth factors for the promotion of accelerated bone and vascularization may also be covalently tethered to the hydrogel phase to allow for enhanced effects upon implantation. The authors have incorporated the features described above and developed a hybrid system comprised of a mechanically load-bearing scaffold infused with a self-assembling peptide hydrogel with tethered bone morphogenetic proteins-2. Moreover, electrospun fiber is also widely used in bone repair and regeneration. Gelatin nanofibers mimic both physical architectures and chemical composition of bone. They show great biocompatibility and adhesion and are the best candidate for osteogenic activities (Aldana and Abraham, 2017). The bone regeneration potential of CS alginate scaffolds was determined using critical size rat calvarial defects. In all the experimental groups no significant bone formation occurred as shown in Fig. 7.13 (Levengood and Zhang, 2014). 7.6.1.2 Nanotechnology in Vascular Cells Tissue Engineering Tissue engineering is defined as a process that improves the biological tissue with the help of a combination of cells, materials, methods, engineering, and other biochemical and

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FIGURE 7.12 Development of a combination of mechanically strong, porous scaffolds and nanofeatured selfassembling peptide hydrogels for bone tissue engineering, (A) Illustration of hybrid scaffolds composed of a mechanically strong component, and a hydrogel phase for enhanced bone regeneration abilities. (B) In vitro release kinetics of biotinylated BMP2. Amount of BMP2 released over time was measured by ELISA. Results show the cumulative release of rhB-MP2 for untethered groups bone morphogenetic proteins (BMP-2-biotin, BMP-2) versus tethered group. (C) Survival of preosteoblastic MC3T3-E1 cells in the hybrid scaffold. Images show live and dead cells cultured on hybrid scaffolds; green represents live cells. BMP2, Bone morphogenetic proteins-2; ELISA, enzyme-linked immunosorbent assay. Adapted with permission from Igwe, J.C., Mikael, P.E., Nukavarapu, S.P., 2014. Design, fabrication and in vitro evaluation of a novel polymer-hydrogel hybrid scaffold for bone tissue engineering. J. Tissue Eng. Regen. Med. 8(2), 131 142 with slight modifications.

physiochemical factors. In vascular tissue engineering four approaches known for tissue assembly: a solid scaffold based approach, in which cells seeded into a porous solid scaffold; a collagen-based approach, in which collagen matrix cells embedded; a cell sheet based assembly approach, in which monolayers of cohesive cells rolled; and acellular type. Drugs like heparin can load in electrospun nanofiber, which shows a great promise in vascular tissue repair (Aldana and Abraham, 2017). Electrospinning is the best modern cost-effective method for a variety of tissue engineering and drug delivery applications, does not cause an immunogenic reaction, and is biodegradable and compatible. However, the mechanism by which nanofiber affects cell behavior and tissue regeneration should be studied. Further study is needed to be carried out for selecting a drug loading capacity, loading and delivery method, dosage, and preclinical development (Aldana and Abraham, 2017). Blood vessel replacement was the earliest application of tissue engineering by ECseeded synthetic graft. Synthetic grafts approach for replacement of small diameter

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FIGURE 7.13 Use of CA scaffolds for cranial defect repair. (A) CA scaffolds were produced as cylinders and cut into slices (scale bar 1/4 10 mm); inset: SEM image of the scaffold showing the porous structure (scale bar 1/4 100 lm). (B) Cross-linked CA scaffolds are flexible and resilient. (C) The cranial defect model is shown during the procedure and (D) illustrated by MicroCT (line 1/4 5.0 mm). CA, Chitosan alginate. Adapted with permission from Levengood, S.K.L., Zhang, M., 2014. Chitosan-based scaffolds for bone tissue engineering. J. Mater. Chem. B 2(21), 3161 3184 with slight modifications.

blood vessel is never used alone. The major limitation of the synthetic graft was there is thrombus formation in the blood and graft surface contact. In this approach, endothelial cells have been seeded onto the synthetic surface. On the graft surface seed, the endothelial cells are harvested from the patient. After harvesting, EC is seeded directly onto the biological sources, commonly dacron or ePTFE; during this process cells will attach to the synthetic material before implantation, and this EC monolayer after implantation with the natural antithrombogenic inner surface will provide the graft. Other limitations include the normal remodeling response of the vascular system blocked by the use of nonbiodegradable synthetic material (Nerem and Seliktar, 2001). Moreover, the collagen-based approach aims to overcome the limitation of the ECseeded synthetic graft approach. In this, in a reconstituted collagen gel matrix, a vascular cell being attached. Collagen gel is used for cell signaling and cell attachment. Benefits include that there is remodeling of the cell-mediated graft as well as vascular activity (Nerem and Seliktar, 2001). In cell sheet based assembly approach, connective and

