Matrices and scaffolds for DNA delivery in tissue engineering

Gene therapy represents a versatile approach for treating genetic disorders by manipulating cell functions. Research at the interface of biomaterials, gene therapy, and drug delivery has identified several design parameters for the non-viral vectors to perform optimum delivery of biologically active material into cells. Progress has been made towards achieving gene delivery, though the design principles for the materials and non-viral vectors that produce efficient delivery require further development. In this paper we summarize our contribution in design and preparation of efficient non-viral vectors by approaching two different strategies: preparation of core-like PEI-based structures and Dynamic Constitutional Frameworks as non-viral vectors. Our efforts resulted in yielding of architectures able to efficiently bind oligonucleotides of different length and even to transfect genetic materials into cells. Both strategies have a great potential in preparation of efficient and even selective gene carriers, and consistent work is being further carried out in our group.

The formulation and fabrication methods for several types of tissue engineering scaffolds with drug delivery capabilities are presented in this review. Tissue engineered constructs are temporary substitutes developed to treat damaged or lost tissue. One key component of such constructs is scaffolds that are often developed to mimic the extra cellular matrix (ECM). As natural ECM contains biomolecules to support proper growth and function of cells, inclusion of these biomolecular cues have been shown to be necessary for proper cell growth and function in tissue engineering. Thus, an effective tissue engineering scaffold should provide such biomolecular cues. This can be achieved through drug delivery in scaffolds. Studies have shown that drug delivery systems are necessary to protect drugs, and provide sustained drug release that is often needed for effective therapy. The tissue engineering features of 4 scaffold types are described, including films, hydrogels, fibers, and microspheres/nanospheres. Fabrication techniques and drug encapsulation methods for these scaffolds are reviewed in addition to some observations arising from the use of these techniques and methods.

Chitin, the second most abundant natural biomaterial composed of β(1→4)-linked 2-acetamido-2-deoxy-β-d-glucose units (or N-acetylglucosamine units), is well recognized as a substance providing many valuable biological properties, namely, biocompatibility, hemostasis, and antibacterial/anti-infectious property (Jayakumar et al., 2011). It is a fairly cytocompatible material that promotes the attachment and spreading of diverse cells such as normal human keratinocytes, broblasts, osteoblasts, and many more. Chitin is highly biodegradable in nature due to the activity of lysozymes present in the human body (Kurita et al., 2000; Noh et al., 2006). It is being used in the fabrication of a variety of medical devices and regenerative medical components due to its high crystallinity, biochemical signicance, and biocompatibility (Yimin et al., 2008). The excellent and unique properties of chitin offer a large and active surface area, making it suitable for different applications, and hence, these properties are being explored in the elds of engineering, technology, and medicine (Muzzarelli, 2011). The main disadvantage of chitin is its insolubility in organic solvents and acids. However, chitin is soluble in saturated CaCl2-methanol solvent system (Tamura et al., 2011). Using this solvent system, chitin solution was prepared (Tamura et al., 2011). This chitin solution can be easily regenerated into gel or nanogel by the addition of water/methanol (Sanoj Rejinold et al., 2011, 2012; Tamura et al., 2011). Chitin hydrogel is subjected to lyophilization to obtain chitin scaffolds (Madhumathi et al., 2010; Jayakumar et al., 2011;CONTENTS34.1 Introduction ........................................................................................................................ 673 34.2 Chitin Nanobers in Tissue Engineering and Wound Dressing ................................ 674 34.3 Chitin Nanocomposite Scaffolds in Tissue Engineering and Wound Healing ........ 675 34.4 Chitin Nanoparticles in Drug Delivery .......................................................................... 676 34.5 Chitin Nanogels in Drug Delivery and Imaging .......................................................... 676 34.6 Conclusions ......................................................................................................................... 678 Acknowledgments ...................................................................................................................... 678 References ..................................................................................................................................... 678Sowmya et al., 2011; Tamura et al., 2011). This chapter will focus on the advantages of chitin nanobers, nanogels, and nanocomposite scaffolds in drug delivery, tissue engineering, and wound healing in detail.

