1 Chapter CHAPTER LITERATURE REVIEW AND INTRODUCTION Tissue engineering is the combination of cell, engineering and material methods, and application of suitable biochemical and physio-chemical factors to improve or replace biological functions. While most definitions of tissue engineering cover a broad range of applications, in practice the term is closely associated with applications that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, etc .). As growth factors are usually expensive and easily damaged, their delivery to specific tissue positions efficiently without losing their bioactivity is a major challenge. Drug delivery refers to the delivery of a pharmaceutical compound to humans or animals. Apart from drug delivery for anticancer agents, drug delivery for tissue regeneration is gaining more attention over past decades. For the case of anticancer drug delivery, the anticancer agents, such as paclitaxel, are employed. As for drug delivery for tissue engineering, proteins, peptides or DNAs are always utilized. Considering that protein, peptide and DNA are very easily digested or damaged in organic solvents and cell plasma, the delivery strategies have been highly emphasized over past years. Most commonly and traditionally used methods of delivery include non-invasive oral (through the mouth), nasal, inhalation, and rectal routes. Protein, peptide and DNA, however, could not be efficiently delivered via these routes as they might be susceptible to degradation and the Chapter most serious weakness is that their concentration in specific tissue might be insufficient to trigger expected biological responses after going through the dilution of circulation system. For this reason, local drug delivery devices have been developed. In this way, high drug concentration can be guaranteed and at the same time systemic toxicity from drug is minimized. In order to achieve the aim of tissue growth and remodeling, correct construction of drug delivery devices for specific tissues is the most crucial challenge, as the interactions between material, drug and cell are extraordinarily complicated. In general, basic requirements for drug delivery devices include negligible toxicity, high concentration of biological factors in tissue, high transfection efficiency of DNA to cells (for the case of gene delivery), suitable degradation rate of devices with adequate sustained drug release (Langer and Vacanti, 1993). Over past decades, many release dosage forms have been developed for drug delivery, such as nanoparticle, microparticle, polymeric disc and film (Freitas et al., 2005). Unfortunately one common problem with such dosage forms is the undesirable release profile of drugs. For nanoparticle and microparticle, a burst release at an early stage together with a very short release course is the major weakness. Xie and Wang reported that paclitaxel loaded nanoparticles caused serious cytotoxicity but the effective stage can not sustain (Xie and Wang, 2005). For disc and film, their release profiles are difficult to tailor and in most cases their release rate is too low (Jackson et al., 2004). As a result, the drug concentration in target tissue may be insufficient to trigger expected biological responses. Recently, some researchers have developed several DNA delivery devices with high gene transcription (Conwell and Huang, 2005; Schreier, 1994; Chapter Tomlinson and Rolland, 1996). There have been two major approaches proposed: the viral mediated and non-viral mediated gene transfection (Ledley and Ledley, 1998). Considering the immunological and safety issues of viral vectors, necessity of the development of non-viral vector systems has been increasingly magnified. Although with several advantages, namely the lower toxicity and immune responses or no integration into the genome, non-viral vectors are always unable to transfect cells efficiently due to the non-optimal device design, in the aspect of interactions between material and gene, material and cells as well as gene and cells. In order to tackle the disadvantages and shortcomings with current drug delivery devices, novel drug delivery devices should be developed. The aim is to obtain and keep high enough concentration of protein delivered or expressed (for the case of gene delivery) in tissue, and pose minimal toxicity to environmental cells. In the following sections, literature reviews on “tissue engineering”, “drug delivery dosage forms”, and “polymeric material for drug delivery” will be interpreted separately in more details. 