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adipose tissue made up of cultured human adipose stem cells and endogenous extracellular matrix component secreted are reconstructed (Mironov et al., 2008). Advantages include immunological mismatch being eliminated, and withstanding the pressure during arterial implantation. Also, in acellular methodology, a noncellular material is implanted and cells from host tissue are replenished. For example, in tissue engineering of acellular lung scaffold, the lung cells are exposed to harsh conditions, which leads to lysis, and the remaining cell debris is removed by a physical method, which acts as acellular lung scaffold from the existing one. The advantage of this kind of scaffold is that they grow as functional tissue cells as they assist the pluripotent stem cells (Nichols et al., 2012). Much attention has been given to tissue-engineered vascular grafts in the last few years, and as an outcome, noteworthy progress has been reported regarding achieving the remodeling of the tissue in the tissue-engineered heart valve (TEHV) constructs similar to the native blood vessels, as presented in Fig. 7.14 (Yamamoto and Shao, 2017). 7.6.1.3 Nanotechnology in Hepatic Cells Tissue Engineering Liver disease is a concern that needs clinical focus as there is an increase in patients suffering from liver disorders, and there is also the emergence of new kinds of diseases. Increase in the demand for donors for orthotopic liver transplantation led to cell-based therapy but due to certain limitations, nanotechnology in cell-based therapy has emerged. The liver has over 500 functions including protein, carbohydrate, and lipid metabolism; detoxification of endogenous and exogenous compounds; production of bile; and secretion of many serum proteins. Each year, over 30,000 people worldwide die due to liver disease, so liver disorder is one of the major concerns that needs special clinical focus. Apart from existing disorders, there is the emergence of new disorders like nonalcoholic fatty liver disease and steatohepatitis (Bhatia et al., 2014), if left untreated these may lead to progressive liver failure, fibrosis, cirrhosis, hypertension in the portal vein, and even cancer. Though prevention and treatment of these conditions is possible, the only curative therapy in end-stage liver disease is orthotropic liver transplantation. The major limitation of this concern is lack of donors, high cost, and permanent immunosuppressive treatment (Pop and Mosteanu; Bhatia et al., 2014). To overcome the major limitations encountered during orthotropic liver transplantation cell-based therapies have emerged. Cell-based therapy is the repair and restoration of cellular-based activity by using living cells, and this therapy has a wide range of advantages over orthotropic liver transplantation as this therapy is concerned with restoration of the damaged tissue by using living cells to produce the cells or tissue with properties mimicking the native tissue (Bhatia et al., 2014). The cells regenerated by cell-based therapy can be cryopreserved, genetically handled to adjust inborn errors of metabolism, or infused so there is no need of surgery, and permanent immunosuppression can be overcome by acquiring cells from the same patient (Pop and Mosteanu). Even though it has a wide range of advantages it has certain limitations like lack of functional cells, low mechanical strength of the newly formed cells, and immune incompatibility with the host cell other than that hepatocyte have special

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FIGURE 7.14 Tissue-engineered blood vessels. (A C) The first clinically used sheet-based tissue-engineered blood vessel tested on three human patients for application as high-pressure arteries. (A) A tissue-engineered graft was implanted between the axillary vein and the humeral artery as an arteriovenous shunt, (B) the vessel exhibited normal suturing and surgical handling properties, (C) the shunt maintained high flow without signs of aneurysm restenosis even after 6 months. (D O) A comparative histological analysis of human pericyte cellseeded TEVGs (D I), unseeded scaffolds (J L), and native rat aorta (M O). The H&E (D, G, J, and M), Masson’s trichrome (E, H, K, and N), and Verhoeff van Gieson (F, I, L and O) stainings demonstrated remodeling of the tissue in the TEHV construct enriched with collagen and elastin similar to the native aorta. (G I) are magnified images for the wall cross-section of (D F). Arrows indicate the remodeled tissue while ES stands for electrospun scaffold layer. ES, Embryonic stem; TEVGs, tissue-engineered vascular grafts. (A C) Reprinted from L’Heureux, N., Dusserre, N., Marini, A., Garrido, S., de la Fuente, L., McAllister, T., 2007. Technology insight: the evolution of tissueengineered vascular grafts—from research to clinical practice. Nat. Clin. Pract. Cardiovasc. Med. 4, 389 395. (D O) Adapted with permission from Yamamoto, K., Shao, Z.J., 2017. Process development, optimization, and scale-up: fluid-bed granulation. Developing Solid Oral Dosage Forms, second ed. Elsevier with slight modifications.