Tissue grafting is mostly used for repair and replacement of severely damaged tissues, the key challenges are compatibility, availability of the grafts, complex surgical process and post-operative complications. Hence, additive technologies such as three-dimensional (3D) bioprinting have emerged as promising alternative for tissue engineering in order to ensure safety, compatibility, and rapid healing. The aim of this chapter is to give an elaborate account of 3D printed scaffolds for bone, cartilage, cardio-vascular and nerve tissue engineering. Various components such as polycaprolactone, poly (lactic-co-glycolic acid), and β-tricalcium phosphate, bioglass 45S5, and nano-hydroxyapatite are combined with collagen and its derivatives to achieve specific pore size in the scaffolds for effective restoration of the defects of soft or hard tissues. Likewise, proanthocyanidin, oxidized hyaluronic acid, methacrylated gelatin, are used in collagen based 3D printed scaffolds for cartilage tissue engineering. Bioink with collagen as active component is also used for developing cardio-vascular implants with recellularizing properties. Collagen in combination with silk fibroin, chitosan, heparin sulphate and others are ideal for fabrication of elastic nerve guidance conduits. In view of the background, collagen-supplemented hydrogels can revolutionize future biomedical approaches for the development of complex scaffolds for tissue engineering.

Adeno-Associated Virus Vectors in Clinical Trials

Introduction and aims: For a scaffold material to be considered effective and efficient for tissue engineering it must be biocompatible as well as bioinductive. Silk fiber is a natural biocompatible material suitable for scaffold fabrication; however, silk is tissue-conductive and lacks tissue-inductive properties. One proposed method to make the scaffold tissue-inductive is to introduce plasmids or viruses encoding a specific growth factor into the scaffold. In this study, we constructed adenoviruses encoding bone morphogenetic protein-7 (BMP-7) and incorporated these into silk scaffolds. The osteo-inductive and new bone formation properties of these constructs were assessed in vivo in a critical-sized skull defect animal model. Materials and methods: Silk fibroin scaffolds containing adenovirus particles coding BMP-7 were prepared. The release of the adenovirus particles from the scaffolds was quantified by tissue-culture infective dose (TCID50) and the bioactivity of the released viruses was evaluated on human bone marrow mesenchymal stromal cells (BMSCs). To demonstrate the in vivo bone forming ability of the virus-carrying silk fibroin scaffold, the scaffold constructs were implanted into calvarial defects in SCID mice. Results: In vitro studies demonstrated that the virus-carrying silk fibroin scaffold released virus particles over a 3 week period while preserving their bioactivity. In vivo test of the scaffold constructs in critical-sized skull defect areas revealed that silk scaffolds were capable of delivering the adenovirus encoding BMP-7, resulting significantly enhanced new bone formation. Conclusions: Silk scaffolds carrying BMP-7 encoding adenoviruses can effectively transfect cells and enhance both in vitro and in vivo osteogenesis. The findings of this study indicate silk fibroin is a promising biomaterial for gene delivery to repair critical-sized bone defects. Introduction Innate bioactivity is one of the most important aspects when considering scaffold materials suitable for tissue engineering designs. Other properties such as biocompatibility, biodegradability, mechanical strength and induction of tissue regeneration, will determine the final potential for clinic application of the materials. A variety of bioactive materials including polymers, collagens, gels, ceramics and metals have been used as scaffolds to carry cells, growth factors, or both, to repair a variety of tissue defects (1-7). Some such materials have deleterious properties for their intended application. For example, some materials require toxic solvents when molded into a scaffold and take long time to degrade in vivo (8). Polymers such as collagens or gels are too soft or fragile to withstand external stresses, whereas ceramics are useful for treating hard tissue defects, but tend to be brittle and fracture easily. With metallic scaffolds biocompatibility and biodegradability are the major concerns, leaving this type of materials unsuitable for many tissue engineering areas. Ideally, scaffolds for effective and efficient tissue engineering should be biocompatible as well as bioinductive, which means the scaffold needs to actively induce the differentiation and regeneration of surrounding cells or tissue, from either the host or donor sources, in the tissue defect areas. And finally, the scaffold material must be non-cytotoxic when applied in vivo. Silk fibroin is a natural, biocompatible material suitable for tissue engineering (9, 10) and has several advantages compared with other biomaterials. The material is naturally water soluble, facilitating easy fabrication; toxic solvents are therefore not required. Silk fibers have greater mechanical strength than collagens or gels and gradually degrade in vivo. In spite of the many advantages of silk fibers as a scaffold material, they are tissue-conductive but not inductive, and this represents its major disadvantage. There are a number of methods for introducing growth factors or bioactive proteins into the tissue defect areas; For example: (i) by direct addition of growth factor into scaffolds (11); (ii) through specific protein-expressing cells delivered by scaffolds; or (iii) through plasmids or viruses encoding a specific protein incorporated within scaffolds. Bone morphogenetic protein-7 (BMP-7) is a growth factor with a proven ability to induce new bone formation both in vitro and in vivo (7, 12-14). The method of adenovirus transfection can produce stable and relatively long-term expression of the protein of interest. Adenovirus is one of the most commonly used viruses to transfect cells and has been proven biosafe when used in vivo (15) In this study, BMP-7 encoding adenoviruses were constructed and impregnated within the silk scaffolds. The osteo-inductivity and new bone formation by this bio-activated silk scaffold were tested in a critical-sized skull defect in mice model. This is the first ever report of gene delivery of BMP-7 using silk fibroin scaffold in bone regeneration. 2.1. Materials CellTiter 96 AQueous One solution reagent (MTS) was purchased from Promega (Promega, Alexandria NSW, Australia). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were from Gibco (Gibco BRL/Life Technologies, Victoria, Australia). All other reagents were analytical grade. Silk fibroin protein was extracted from mulberry silk cocoons following standard extraction procedures (16). Cocoons were cut into 1cm pieces, then boiled in an aqueous solution of 20 mM Na2CO3 for 30 min (~ 10% w/v of silk cocoons and Na2CO3) to remove all traces of sericin to completely degum the samples. The cocoon pieces were then washed thoroughly in distilled water and dried for 1 h at 50°C. Degummed shell pieces were dissolved in 9.3 M lithium bromide for 4 h at 60°C and then centrifuged to remove the insoluble material. The supernatant was dialyzed extensively for 3 days against deionized water with frequent changes. The protein concentration was then measured using bicinchoninic acid (BCA) protein assay (Progen Industries Ltd, Australia) (17). Recombinant adenoviruses were created using the Ad-Easy system (Stratagene, La Jolla, CA, USA). Production of the recombinant viruses (Ad-BMP-7) were performed as described in detail by our previous study (7). Adenovirus purification and concentration were done using the method of Ugai et al (18), and viral titers were estimated by the tissue-culture infective dose (TCID50), which obtained 1.8 ×10