1.1 Tissue engineering In 2003, the National Science Foundation (NSF)* published a report entitled "The Emergence of Tissue Engineering as a Research Field" (Langer and Vacanti, 1993), which gives a thorough description of the history of this field. A commonly applied definition of tissue engineering, as stated by Langer and Vacanti (Langer and Vacanti, * Please refer to http://www.nsf.gov and http://en.wikipedia.org/wiki/Tissue_engineering Chapter 1993), is "an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ"(Langer and Vacanti, 1993). Tissue engineering has also been defined as "understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use" (MacArthur, 2005). A further description goes on to say that an "underlying supposition of tissue engineering is that the employment of natural biology of the system will allow for greater success in developing therapeutic strategies aimed at the replacement, repair, maintenance, and/or enhancement of tissue function" (Murray et al., 2007). Powerful developments in the multidisciplinary field of tissue engineering have yielded a novel set of tissue replacement parts and implementation strategies. Scientific advances in biomaterials, stem cells, growth and differentiation factors, and biomimetic environments have created unique opportunities to fabricate tissues in the laboratory from combinations of engineered extracellular matrices ("scaffolds"), cells, and biologically active molecules (Langer and Vacanti, 1993). Among the major challenges now facing tissue engineering is the need for more complex functionality, as well as both functional and biomechanical stability in laboratory-grown tissues destined for transplantation. The continued success of tissue engineering, and the eventual development of true human replacement parts, will grow from the convergence of engineering and basic research advances in tissue, matrix, growth factor, stem cell, and developmental biology, as well as materials science and bioinformatics (Donald and Mohammad, 2001). Chapter 1.1.1 Cells Tissue engineering utilizes living cells as engineering materials. Examples include using living fibroblasts in skin replacement or repair, cartilage repaired with living chondrocytes, or other types of cells used in other ways (Weng and Wang, 2001). Cells became available as engineering materials when scientists at Geron Corp.* discovered how to extend telomeres in 1998, producing immortalized cell lines. Before this, laboratory cultures of healthy, noncancerous mammalian cells would only divide a fixed number of times, up to the Hayflick limit (Hayflick, 1965). Cells are often categorized by their source in the following forms: Autologous cells are obtained from the same individual as that to which they will be reimplanted. Autologous cells have the fewest problems with rejection and pathogen transmission, however in some cases might not be available. For example in genetic disease suitable autologous cells are not available. Also very ill or elderly persons, as well as patients suffering from severe burns, may not have sufficient quantities of autologous cells to establish useful cell lines. Allogeneic cells come from the body of a donor of the same species. While there are some ethical constraints to the use of human cells for in vitro studies, the employment of dermal fibroblasts from human foreskin has been demonstrated to be immunologically safe and thus a viable choice for tissue engineering of skin. * Please refer to http://www.geron.com/ Chapter Xenogenic cells are those isolated from individuals of another species. In particular animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models. Primary cells are from an organism. Secondary cells are from a cell bank. Stem cells are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. According to their source stem cells are divided into "adult" and "embryonic" stem cells, the first class being multipotent and the latter mostly pluripotent; some cells are totipotent, in the earliest stages of the embryo. While there is still a large ethical debate related with the use of embryonic stem cells, it is thought that stem cells may be useful for the repair of diseased or damaged tissues, or may be used to grow new organs. 1.1.2 Growth factors Growth factor refers to a naturally occurring protein capable of stimulating cellular proliferation and cellular differentiation. Growth factors are important for regulating a variety of cellular processes. Typically they act as signaling molecules between cells. They often promote cell differentiation and maturation, which varies between growth factors. For example, bone morphogenic proteins stimulate bone cell differentiation, Chapter while fibroblast growth factors and vascular endothelial growth factors stimulate blood vessel differentiation (angiogenesis). 1.1.2.1 Definition of growth factors Growth factor is sometimes used interchangeably among scientists with the term cytokine. Historically, cytokines were associated with hematopoietic (blood forming) cells and immune system cells (e.g., lymphocytes and tissue cells from spleen, thymus, and lymph nodes). For the circulatory system and bone marrow in which cells can occur in a liquid suspension and not bound up in solid tissue, it makes sense for them to communicate by soluble, circulating protein molecules. However, as different lines of research converged, it became clear that some of the same signaling proteins the hematopoietic and immune systems used were also being used by all sorts of other cells and tissues, during development and in the mature organism. While growth factor implies a positive effect on cell division, cytokine is a neutral term with respect to whether a molecule affects proliferation. In this sense, some cytokines can be growth factors, such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). However, some cytokines have an inhibitory effect on cell growth or proliferation. Yet others, such as Fas ligand (FasL) are used as "death" signals; they