limitation of trans diffusion to hepatocytes which leads to the loss of function and this lead nanotechnology to enter the field of health. Nanotechnology has proven to be an effective method for reproducing and repairing the cells several times by improving cell properties and this can be achieved by providing

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the cell with the 3D microenvironment with the help of the nanostructured collagen, matrix, elastin, and lamin; at this nanoscale level there is more evidence of interaction between the cell and the microenvironment provided and this helps the cells to differentiate from others (Montaser and Fawzy, 2015). So, the scaffold of biochemical, mechanical, and electrical properties manipulating the native tissue is nanoengineered and this enhances the differentiation, proliferation, and maturation and fosters the cell growth and function; moreover the microenvironment provided by the nanotechnology prevents the hepatocytes from transdiffusing into the fibroblast and helps in retaining spheroidal morphology, which is the major challenge in the cell-based culture of hepatocytes (Krishnan and Sethuraman, 2013). Commonly used in fabricating materials are graphene, CNTs, and molybdenum and tungsten. Electrospinning technique, self-assembly, cell printing technique, solvent casting, freeze-drying, particulate leaching, microsphere-based sintering, and phase separation are some of the techniques used for fabrication of nanofibers to provide a physiological environment that mimics the physiological environment of the native tissue (Krishnan and Sethuraman, 2013). However, the most commonly employed techniques are electrospinning technique and freeze-drying. 7.6.1.4 Nanotechnology for Stem Cell Engineering The core triumph of engineered tissue by in vitro method is to use the patient’s own primary cells (autologous cells), which are further introduced in scaffolds to reimplant, but this strategy can suffer from severe drawbacks like invasive nature of cells due to unhealthy conditions. Therefore, stem cells are given more emphasis, including bone marrow mesenchymal stem cells (MSCs), embryonic stem cells, and umbilical cord derived MSCs (Howard et al., 2008). The roots of cellular genetics can be modified by incorporating or eliminating one or the more genetic fragments in need of generating new and useful organisms. Currently, researchers are focusing on how they can engineer stem cells to increase the immune sensitization against a tumor and increase lifespan in animals (Aten et al., 2012). The combination of genetic engineering with nanotechnology is proving to have good potential in the field of agriculture. Nanoinjection based on microelectromechanical systems (MEMS) fragment with moveable, nanometer-sized cut is reported to be able to hold DNA on the basis of its electrical charge. Nanotechnology is playing a vital role in genomics for improving crop features such as an increase in yield and resistance to infection, to overcome malnutrition and food insecurity (Coccia, 2012). Clinical practices in diagnostic therapies and therapeutics are exploring innovative applications by the convergence of the nanotechnology and other biomedical sciences. In addition to microenvironmental control of MSC behaviors, MSCs have shown to modulate the microenvironmental characteristic via cell cell contact and more interestingly, paracrine secretion of some cytokines and extracellular vesicles (Sui et al., 2019). Moreover, the reciprocal regulations between MSCs and microenvironments, particularly those connecting MSCs with diseased recipient microenvironmental factors, provide crucial mechanisms determining the efficacy of transplanted MSC-based bone regeneration (Fig. 7.15).

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FIGURE 7.15 The interplay between MSCs and microenvironments in bone tissue engineering and cytotherapy. MSCs, Mesenchymal stem cells. Adapted with permission from Sui, B.-D., Hu, C.-H., Liu, A.-Q., Zheng, C.-X., Xuan, K., Jin, Y., 2019. Stem cell-based bone regeneration in diseased microenvironments: challenges and solutions. Biomaterials 196, 18 30.