In this paper, a comprehensive review of recent patents concerning the molecular self-assembly of peptides, peptide amphiphiles and peptidomimetics into molecules through nanoarchitectures to hydrogels is provided. Their potential applications in the field of drug delivery and tissue engineering have been highlighted. The design rules of this rapidly growing field are centered mainly on the construction of peptides in the form of peptide amphiphiles, aromatic short peptide derivatives, all-amino acid peptide amphiphiles, lipidated peptides with single and multiple alkyl chains and peptide-based block copolymers and polymer peptide conjugates. The interest in patenting of self-assembling peptides is also driven by their type (I, II, III and IV) and their ability to form well-regulated highly-ordered structures such as β-sheets/β-hairpins, α-helices/coiled coils and to hierarchically self-organize into supra-molecular structures. The applicability of these systems in cell culture scaffolds for tissue engineering, drug and gene delivery and as templates for nanofabrication and biomineralization has inspired various groups over the globe. This resulted in development of self-assembling peptides as synthetic replacements of biological tissues, designing materials for specific medical applications, and materials for new applications such as diagnostic technologies. Furthermore, biologically derived and commercially available systems are also discussed herein along with a brief account of various awarded and pending patents in the past 10 years. An overview of the diversity of the patent applications is also provided for self-assembling systems based on nano- and/or micro-scale such as fibers, fibrils, gels, hydrogels, vesicles, particles, micelles, bilayers and scaffolds.

Tissue engineering has tremendous potential for the treatment of a wide range of medical conditions. In combination with this, the developments in the fields of embryonic stem cells (ES) and adult stem cells are leading to a whole new generation of therapies. However, for any treatment to be successful, a number of key issues must be addressed; in particular the choice of cell and scaffold types used and the methods required to overcome immune rejection of any transplant. The 1-day EuroSciCon meeting, at the MI Centre in London, UK, on November 17, 2006, covered the recent advances in the fields of tissue engineering and regenerative medicine. Entitled ‘Tissue Engineering Today, Not Tomorrow’, the meeting drew presenters from throughout Europe and as far afield as Japan. It focused on the key areas of development in stem cell differentiation, scaffold development and immune modulation, each with a clinical setting or scenario in mind. The conference was opened by Dame Julia Polak (Imperial college, London, UK), who gave an interesting overview of the processes involved in developing tissue engineering from the laboratory into the clinic. Specific examples were given for the importance of biochemical and physical microenvironments on ES cell differentiation and how gene overexpression of osterix can induce bone formation. The use of bioglass for bone regeneration and collagen–polyglycolic acid composite scaffolds for bladder tissue engineering were also given. In conclusion, Dame Polak discussed how the fields of stem cells, tissue engineering, gene therapy, nanotechnology and regenerative medicine are all converging for clinical application to save lives. This gave a good introduction to the themes of the remaining talks.