cause target cells to undergo programmed cell death or apoptosis. Chapter 1.1.2.2 Examples of growth factors* Individual growth factor proteins tend to occur as members of larger families of structurally and evolutionarily related proteins. There are dozens of growth factor families such as TGF-beta (transforming growth factor-beta), BMP (bone morphogenic protein), neurotrophins (NGF, BDNF, and NT3), fibroblast growth factor (FGF), and so on. Several well known growth factors are: * • Transforming growth factor beta (TGF-β) • Granulocyte-colony stimulating factor (G-CSF) • Granulocyte-macrophage colony stimulating factor (GM-CSF) • Nerve growth factor (NGF) • Neurotrophins • Platelet-derived growth factor (PDGF) • Erythropoietin (EPO) • Thrombopoietin (TPO) • Myostatin (GDF-8) • Growth differentiation factor-9 (GDF9) • Acidic fibroblast growth factor (aFGF or FGF-1) • Basic fibroblast growth factor (bFGF or FGF-2) • Epidermal growth factor (EGF) • Hepatocyte growth factor (HGF) Please refer to http://www.med.unibs.it/~marchesi/growfact.html. Chapter 1.1.2.3 Bone morphogenetic proteins (BMPs) BMPs are a group of growth factors and cytokines known for their ability to induce the formation of bone and cartilage (Chen et al., 2004). Originally, seven such proteins were discovered. Of these, six of them (BMP-2 through BMP-7) belong to the Transforming growth factor beta superfamily of proteins. Since then, thirteen more BMPs have been discovered, bringing the total to twenty. BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs results in mobilization of members of the SMAD family of proteins. The signaling pathways involving BMPs, BMPRs and Smads are important in the development of the heart, central nervous system, and cartilage, as well as post-natal bone development. They have an important role during embryonic development on the embryonic patterning and early skeletal formation. As such, disruption of BMP signaling can affect the body plan of the developing embryo. For example, BMP-4 and its inhibitors noggin and chordin help regulate polarity of the embryo (i.e. back to front patterning). Mutations in BMPs and their inhibitors (such as sclerostin) are associated with a number of human disorders which affect the skeleton. Several BMPs are also named “cartilage-derived morphogenetic proteins” (CDMPs), while others are referred to as “growth differentiation factors” (GDFs). Chapter 10 Figure 1.1 Model of human bone morphogenetic protein created using Cn3D* Bone morphogenetic protein or BMP-2 (Nickel et al., 2001; Kawamura et al., 2003; Marie et al., 2003) is a protein that belongs to the TGF-β superfamily of proteins. It, like other bone morphogenetic proteins, plays an important role in the development of bone and cartilage. It is involved in the Hedgehog pathway, TGF-beta signaling pathway, and the Cytokine-cytokine receptor interaction. 1.2 Polymeric materials for drug delivery 1.2.1 Non-degradable materials The production of drug-loaded (drug in a broad sense including chemical drugs, protein, DNA, et al.) polymeric pellets and microspheres introduced a new concept in drug delivery: Drugs can be delivered to tissues in a sustained, continuous and predictable fashion using polymers as delivery devices. Since the discovery of the first controlledrelease polymer systems in 1960s, new drug delivery devices have become available for clinical use, including steroid-releasing reservoirs for contraception (Norplant® and * Please refer to www.cytok.com/structure.php?start=120 16 Chapter Table 1.1 Properties of some biodegradable polymers (adapted from Daniels et al., 1990) Polymer Melting Point Glass-transition Modulus Degradation (ºC) Temperature (ºC) (Gpa)a Time (month)b PGA 225-230 35 ~ 40 7.0 ~ 12 L-PLA 173-178 60 ~ 65 2.7 > 24 DL-PLA Amorphous 55 ~ 60 1.9 12 ~ 16 PCL 58-63 -65 ~ -60 0.4 > 24 PDO N/A -10 ~ 1.5 ~ 12 PGA-TMC N/A N/A 2.4 ~ 12 85/15 DL-PLGA Amorphous 50 ~ 55 2.0 5~6 75/25 DL-PLGA Amorphous 50 ~ 55 2.0 4~5 65/35 DL-PLGA Amorphous 45 ~ 50 2.0 3~4 50/50 DL-PLGA Amorphous 45 ~ 50 2.0 1~2 a: tensile strength or flexural modulus b: time to complete mass loss. Rate also depends on part geometry 1.2.3 Poly(DL-lactide-co-glycolide) Using the polyglycolide and poly(L-lactide) properties as a starting point, it is possible to copolymerize the two monomers to extend the range of homopolymer properties. Copolymers of glycolide with both L-lactide and DL-lactide (Figure 1.2) have been developed for both drug delivery and tissue engineering applications. It is important to note that there is not a linear relationship between the copolymer composition and the mechanical and degradation properties of the materials. For example, a copolymer of 50% glycolide and 50% D,L-lactide degrades faster than either homopolymer. Copolymers of L-lactide with 25-70% glycolide are amorphous due to the disruption of the regularity of the polymer chain by the other monomer. A copolymer of 90% glycolide Chapter 17 and 10% L-lactide was developed by Ethicon as an absorbable suture material under the trade name Vicry1. It is absorbed within 3-4 months but has a slightly longer strengthretention time. It is proved that PLGA 50:50 (50/50 DL-PLG) is the best for drug delivery due to its favorable degradation