7.6.2 Nanotechnology-Based Tissue Engineering for Cell Labeling, Purification, Detection, and Suicide Bombing Nanoparticles, such as quantum dot (CdSeB8 nm), can be used to label a variety of cellular targets. For example, allowing cells to ingest quantum dot dyes, the cell movement can be monitored for days without photobleaching the quantum dot dyes. Cell migration behavior can be studied by watching the quantum dot ingestion behavior, so that the cell types may be distinguished. Invasive cancerous cells and immotile nontumor cells can be differentiated by seeding them on a quantum dot bed. Cells ingest quantum dots and leave a so-called phagokinetic track behind. Cancer cells exhibit diverse uncertain behavior, and some nontumor cells show specific endocytosis and migration patterns. Quantum dots can also be used to trace cells for up to 10 generations because quantum dots are distributed during dividing. Therefore dots can be used to trace cancer cells and stem cells. Moreover, nanotechnology -enabled measurement of the electric properties of a single individual cell, which is important in cell purification. The scientist is able to sort/purify cells through their different intrinsic electric properties. This application will be very

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important in stem cell purification. Currently, we do not have the techniques to get purified stem cells; with the aid of nanotechnology, large quantities of purified stem cells may be obtained in the very near future. Furthermore, cell detection is also possible by employing nanotechnology. About a thousand wires, each about 8 nm in diameter, coated with different antibody or nucleic acid, can be used to detect thousands of genes in a cell and proteins synthesized by a cell. Silicon nanowires tethered with biomolecules specifically bond to a protein expressed by cancer cells. When there is bonding, an electrical signal can be detected. The detectors made of nanowires are very sensitive and required minimal sample preparation; several bonds can be precisely detected. Moreover, the detectors are very cheap and patients can use them at home. However, the ions in the media for maintenance of the normal life of the cell may interfere with the biochemical reactions. Despite significant progress of tissue engineering in both academic research and in industry, some issues have arisen that have forced the research progress and commercial procedures to slow down. The biggest setback that the field of tissue engineering faces now is the unknown fate of tissue-engineered analogs inside the human body. That is why the FDA has approved very few tissue-engineered products so far. Before any further breakthroughs can be made, the following questions must be answered. Where will the transplanted cells go? How will they grow? What will they differentiate into? How will they be eliminated if an unexpected event occurs, such as tumor genesis? Unfortunately, most scientists are concentrating on regenerating all kinds of tissues and organs but are ignoring the control issues for proper regeneration and the fate of tissue-engineered grafts in vivo. Before these questions can be completely answered, one temporary solution called “suicide bombing” may be used. A “smart” agent in nanoscale can be taken up by all cells in the tissue-engineered grafts. The agent is inactive during a normal condition and is activated only by specific enzymes that are expressed in the cell under certain pathologic states. The activation of the agent will kill the abnormal cells. This idea is not only good for killing the abnormal cells but also suitable for the treatment of specific diseases if a particular therapeutic agent is used.

7.7 RECENT PATENTS OVERVIEW Biotechnology-based tissue engineering is widely emerging field nowadays. Tissue engineering is engineering of cells using physicochemical and biochemical parameters to improve or replace biological tissue. The areas of application of tissue engineering are cartilage, heart, bone, cancer, pancreas, etc. (Castells-Sala et al., 2013). Bone scaffolds, cartilage scaffolds, heart scaffolds have been developed using biotechnology-based tissue engineering. Recently, there has been a focus to produce regenerative heart valve as the mechanical heart valve has the limitation of thromboembolism, which can be overcome by a tissueengineered regenerative valve (Zhu and Grande-Allen, 2018). Much biotechnology-based tissue engineering has been patented.

