Applications of electrospinning and supercritical carbon dioxide foaming techniques in controlled release and bone regeneration 1

As growth factors are usually expensive and easily damaged, their delivery to specific tissue positions efficiently without losing their bioactivity is a major challenge.. In order to ac

CHAPTER 1

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

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;

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

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)

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/

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,

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

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)

• 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

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)

Figure 1.1 Model of human bone morphogenetic protein 2 created using Cn3D*

Bone morphogenetic protein 2 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 release 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

Progestasert®), pilocarpine-releasing devices for glaucoma therapy (Ocusert®) and a host of new delivery systems for the treatment of cancer Drug delivery devices are based

on biocompatible polymers, a subset of polymer materials with sufficient biocompatibility and appropriate physical properties to provide controlled delivery (Fung and Saltzman, 1997)

The first polymeric controlled-release devices were based on non-degradable polymers, principally silicone elastomers In 1964, researchers recognized that certain dye molecules could penetrate through the walls of silicone tubing (Folkman and Long, 1964; Folkman and Long, 1966), an observation that leads to the development of reservoir drug

delivery devices, which are hollow polymer tubes filled with a drug suspension The drug

is released by dissolution into the polymer and then diffusion through the polymer wall, a mechanism that works for any agent that can dissolve and diffuse through either silicone

or poly(ethylene-co-vinylacetate) (EVAc), the two most commonly used nondegradable

polymers The Norplant® 5-year contraceptive delivery system, approved by use by women in the United States since 1990, is based on this technology

Solid matrices of non-degradable polymers can also be used for long-time drug release

In comparison to reservoir systems, these devices are simpler (since they are homogenously and, hence, easier to produce) and potentially safer (since a mechanical defect in a reservoir device, but not a matrix, can lead to dose dumping) On the other hand, it is more difficult to achieve constant rates of drug release with non-degradable

matrix; for example, the rate of release of carmustine from an EVAc matrix device drops

continuously during incubation in buffer water Constant release can sometimes be achieved by adding rate limiting membranes to homogenous matrices, yielding devices in

which a core of polymer/drug matrix serves as the reservoir In other cases, water-soluble crosslinked polymers can be used as matrices (Kim et al., 1992); release is then activated

by swelling of the polymer matrix after exposure to water

Although non-degradable materials are very stable and can make specific structures easily, their applications in medical field are strongly hindered for several reasons First, when the field expanded from research to application, it was recognized that surgical removal of drug-depleted delivery systems was difficult, yet leaving non-degradable

foreign materials in the body for an indefinite time period constituted an undesirable toxicological hazard Therefore, the physician’s simple desire to have a device that can be used as an implant and will not require a second surgical intervention for removal constitutes the most basic reason for the interests in biodegradable polymers Secondly,

although diffusion-controlled release is an excellent means of achieving predefined rates

of drug delivery, it is limited by the polymer permeability and the characteristics (molecular weight, solubility) of the drug because diffusion follows Fick’s first law, which states that the rate of diffusion is proportional to the concentration gradient of the

drug and the diffusion coefficient Therefore, drugs that have either high molecular weight (>500 daltons) or poor solubility in the polymer are not amenable to diffusion-controlled release Besides these reasons, biodegradation may offer other advantages, For example, a fractured bone that has been fixated with a rigid, non-biodegradable stainless

implant has a tendency to re-fracture upon removal of the implant Because the stress is

borne by the rigid stainless steel, the bone has not been able to carry sufficient load during the healing process However, an implant prepared from biodegradable polymer

can be engineered to degrade at a rate that will slowly transfer a load to the healing bone

1.2.2 Biodegradable materials

Biodegradation is defined as the conversion of materials into less complex intermediates

or end products by solubilization, simple hydrolysis, or the action of biologically formed entities that may be enzymes and other products of the organism Polymer molecules may, but not necessarily, break down to produce fragments in this process, and the integrity of the material decreases as a result of this process (Albertsson and Karlsson, 1990; Vert,

1989; Anderson, 1989) The formed fragments can move away from their site of action but not necessarily from the body

In the first half of 20th century, research on materials synthesized from glycolic acid and

other hydroxyl acids was abandoned for further development because the resulting polymers were too unstable for long-term industrial uses Consequently, the pioneering studies in the field of controlled drug delivery, begun in the 1960s, used bio-stable commercial polymers such as polyethylene and silicone rubbers In these systems, the

release rate of the drug from the polymeric matrix or reservoir device was determined solely by diffusion; biodegradation of the polymer was not considered at that time However, this very instability of early materials-leading to biodegradation-has been proven to be immensely important in medical applications over the last five decades

Therefore, much research has been conducted on biodegradable polymer thereafter

With the development of biodegradable polymers, great progress has been achieved For

example, polymers prepared from glycolic acid and lactic acid have found a multitude of uses in the medical industry, beginning with the biodegradable sutures first approved in the 1960s Since that time, diverse products based on lactic and glycolic acid-and on other materials, including poly(dioxanone), poly(trimethylene carbonate) copolymers,

and poly(caprolactone) homo-polymers and copolymers have been accepted for use as medical devices In addition to these approved devices, a great deal of research continues

on polyanhydrides, polyorthoesters, polyphosphazenes, and other biodegradable polymers

Biodegradable polymers can be either natural or synthetic In general, synthetic polymers offer greater advantages than natural materials in that they can be tailored to give a wider range of properties and more predictable uniformity than materials from natural sources

However, there are several points to be considered in choosing biodegradable polymers for medical applications The general criterion for selecting polymeric biomaterial is to match the mechanical properties and the time of degradation according to the needs of the application An ideal polymer for a particular application would be configured so that it:

• has mechanical properties that match the application, remaining sufficiently strong until the surrounding tissue has get healed and remodeled;

• does not invoke an inflammatory or toxic response;

• is metabolized in the body after fulfilling its purpose, leaving no trace;

• is easily processable into the final product form;

• demonstrates acceptable shelf life;

• is easily sterilized

A number of typical properties of biodegradable polymers have been studied (see Table

1.1) The factors affecting the mechanical performance of biodegradable polymers are those that are well known to the polymer scientists, and include monomer selection, initiator selection, process conditions, and the presence of additives These factors in turn influence the polymer’s hydrophilicity, crystallinity, melting and glass-transition temperatures, molecular weight, molecular-weight distribution, end groups, sequence

distribution (random versus blocky), and presence of residual monomer or additives In addition, the polymer scientist working with biodegradable materials must evaluate each variable for its effect on biodegradation (Daniels et al., 1990)

Biodegradation has been accompanied by synthesizing polymers that have hydrolytically unstable linkages in the backbone The most common chemical functional groups with this characteristic are esters, anhydrides, orthoesters, and amides The synthetic

biodegradable polymers are currently being used or investigated for use in wound closure (sutures, staples), orthopedic fixation devices (pin, rods, screws, tacks, ligaments), 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