rate. Figure 1.2 Molecular formula of poly(DL-lactide-co-glycolide) Although degradation and mechanical strength of polymeric materials can be tailored to some extent according to customer’s requirements, no optimal material has been developed for bone tissue engineering. PLGA 50:50 (50/50 DL-PLG, Table 1.1) is widely used in drug delivery applications due to its favorable degradation rate, but its mechanical properties are far from meeting requirements for bone tissue engineering. Synthetic bone grafts based on metals and some ceramics, like hydroxylapatite and tricalcium phosphate, have different mechanical properties when compared to human bone tissue, and this incompatibility often results in implant failure and consequently, revision surgery (Lane and Bostrom, 1998; Lane et al., 1999). However, the mechanical strength of HAp may be a good supplement to PLGA-based delivery devices. One of the purposes of this thesis is to develop PLGA-based novel drug delivery devices, which can achieve and maintain sufficiently high concentrations of protein or DNA in tissue and have acceptable mechanical properties. These devices developed in this thesis may be of Chapter 18 importance in combining tissue engineering and drug delivery nicely, with potential in clinical applications. 1.3 Drug delivery dosage forms In addition to the attachment of growth factors to polymeric scaffolds, growth factors can be directly incorporated within scaffolds during the fabrication process. These methods generally involve the mixing of the polymers with the growth factor before processing to form the scaffold. The main challenge of this set of methods is to ensure that the processing conditions not significantly denature the incorporated growth factors while allowing the secretion of the growth factor in a sustained manner (Sokolsky-Papkov et al., 2007). 1.3.1 Hydrogel scaffolds Hydrogel matrices are physically or chemically crosslinked water-soluble polymers, which swell to form a gel like substance on exposure to water (Drury and Mooney, 2003). Hydrogels are formed by the crosslinking of polymer chains to form a scaffold made up of connected polymer chains. Crosslinking can be done either through physical (UV irradiation, freeze drying and heating) and chemical means such as ionic crosslinking in presence of divalent ions or utilization of chemical crosslinkers such as glutaraldehyde and carbodiimide. Hydrogels can be made from synthetic polymers such as poly(ethylene glycolide), poly vinyl alcohol, or naturally occurring polymers such as collagen, chitosan and gelatin. Release of growth factors from hydrogels is believed to be either through diffusion of the growth factor, mechanical stimulation, or hydrolytic degradation of the Chapter 19 scaffold (Drury and Mooney, 2003). Some examples of hydrogels are reported by Chenite and coworkers (Chenite et al., 2000). In this particular work hydrogel formation was induced by temperature, with bone morphogenic protein (rhBMP) introduced into the hydrogel material (temperature sensitive chitosan-polyol salt combination) during fabrication and shown to be effective in promoting de-novo bone and cartilage formation in vivo. However due to the release kinetics of growth factor through hydrogels being mainly diffusion controlled through the numerous aqueous channels within the hydrogels, extended release of the proteins is not easily achieved. Immobilization of the growth factor within the biodegradable hydrogel seems to improve the release kinetics, with release of the immobilized factor being controlled by the degradation of the hydrogel. Several chemical and physical methods exist for the immobilization of growth factors in hydrogels. Chemical methods involving attachment of reactive groups to the growth factor for crosslinking with hydrogel polymers have seen increasing use within cartilage tissue engineering (Holland et al., 2007). Physical methods of immobilization of growth factors within scaffolds exploits electrostatic interactions that might occur between charges on growth factors and that on the polymer chains of the hydrogel (Tabata, 2005). 1.3.2 Microspheres based scaffolds Polymer scaffolds can also be manufactured from growth factor loaded microspheres. Scaffold fabrication from microspheres could range from fusing of microspheres to form scaffolds, to formation of microsphere containing composite scaffolds. Growth factor Chapter 20 loaded microsphere can be formed either through chemical cross-linking of aqueous solutions of natural polymers in the presence of growth factors or by solvent extraction. The solvent extraction method is mainly used for formation of microspheres from synthetic polymers. It involves the evaporation of an organic solvent such as dichloromethane (chloroform and ethyl acetate have also been used), from dispersed oil droplets containing both the polymer and the growth factor (Freiberg and Zhu, 2004). Two popular variations of this technique are currently used and are described below. The double emulsion method involves the dispersion of an aqueous solution of the growth factor in an organic solution containing the polymer - leading to the formation of the primary water in oil (W/O) emulsion. A secondary oil in water (O/W) emulsion is formed by dispersing