Chapter 22 - Musculoskeletal tissue engineering

Viral delivery and therapeutic strategies: retroviral vectors for gene therapy adenovirus vectors for gene therapy adeno-associated virus and adeno-associated virus vectors for gene delivery ex vivo gene therapy using myoblasts and regulatable retroviral vectors design and use of herpes simplex viral vectors for gene therapy alphavirus-based vectors for vaccine and gene therapy applications. Nonviral delivery and therapeutic strategies: gene delivery with polyethylenimine receptor-mediated gene transfer modification of melanoma cells via ballistic gene therapy for vaccination polymer-encapsulated cells for gene therapy molecular interactions in lipids, DNA, and DNA-lipid complexes bio-organic colloids - macromolecules, DNA, self-assembled particles and their complexes synthetic virus systems for systemic gene therapy. Other therapeutic strategies and regulatory aspects: gene therapy applications of ribozymes antisense oligonucleotide-based therapeutics selectable markers for gene therapy suicide gene therapy regulatory aspects of gene therapy. Disease targets and therapeutic strategies: gene transfer into haematopoietic stem cells gene therapy for haematopoietic disorders gene therapy for cardiovascular disease and vascular grafts gene therapy for cancer barriers to efficient airway epithelial gene transfer -lessons from cystic fibrosis gene transfer to the central nervous system principles of gene therapy for inborn errors of metabolism gene therapy and HIV-1 infection gene delivery to the skin DNA vaccines.

Musculoskeletal conditions, including arthritis, back pain, and skeletal injuries/trauma, affect 50 % of the adult population in the U.S.A., and account for the second greatest cause of disability worldwide (www.bjdonline.org). The aggregate total annual cost of musculoskeletal diseases was $796 billion in 2011. While most musculoskeletal injuries and diseases have lowmortality, they have a tremendous impact of quality of life and morbidity. Biomaterials have a long history in orthopedics—metal and plastic implants have been used over the last 60 years to treat end-stage joint degeneration and skeletal tissue loss, and alleviate the resulting pain and disability. First-generation orthopedic implants were designed to be biologically inert and selected primarily for their mechanical properties. However, they do not replicate the function of normal tissue and have a limited life span in the body, which is especially problematic in younger patients. Recent decades have focused on the development of biomaterials that are biodegradable and incorporate tissue-specific physicochemical cues to guide cell processes and tissue morphogenesis, with the goal of restoring natural tissue structure and function. These advances in biomaterials provide critical enabling technologies for the delivery of stem cells, gene therapies, and new biologics in the orthopedic field. This special issue highlights the recent advances and future directions in orthopedic biomaterials and drug delivery systems, and showcases emerging pharmaceutical and regenerative approaches to treat injuries, diseases, and disorders of the musculoskeletal system. Non-union bone fractures are a significant clinical challenge, and bone tissue engineering could have a key impact in improving current clinical options. Telvin et al. review the application of stem cells from various sources towards bone tissue engineering. The ability of transplanted stem cells to survive, continue to differentiate, and form bone tissue is key to making stem cell-mediated bone tissue regeneration approaches successful. The research article by Wen et al. precisely address this challenge. Their study compares the in vivo bone tissue forming ability of human bone marrow derived stem cells and human embryonic stem cells using osteoinductive mineralized materials as a scaffold. The article by Nyberg et al. reviews progress and challenges in achieving precise temporal and spatial control of growth factor presentation for functional bone tissue engineering ex vivo and in vivo. Ultimately, successful bone regeneration requires integration between the repair and native bone tissue. Botchway and colleagues address this critical issue, by modifying the surface of a bone allograft with FTY720, a synthetic analog of sphingosine 1-phosphate, to promote endogenous bone formation and osseous integration at the interface. Another key hurdle for the success of bone tissue engineering is vascularization in vivo. The review by Garcia and Garcia summarizes the current strategies and new developments targeted at vascularization of engineered bone, including growth factor and cell delivery, gene therapy, biomaterials, and combination approaches. The article by Tonello et al. demonstrates the potential of human placental matrix for promoting angiogenesis and its controlled release from biodegradable microparticles. In addition to bone, this special issue contains articles that highlight advances in cartilage tissue regeneration and osteoarthritis (OA) therapies. Osteoarthritis is the leading cause of * Blanka Sharma blanka.sharma@bme.ufl.edu