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7.7.1 Magnetic Pole Matrices This invention regards targeted treatment of cardiovascular system (CVS) disease by magnetic polymer nanoparticle drug. Aiming at a particular site of the body is a critical issue in drug administration. To achieve a therapeutic level at the target site, a high dose of the drug has to be administered as only part of it reaches the desired target site (Steinhoff et al., 2006). High dose may also cause side effects on sites other than the target site (Steinhoff et al., 2006). Targeted drug delivery may be the solution for the side effect because it reduces the concentration at other sites (Steinhoff et al., 2006). Magnetic targeting is the one way of targeting the desired site of the organ. It involves the addition of magnetic particle into the drug carrier, and the drug is directed to the targeted site after being administered into the bloodstream using the external magnetic field (Steinhoff et al., 2006). Furthermore, these magnetic particles are compatible with the human body (Steinhoff et al., 2006). However, one of two limitations is that an accumulation of magnetic particle is affected by blood flow rate and the site having higher blood flow rate such as large arteries may require strong external magnetic field (Steinhoff et al., 2006). The second limitation is the site more than 2 cm deeper in the body is not easy to target with external magnetic field and there is also the chance of aggregate (Steinhoff et al., 2006). Biomedical devices with endothelial seeding can address the problem of thrombosis (Steinhoff et al., 2006). However, in vitro seeding is time-consuming and the risk of cell culture contamination is greater (Steinhoff et al., 2006). In in vivo seeding of magnetically modified cell to the device, the surface has also a limitation of aggregation (Steinhoff et al., 2006). This invention utilizing magnetic polymer nanoparticle solved the issues described above (Steinhoff et al., 2006).

7.7.2 Differentiable Human Mesenchymal Stem Cells This invention is related to the recipe and procedure of synthetic polymer’s nanofiber matrix which is used for the delivery of human mesenchymal cells as a scaffold application of tissue engineering (Arinzeh et al., 2006). The isolated differentiable human mesenchymal cells are dispersed in the matrix prepared using the synthetic polymer nanofiber and it gives support to the maturing human mesenchymal cells (Arinzeh et al., 2006). The polymer used in the synthesis of nanofiber is 3D as well as biocompatible (Arinzeh et al., 2006). As per a different embodiment, the polymer contains poly D, L lactide glycolide, and the preparation method is electrospinning (Arinzeh et al., 2006).

7.7.3 Degradable Polyurethane Foams Allograft bone was mainly used to cure the bone defect. However, now its use is limited due to the spreading of disease and immune response (Guelcher et al., 2012). Here, one or more biocompatible polyol and water, one or more stabilizer, and one or more cell opener are used for the preparation of resin mix (Guelcher et al., 2012). The advantages of this invention are highly stable, very porous, nonharmful to a biological system, and most

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importantly biodegradable. The binding and growth of cells in vitro is aided by the foams of the invention (Guelcher et al., 2012). This invention serves as a scaffold for the growth of the cell (Guelcher et al., 2012). Foam leakage is also present in this invention. The biodegradability of this polyurethane foam is due to its ability to undergo hydrolysis and it does not form any noxious, harmful, injurious substance that would cause an immunological response (Guelcher et al., 2012). The application of this invention is very wide. It is used in an injectable scaffold for bone and in drug and gene delivery. In addition to that, it can be applied to the surface of bone that is damaged or has a hole, can be put into the two pieces of the bone, and can also be applied to skin that has been damaged (Guelcher et al., 2012). Moldability of this invention is very wide as it can be molded into a plate or screw (Guelcher et al., 2012).

7.7.4 Multilayer Polymer Scaffolds This invention is a device and method applicable to tissue engineering (Hansford et al., 2005). The invention is a multilayer thermal plastic polymer scaffold, and helps in the curing of damaged tissue; in other words, this invention is useful for tissue regeneration (Hansford et al., 2005). The procedure for the fabrication of scaffolds is described in the patent (Hansford et al., 2005). The scaffold is 3D as well as multilayer or single layer (Hansford et al., 2005). Various ongoing/completed patents in these fields have been discussed, providing a means for further developments and possible commercialization of these treatment modalities, in Table 7.1.

7.8 CLINICAL TRIAL STATUS The loss of tissue or total organ failure is one frequent and costly proposition in healthcare. Millions of people worldwide are suffering from disability because of congenital deformity or organ loss either due to trauma, postoperatively after diseases like cancer, or pathological degeneration (Fisher and Mauck, 2013). Tissue engineering technology employing principles of bioengineering has revolutionized the treatment in such cases by providing methods to design and fabricate robust scaffolds from biocompatible materials having applications in tissue repair and regeneration. Tissue engineering enables a substantial savings in treatment cost as the in vitro substitutes for in vivo tissue repair are much cheaper than organ transplantation (Aljohani et al., 2017). Today, there are many clinical trials being undertaken in this new and interdisciplinary field aiming to provide devices that functional substitutes that can replace a damaged organ or tissue with some successful attempts having achieved patentability. This section aimed to provide insight for scientists with research interest in the areas of tissue engineering and regenerative medicine employing novel nanoscaled scaffolds. Various ongoing/completed clinical trials in these fields have been discussed providing a means for further developments and possible commercialization of these treatment modalities in Table 7.2.