under continuous mechanical agitation of the primary W/O emulsion in an aqueous medium containing stabilizers such as poly(vinyl alcohol) (PVA) or poly(ethylene glycol) (PEG). Microspheres are produced by the evaporation of the organic solvent from the emulsion droplets. The microspheres are then collected either by centrifuging or filtration, washed and lyophilised to obtain the free flowing and dried microspheres. Microspheres prepared from poly(DL-lactic-co-glycolic acid) (PLGA) using the double emulsion method have been used for incorporation of BMP-2 (Hiraoka et. al., 2006), TGF-β1 (Tabata et al., 1999) and PDGF-BB (Nakahara et al., 2003). The single emulsion method involves bypassing the primary W/O emulsion stage, by dispersing or dissolving the growth factor directly into the organic solution of the polymer. Microspheres are then formed by the subsequent formation of the O/W emulsion, evaporation of the organic solution from the emulsion droplets. The growth factor could be micronized by lyophilisation with PEG, CDs etc before dispersing it into the organic 21 Chapter polymer solution to improve its entrapment efficiency (Morita et al., 2000). In the work carried out investigating the stimulation of angiogenesis within engineered tissues, King and Patrick (King and Patrick, 2000) were able to produce using the single emulsion method vascular endothelial growth factor (VEGF) loaded microspheres with a sustained release of the active growth factor over a 28 day period. Growth factor released from these microspheres was shown to increase the proliferation of human umbilical vein endothelial cells in culture. Growth factor loaded microspheres can be used for tissue engineering applications either by fusing of microspheres directly to form scaffolds, or combination with other scaffold forming materials to construct composite scaffolds. In work described by Wei and coworkers (Wei et al., 2006), PDGF-BB (platelet derived growth factor) loaded microspheres were incorporated in poly(lactic acid) (PLLA) nanofibrous scaffold. Due to its structural similarity to collagen (which is a major extracellular component of bone, cementum, and periodontal ligament (PDL)), the nano-fibrous structure has been demonstrated to improve cell attachment (Woo et al., 2003), and possibly to stimulate cell proliferation and differentiation as well (Xu et al., 2004). Sustained release for days to months of bioactive PDGF-BB was achieved by the microspheres in scaffold. The authors of this work postulated that composite scaffold based on 3D nano fibrous material can be used for complex tissue regeneration. Kempen and Hedberg and their coworkers (Kempen et al., 2003; Hedberg et al., 2002) showed the application of PLGA and poly(propylene fumarate) (PPF) microspheres in microsphere/scaffold composite for controlled release of growth factors for bone tissue engineering. Chapter 22 1.3.3 Porous solid scaffolds Formation of growth factor loaded porous solid scaffolds can be achieved through several techniques. The main techniques involve either one-step direct formation of growth factor loaded scaffolds or formation of growth factor loaded microspheres which can then be fabricated to form scaffolds. 1.3.3.1. Solvent casting/particulate leaching Solvent casting-particulate leaching is one of the first techniques that have been utilized in formation of porous scaffolds for tissue engineering applications, although it is being replaced by more sophisticated techniques. This technique involves the pouring of the polymer solution into a bed of salt particles with defined size. Precipitation of the polymer by evaporation of the polymer solvent under vacuum, followed by leaching of the salt particles in distilled water leads to the formation of a highly porous scaffold with well defined pores (Hile et al., 2000). This process of scaffold production is a popular technique and has been used in the production of angiogenic factors loaded scaffolds for the induction of angiogenesis (Hile et al., 2000). These factors are emulsified within the polymer solution prior to the solvent casting-salt leaching process. As the organic solvent is removed, the polymer precipitates and encapsulates the angiogenic factors. 1.3.3.2. Supercritical processed foam Denaturation of growth factors during scaffold fabrication has been indicated as the main cause of activity loss of protein released from scaffold. The need to improve on the current fabrication methods has led to the creation of scaffolds using supercritical fluids. Chapter 23 The benefits of using these process methodologies are due to the ability to form growth factor loaded scaffolds in conditions that are favourable to growth factors, avoiding the need for using organic solvents and harsh process conditions (Kanczler et al., 2007). Supercritical fluids (SCF) are substances that exist at temperatures and pressures that exceed its critical temperature and pressure. SCFs combine the properties of the two phases from which they are formed; they have densities and solvating properties similar to those of liquids, alongside diffusion and viscosity properties of a gas. CO2 is the most common candidate for use as a SCF due to its low toxicity, ease of use and low cost. Scaffold fabrication using