Microneedles for gene and drug delivery in skin cancer therapy

Tissue engineering (TE) is a modern approach to improve or restore tissues that has been diseased or damaged by combining the factors in growth signaling and appropriate cells to form compatible biomaterial scaffold. Bone repair has been a major global health challenge in recent decades. Bone transplantation is widely used as an effective clinical treatment for this purpose. However, there are several serious issues with transplantation, including a shortage of autologous bone, immune rejection, the risk of virus transmission with allogeneic bone, and postoperative complications. In recent years, scaffolds for bone tissue engineering have emerged as a promising alternative for bone repair. These scaffolds have porous structures that mimic the extracellular matrix, which can enhance the migration, proliferation, and differentiation of osteoblasts. This process accelerates the repair of bone defects.The usage of bioactive materials has become important tool in tissue engineering and regenerative medicine application as they are to mimic tissues mechanically, chemically, and physically. Polymeric scaffold provides numerous advantageous for tissue engineering since the physicochemical properties such as porosity, pore size, solubility and biocompatibility can be controlled. Therefore, research works were carried out to explore the potential of various materials in producing bone scaffolds in tissue engineering technology. As for both newcomers and experts, this review paper will help them to find information on various types of biomaterials in imparting or enhancing their biological properties such as biocompatibility, bacterial inhibition, tissue regeneration and cell growth, bio inertness, bioactive and resorbability for bone scaffold tissue engineering application.

Calcium phosphate ceramics have been widely considered as scaffolds for bone tissue engineering. Selection of the best support for cultured cells, crucial for tissue engineered systems, is still required. We examined three types of calcium phosphate compounds: α-tricalcium phosphate - the most soluble one, carbonate hydroxyapatite - chemically the most similar to the bone mineral and biphasic calcium phosphate - with the best in vivo biocompatibility in order to select the best support for osteoblastic cells for tissue engineered systems. Human osteoblasts were tested in direct contact with both dense samples and 3D scaffolds in either static or dynamic culture. Cell viability, cell spreading, osteogenic cell capacity, and extracellular matrix production were examined. The obtained data indicate that biphasic calcium phosphate is the optimal cell-supporting material. In addition, dynamic culture improved cell distribution in the scaffolds, enhanced production of the extracellular matrix and promoted cells osteogenic capacity. Biphasic calcium phosphate should be recommended as the most suitable matrix for osteogenic cells expansion and differentiation in tissue engineered systems.

Coordinated investigations into the interactions between biologically mimicking (biomimetic) material constructs and stem cells advance the potential for the regeneration and possible direct replacement of diseased cells and tissues. Any clinically relevant therapies will require the development and optimization of methods that mass produce fully functional cells and tissues. Despite advances in the design and synthesis of biomaterial scaffolds, one of the biggest obstacles facing tissue engineering is understanding how specific extracellular cues produced by biomaterial scaffolds influence the proliferation and differentiation of various cell sources. Matrix elasticity is one such tailorable property of synthetic scaffolds that is known to differ between tissues. Here, we investigate the interactions between an elastically tailorable polyethylene glycol (PEG)-based hydrogel platform and human bone marrow-derived mesenchymal stem cells (hMSCs). For these studies, two different hydrogel compositions with elastic moduli in the ranges of 50–60 kPa and 8–10 kPa were implemented. Our findings demonstrate that the different elasticities in this platform can produce changes in hMSC morphology and proliferation, indicating that the platform can be implemented to produce changes in hMSC behavior and cell state for a broad range of tissue engineering and regenerative applications. Furthermore, we show that the platform’s different elasticities influence stem cell differentiation potential, particularly when promoting stem cell differentiation toward cell types from tissues with stiffer elasticity. These findings add to the evolving and expanding library of information on stem cell–biomaterial interactions and opens the door for continued exploration into PEG-based hydrogel scaffolds for tissue engineering and regenerative medicine applications.