BIOMATERIALS AND BIONANOTECHNOLOGY

TABLE 7.1 Various Patents and Ongoing Clinical Trials in the Field of Tissue Engineering. Patent No.

Publication Status

Publication Date

US 9,012,415

Grant

April 21, 2015

Stemmatters, Biotechnologie The patented invention comprises a gellan gum based photocross-linkable hydrogel Medicina Regenerativa (Barco, that has applications in tissue regeneration in addition to drug delivery. A preferred Portugal) embodiment narrates that the monomeric subunit of acyl gellan gum is substituted with a polymerizable moiety and a free radical based photoinitiator, which photopolymerizes in ultraviolet light. According to another embodiment, this hydrogel can also be formulated as a micro or a nanoparticle incorporating a bioactive agent in amounts that show a therapeutic effect. The invention has applications as an injectable hydrogel, biosensors, implants, medical procedures involving radio-frequency ablation, etc.

US 8,999,916

Grant

April 7, 2015

Hauser C, Seow WY

US 8,992,99

Grant

March 31, 2015

The Johns Hopkins University The scope of the appended claims includes the disclosed subject matter as well as (Baltimore, MD, United States) some modifications therewith. The derived biodegradable multicomponent cationic polymer that gets self-assembled with DNA is devised as a nanoparticle with bioactive materials useful in tissue regeneration and cancer.

US8691974 B2 Grant

April 8, 2014

Virginia Tech Intellectual Properties, Inc.

US8614189 B2 Grant

Dec 24, 2013 University of Connecticut

Original Assignee

Refer https://clinicaltrials.gov for more details. BC, Biosynthetic cellulose.

Description of Patented Technology

The scope of this patent includes an amphiphilic peptide or peptide that undergoes a conformational change for self-assembling to form a macromolecular 3D nanofibrous network, which entraps water and converts into a hydrogel consisting of at least one chemically cross-linked peptide. This hydrogel has applications in tissue replacement and regeneration, drug delivery, gene delivery, pharmaceutical formulations, cosmetic compositions, etc.

This invention describes a novel technique for the production of BC via bacterial fermentation of culture medium using a microfluidic system. The medium flows into porous molds resulting in 3D-BC nanostructure of the desired shape. The porosity of the structure is controlled using 3D printing employing ink-jet printer technology. This structure can be used as biomedical implants or scaffolds supporting cell growth. The claims of this investigation include a composite biocompatible material having sufficient mechanical strength. The scaffold consists of polar functionalized multiwalled carbon nanotubes to impart water dispersibility. PGLA is incorporated with one or a combination of the bioactive agent is the biodegradable polymer having the shape of a microsphere. The nanotubes are sintered on the microsphere at the junction of two microspheres. An embodiment therein mentions this scaffold bears suitable properties for cortical bone regeneration.

TABLE 7.2 A Brief Insight Into Clinical Trials was Undertaken to Amalgamate Tissue Engineering and Nanotechnology.

Subject/NCT No.

Location/ Sponsor

Start Year/End Year

Type/Phase

Enrollment/ Eligibility

Gender/ Healthy Primary Outcome/ Volunteer Secondary Outcome

Nanostructured allogeneic artificial human cornea for corneal trophic ulcers in patients postconventional ophthalmic treatment/ NCT0176524

Spain/ Andalusian Initiative for Advanced Therapies

2014/2019 Interventional/ 20/Adult, suffering I and II from corneal Mackie third stage

Evaluation of nanobone, a nanocrystalline hydroxyapatite silica gel synthetic bone graft for the management of intrabony periodontal defects/NCT02507596

Egypt/Cairo University

2015/2017 Interventional/ 30/30 55 years, bone All/Yes Unavailable loss with one or more pocket depth $ 6 mm, level of clinical attachment $ 5 mm detected by periapical radiographs

Chitosan nanoscaffolds Unavailable/ 2017/2019 Interventional/ seeded with Assuit Phase I mesenchymal stem cells University derived from adipose tissue in the treatment of diabetic foot ulcer/ NCT03259217

OFD, Open flap debridement.

All/No

All/No 40/Child, adult, senior, patients with grade 1 or 2 neuroischemic or neuropathic diabetic foot ulcer on Wagner scale and HbA1c # 7.5%

Study

Adverse events related to treatment/corneal stoma repair, visual acuity and corneal transparency.