SCF technology (RESS technique) normally involves the dissolution of the polymer/growth factor complex in supercritical CO2 (scCO2). This is followed by a rapid expansion of the mixture into a low temperature and pressure environment which leads to scaffold formation (Vasita and Katti, 2006). Another technique utilizing the scCO2 is the PGSS technique, which involves using the scCO2 to plasticize the polymer by lowering the glass transition temperatures (Tg) of the polymer. This technique involves the application of the SCF under pressure into the mixture of polymer and growth factor until the mixture is saturated. This is followed by depressurization through a nozzle, leading to the formation of highly porous scaffolds as the gas comes out of liquefied polymer. This process has the advantages that the starting material does not have to be soluble in the SCF, and that no organic solvents are required during processing (Quirk et al., 2004). Mooney and coworkers first showed the possibility of forming scaffolds using CO2, by producing porous polymeric matrices with carbon dioxide (Mooney et al., 1996; Harris et al., 1998). In further experiments they incorporated vascular endothelial growth factor (VEGF) during the scaffold fabrication, Chapter 24 and were able to show the release of the active growth factor over a 12 day period (Murphy et al., 2000). VEGF encapsulated PLA scaffolds have also been shown to stimulate the formation of blood vessel network in an ex vivo chick chorioallantoic membrane (CAM) angiogenesis model (Kanczler et al., 2007). In a study carried out by Yang and colleagues, the effects of recombinant human pleotrophin (PTN) loaded PLGA scaffolds prepared using the supercritical fluid technology, was compared to a PLGA scaffold without the loaded growth factor on primary human bone marrow cells. Negligible cellular growth was observed on PLGA scaffold alone while the PTN loaded scaffold was shown to induce proliferation of the cell line with total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase-specific activity observed with increasing growth factor concentrations (Yang et al., 2003). In another study carried out by the same group, porous BMP-2-encapsulated poly(DL-lactic acid) (PLA) scaffolds generated by the supercritical fluid process was shown to promote adhesion, migration, proliferation, and differentiation of human osteoprogenitor cells on three-dimensional scaffolds. The BMP-2-encapsulated polymer scaffolds showed evidence of new bone matrix and cartilage formation after subcutaneous implantation into athymic mice (Yang et al., 2004). 1.3.4 Electrospun fiber Recently, natural and synthetic polymers have been processed into porous fibrous scaffolds for tissue engineering applications using the electrospinning process. Scaffolds generated by electrospinning contain nanoscale fibers with microscale-interconnected pores, resembling the extracellular matrix (ECM) (Li et al., 2006) and the three- Chapter 25 dimensional nature of the scaffold allows for cells to infiltrate the matrix and proliferate. Collagen, fibrinogen, chitosan, poly(lactic acid), poly(L-lactide-co-caprolactone), and poly(D,L-lactide-co-glycolide) are just a few of the polymers being investigated for use in electrospun tissue engineering constructs. The electrospinning process produces fibers with nanoscale diameters through the action of a high electric field (Li et al., 2006). This process involves the subjecting of a polymer solution in a capillary to an electric field generated by high voltage differences. When the generated electric field exceeds the surface tension of the polymer solution, ejection of a polymer jet occurs. This polymer jet is targeted towards a grounded collector. Enroute to the collector plate, the polymer jet looses stability, leading to the stretching of the jet, with solid fibers deposited on the collector in the form of a nonwoven fabric. By the adjustment of solution and operating parameters, fiber diameter and porosity of fiber mats can be controlled during electrospinning (Zong et al., 2002; Zong et al., 2003), which makes this a promising technique for fabrication of tissue-engineered scaffolds. Li and coworkers produced silk fibroin fiber scaffolds containing bone morphogenetic protein (BMP-2) and/or nanoparticles of hydroxyapatite (nHAp) using electrospinning, and showed its ability in inducing in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs) (Li et al., 2006). The electrospun scaffolds containing BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers than the electrospun scaffolds without BMP-2, indicating that the scaffolds were an efficient delivery system for BMP-2. Chapter 26 1.4 Summary Electrospinning is the most widely used method for fabrication of nanofiber non-woven matrices, due to its simplicity and efficiency. Supercritical CO2 foaming is a very friendly technique for fabricating growth factor loaded scaffolds as the operating parameters are favourable to growth factors, avoiding the need for use of organic solvents and harsh process conditions. Moreover, the microporous and macrofibrous 3-D scaffolds from electrospining and supercritical CO2 foaming make good preparation for blood circulation, tissue regeneration and subsequent remodeling. Among all the polymers which are biocompatible, biodegradable, bioresorbable and approved by the Food and Drug Administration (FDA), poly(DL-lactide-co-glycolide) (PLGA) is advantageous due to its controlled degradation behavior and tunable mechanical properties according to the specific requirements for the target tissue. The rate of polymer degradation should be carefully controlled to synchronize with the rate of tissue formation and in-growth to achieve successful regeneration or repair within a desired time frame. However, these hydrophobic polymers (PLGA 50:50, PLGA 65:35, PLGA 85:15 et al) degrade into acidic by-products, which might cause negative effects on cell adhesion and growth. Moreover, the devices made of PLGA are soft and their mechanical properties are not sufficient for tissue support and remolding. 27 Chapter CHAPTER OBJECTIVES AND ORGANIZATION OF THESIS This chapter describes the main objectives and motivation for the thesis and highlights the organization of the thesis. 2.1 Objectives and motivation of thesis Though widely used, PLGA-based drug delivery devices have several drawbacks such as the following: • Mechanical properties of PLGA copolymers are not sufficient for tissue support and remolding. • Negatively charged surface of PLGA matrix is unfavorable for cell adherence and subsequent cell proliferation. • Acidic by-products of PLGA copolymer might cause negative effects on cell adhesion and subsequent cell proliferation. • High delivery efficiency and low cytotoxicity are difficult to achieve simultaneously. • The integrity and bioactivity of protein or DNA is difficult to maintain in the fabrication of drug delivery devices. Chapter 28 Due to the abovementioned disadvantages, there is an intense need of developing new drug delivery devices in the aspect of improving the mechanical and medical performances of PLGA matrix. The overall aim of this research was to develop various biodegradable polymer matrix-based controlled drug (BMP-2 protein or DNA) delivery devices by extending the applications of electrospinning and supercritical fluid techniques to biomedical field and characterize the devices using various state-of-the-art analytical techniques. Specifically, the research areas of this research include: • Developing BMP-2 protein loaded PLGA/hydroxylapatite composite fibrous scaffolds for bone regeneration. • Characterizing the performance of BMP-2 loaded PLGA/hydroxylapatite composite fibrous scaffolds in vivo. • Optimizing the delivery of plasmid DNA encoding bone morphogenetic protein-2 (BMP-2). • Probing the performances of three kinds of delivery methods of plasmid DNA encoding BMP-2 in vivo. • Developing PLGA/chitosan composites from a combination of spray drying and supercritical fluid foaming techniques for DNA delivery. • Developing lysine-based peptides functionalized PLGA foams for controlled DNA delivery. Chapter 29 These studies focused on the applications of electrospinning and supercritical CO2 foaming techniques. With the aid of physical and chemical modifications on mechanical properties and surface charge, the applications of these two techniques were successfully extended to the biomedical fields, such as bone regeneration and development of novel DNA delivery devices. These biodegradable polymer matrix-based controlled drug delivery devices would avoid frequent administration of drugs (protein or DNA). Therefore, the devices should help minimize drug waste and maintain high level of growth factor in site. Moreover, the dosage forms developed in this research may provide alternative ways to treat bone fractures and bone defects, which can eventually improve the life quality of patients. In addition, several general studies about DNA adsorption and desorption on PLGA matrixes may improve the current understanding on the interactions between polymeric matrix and DNA, which can help design more favorable DNA delivery systems in the future. 2.2 Organization of thesis The organization of the thesis is described by the flowchart in Figure 2.1. The general introduction is presented in Chapter 1, and the objectives and motivation of the thesis is presented in Chapter 2. In the following chapters, each kind of device developed in this research will be described in details in one chapter, with a specific introduction and objectives presented in the beginning. In Chapter 3, two kinds of protein loading methods into PLGA/HAp composite scaffolds are employed to study the parameters affecting the release rates and bioactivity. In a similar manner, three kinds of DNA loading methods into PLGA/HAp composite scaffolds are investigated in Chapter 5. Chapters and Chapter 30 demonstrate the performances of BMP-2 protein and BMP-2 plasmid loaded PLGA/HAp composite scaffolds in vivo, respectively. Chapters and illustrate the development of PLGA foams by supercritical CO2 foaming technology designed for DNA delivery. They are modified by the use of chitosan and L-lysine, respectively. Finally, Chapter gives general conclusions and recommendations for future studies. 31 Chapter 1. Introduction 2. Objectives and Organization of thesis Bone Regeneration in vitro in vivo Gene Delivery 3. 5. 7. 