Five patients implanted with the nanostructured artificial human cornea with allergenic cells of dead donors and other biomaterials for a duration of 5 years with first 36 months of inclusion and next 24 months of follow-up versus the randomized control group subjected to conventional transplantation of amniotic membrane.

Shift from baseline in the level of clinical attachment in mm in patients with chronic periodontitis after an interval of 6 months/ estimation of pocket depth in mm and bone fill defects in mm2 both at baseline and after the 6 months interval.

A comparison between the nanobone and conventional OFD surgical procedure as a negative control was conducted in 30 patients. Both the experimental and negative control groups were assigned an equal number of patients. Patients were assessed via clinical as well as radiographic parameters for a shift from baseline at 3 and 6 months interval postoperatively.

Complete epithelialization of chronic ulcer/50% healing rate within 6 months and the rate of ulcer recurrence within 1 year.

Collagen alginate consisting of curcumin loaded chitosan NP a biocompatible as well collagen as the biodegradable material may have applications in regenerative efficiency in diabetic foot ulcers and ischemic wound healing.

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257

7.9 CONCLUSION The application of nanotechnology in tissue engineering is growing rapidly, as it can help to create, repair, and/or replace cells, tissues, and organs in combinations with nanoscale materials. By applying nanotechnology in tissue engineering based treatments/therapies millions of patients can benefit. As a general protocol in tissue engineering, firstly, cells are subjected to seeding on biomimicked platform providing adhesive surfaces, followed by the cellular deposition in their own protein to make them more compatible with biological substrates. For this purpose a variety of materials have been described in this chapter with diverse characteristics to support a particular type of regeneration of cells/ tissues. These materials include CS, albumin, alginates, silica, dendrimers, PLGA, PEG, PLA, etc. This chapter successfully described that advantages of nanotechnology-based tissue engineering using electrospinning nanofibers, nanogels, and nanoparticles in comparison with traditional approaches and are employed in many of the fields for different purposes.

Acknowledgments RKT would like to acknowledge Science and Engineering Research Board (statutory body established through an Act of Parliament: SERB Act 2008), Department of Science and Technology, Government of India for the grant (Grant #ECR/2016/001964) and N-PDF funding (PDF/2016/003329) for work on targeted cancer therapy in Dr. Tekade’s Laboratory.

ABBREVIATIONS CSH DMP1 ECM Hap ICs PLGA PBT PGA PLA RNAi

calcium sulfate α-hemihydrate dentin matrix protein 1 extracellular natural matrix hydroxyapatite integrated circuits poly(lactic acid-co-glycolic acid) polybutylene terephthalate polyglycolic acid polylactic acid RNA interference

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Walmsley, G.G., McArdle, A., Tevlin, R., Momeni, A., Atashroo, D., Hu, M.S., et al., 2015. Nanotechnology in bone tissue engineering. Nanomedicine 11 (5), 1253 1263. Werani, J., Gru¨nberg, M., Ober, C., Leuenberger, H., 2004. Semicontinuous granulation—the process of choice for the production of pharmaceutical granules? Powder Technol. 140 (3), 163 168. Yamamoto, K., Shao, Z.J., 2017. Process development, optimization, and scale-up: fluid-bed granulation, Developing Solid Oral Dosage Forms, second ed. Elsevier. Zhu, A.S., Grande-Allen, K.J., 2018. Heart valve tissue engineering for valve replacement and disease modeling. Curr. Opin. Biomed. Eng. 5, 35 41.

Further reading Chaudhari, A.A., Vig, K., Baganizi, D.R., Sahu, R., Dixit, S., Dennis, V., et al., 2016. Future prospects for scaffolding methods and biomaterials in skin tissue engineering: a review. Int. J. Mol. Sci. 17 (12), 1974. Gautam, S., Chou, C.-F., Dinda, A.K., Potdar, P.D., Mishra, N.C., 2014. Surface modification of nanofibrous polycaprolactone/gelatin composite scaffold by collagen type I grafting for skin tissue engineering. Mater. Sci. Eng. C 34, 402 409. Mohamed, A., Xing, M.M., 2012. Nanomaterials and nanotechnology for skin tissue engineering. Int. J. Burns Trauma 2 (1), 29.

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