8. Three-dimensional Fibrous PLGA/HAp Composite Scaffold for BMP-2 Protein Delivery Fabrication and Characterization of PLGA/HAp Composite Scaffolds for Delivery of BMP-2 Plasmid PLGA/Chitosan Composites from a Combination of Spray Drying and Supercritical Fluid Foaming Lysine-Based Peptide Functionalized PLGA Foams for Controlled DNA Delivery 4. 6. Optimized Bone Regeneration Based on Sustained Release of BMP-2 Protein from Fibrous PLGA/HAp Composite Scaffolds BMP-2 Plasmid Loaded PLGA/HAp Composite Scaffolds for Treatment of Bone Defects in Nude Mice 9. Conclusion and Recommendation Figure 2.1 Flowchart summarizing the organization of thesis. [...]... mats can be controlled during electrospinning (Zong et al., 2002; Zong et al., 2003), which makes this a promising technique for fabrication of tissue-engineered scaffolds Li and coworkers produced silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and/ or nanoparticles of hydroxyapatite (nHAp) using electrospinning, and showed its ability in inducing in vitro bone formation... Characterizing the performance of BMP-2 loaded PLGA/hydroxylapatite composite fibrous scaffolds in vivo • Optimizing the delivery of plasmid DNA encoding bone morphogenetic protein-2 (BMP-2) • Probing the performances of three kinds of delivery methods of plasmid DNA encoding BMP-2 in vivo • Developing PLGA/chitosan composites from a combination of spray drying and supercritical fluid foaming techniques. .. The general introduction is presented in Chapter 1, and the objectives and motivation of the thesis is presented in Chapter 2 In the following chapters, each kind of device developed in this research will be described in details in one chapter, with a specific introduction and objectives presented in the beginning In Chapter 3, two kinds of protein loading methods into PLGA/HAp composite scaffolds are... devices, which can achieve and maintain sufficiently high concentrations of protein or DNA in tissue and have acceptable mechanical properties These devices developed in this thesis may be of Chapter 1 18 importance in combining tissue engineering and drug delivery nicely, with potential in clinical applications 1. 3 Drug delivery dosage forms In addition to the attachment of growth factors to polymeric... matrix-based controlled drug (BMP-2 protein or DNA) delivery devices by extending the applications of electrospinning and supercritical fluid techniques to biomedical field and characterize the devices using various state -of- the-art analytical techniques Specifically, the research areas of this research include: • Developing BMP-2 protein loaded PLGA/hydroxylapatite composite fibrous scaffolds for bone regeneration. .. scaffolds 1. 3.3 .1 Solvent casting/particulate leaching Solvent casting-particulate leaching is one of the first techniques that have been utilized in formation of porous scaffolds for tissue engineering applications, although it is being replaced by more sophisticated techniques This technique involves the pouring of the polymer solution into a bed of salt particles with defined size Precipitation of the polymer... Developing lysine-based peptides functionalized PLGA foams for controlled DNA delivery Chapter 2 29 These studies focused on the applications of electrospinning and supercritical CO2 foaming techniques With the aid of physical and chemical modifications on mechanical properties and surface charge, the applications of these two techniques were successfully extended to the biomedical fields, such as bone regeneration. .. improve the release kinetics, with release of the immobilized factor being controlled by the degradation of the hydrogel Several chemical and physical methods exist for the immobilization of growth factors in hydrogels Chemical methods involving attachment of reactive groups to the growth factor for crosslinking with hydrogel polymers have seen increasing use within cartilage tissue engineering (Holland et... use of chitosan and L-lysine, respectively Finally, Chapter 9 gives general conclusions and recommendations for future studies  31 Chapter 2 1 Introduction 2 Objectives and Organization of thesis Bone Regeneration Gene Delivery 3 in vivo 7 8 Fabrication and Characterization of PLGA/HAp Composite Scaffolds for Delivery of BMP-2 Plasmid PLGA/Chitosan Composites from a Combination of Spray Drying and Supercritical. .. dental applications (guided tissue regeneration) , cardiovascular applications (stents, grafts), and intestinal applications (anastomosis rings) Most of the commercially available biodegradable devices are polyesters composed of homo-polymers or copolymers of glycolide and lactide 16 Chapter 1 Table 1. 1 Properties of some biodegradable polymers (adapted from Daniels et al., 19 90) Polymer Melting Point . Chapter 1 1 CHAPTER 1 L ITERATURE REVIEW AND INTRODUCTION Tissue engineering is the combination of cell, engineering and material methods, and application of suitable biochemical and physio-chemical. developed in this thesis may be of Chapter 1 18 importance in combining tissue engineering and drug delivery nicely, with potential in clinical applications. 1. 3 Drug delivery dosage forms In. cell, and developmental biology, as well as materials science and bioinformatics (Donald and Mohammad, 20 01) . Chapter 1 5 1. 1 .1 Cells Tissue engineering utilizes living cells as engineering