DRUG AND GENE DELIVERY STUDIES UTILISING PLGA SCAFFOLD

Yekbun ADIGUZEL

Contents

Introduction -Overall info.
Roots of the field – biomaterials
History
Tissue Engineering in the World Market
Table 1. Tissue Engineering in the World Market
Tissue Engineering and Biomaterials science
Tissue Engineering
Biomaterials science
Recent Progress
An area under development
Polymers in Controlled Drug Delivery
Table 2. Environmental factors that induce change in polymer
Case:
Successful Delivery of Active BMP-2 Using Bone Osteoprogenitors on Porous Biodegradable Scaffold
Bone healing, replacement, and problems
Current approaches                                                                                                                         Growth factors and gene teraphy           Scaffolds
Discussion and Conclusion
References

               Introduction

The roots of the field - biomaterials

The term biomaterials usually refer to systemically and pharmacologically inert substances designed for implantation to restore the function of natural tissues and organs in the body. (ES 704)

            Man-made materials and devices have been developed to the point at which they can be used succesfully to replace parts of living systems in the human body. These special materials are able to function in intimate contact with living tissue, with minimal adverse reaction or rejection in the body. (ES 704)

Bone plates were introduced in the early 1900s to assist in the healing of skeletal fractures, were among the earliest succesful biomedical implants. The plate is generally removed once the bone is healed and become able to support loads, without refracturing. (ES 704)

Artificial knee joints were implanted in patients with a diseased joint to alleviate pain and restore function. After about 10 years of use, these artificial joints often need to be replaced because of wear and fatique-induced delamination of the polymaric component; development of improved materials to extend the lifetime of orthopedic implants such as knees and hips are under consideration of biomaterials ans tissue engineering. (ES 704)

History

“Staudinger initiated a synthetic macromolecular revolution 65 years ago, with the introduction of his ‘macromolecular hypothesis’. This seminal event has led to the evolution of three major macromolecular architectures, namely: linear (class I), cross-linked (bridged; class II) and branched types (class III). These three architectural classes are recognized as traditional synthetic polymers. In all these classes, structures or architectures are produced by largely statistical polymerization processes, rather than exact distribution processes. These processes produce polydispersed (i.e. Mw/Mn >2.10) products of many different molecular weights. In general, these are not structure-controlled macromolecular architectures such as those observed in biological systems. However, considerable progress has occurred recently in the areas of living anionic, cationic and radical polymerizations.

         As early as 1979 the first synthetic strategies to produce monodispersed, structure-controlled, dendritic macromolecules in ordinary laboratory glassware were initiated. Although dendrimer structures exhibit structural control reminiscent of biological systems, the synthetic approaches did not require biological components. They did, however, involve significant innovation and digression from classical organic synthesis methods. Commercial quantities (kg) of controlled macromolecular structures with polydispersities of 1.0005.1.10 are now routinely synthesized using traditional organic reagents and monomers, such as ethylenediamine and alkyl acrylates. These new structures are referred to as dendrons or dendrimers. Since 1979, two major strategies have evolved for dendrimer synthesis. The first was the divergent method in which growth of a dendron (molecular tree) originates from a core site (root). During the 1980s, virtually all dendritic polymers were produced by construction from the root of the molecular tree. This approach involved assembling monomeric modules in a radial, branch-upon-branch motif according to certain dendritic rules and principles. This divergent approach is currently the preferred commercial route used by worldwide producers including Dendrimax (Ann Arbor, MI, USA), DSM Fine Chemicals (Geleen, The Netherlands) and The Perstorp Group (Perstorp, Sweden).

         A second method that was pioneered by Fréchet and colleagues is the convergent growth process. It proceeds from what will become the dendron molecular surface (i.e. from the leaves of the molecular tree) inward to a reactive focal point at the root. This leads to the formation of a single reactive dendron. To obtain a dendrimer structure, several dendrons are reacted with a multi-functional core to yield such a product. Using these two synthetic strategies, >100 compositionally different dendrimer families have been sysnthesised.”

(Esfand and Romalia, 2001)

Tissue Engineering in the World Market

Table 1. Tissue Engineering in the World Market

ORGANS		SOME APPLICATIONS		COMPANIES
Bone	Bone-growth factors or stem cells are inserted into a porous material, which is cut into a specific shape, to create new jaws or limbs. A product that creates shinbones is in clinical trials.		Creative Biomilecules, Orquest, Sulzer Orthopedics Biologics, Genetics Institute, Osiris Therrapeutics, Regeneration.
Skin	Organogenesis’ Apligraf, human-skin equivalent, is the first engineered body part to win FDA approval, initially for leg ulcers. Dermagraft, skin substitutes. Other skins are in the works for foot ulcers and burns.		Organogenesis (e.g. Vitrix, Apligraf), Advanced Tissue Sciences (e.g. TransCyte, Dermagraft), Integra LifeSciences, LifeCell (e.g. Alloderm), Ortec International.
Cartilage	Genzyme Biosurgery’s cartilage graft (Carticel II); also it’s cartilage cells (Carticel) is already on the market that regrows knee cartilage. A chest has been grown for a boy and a human ear on mouse. Curis’ Chondrogel, is cartilage gel to prevent urinary reflux.		Genzyme Biosurgery (e.g. Carticel, Carticel II), Biomatrix, Integra LifeSciences, Advanced Tissue Sciences, ReGen Biologics, Osiris Theurapeutics, Curis (e.g. Chondrogel).
Bladder	Doctors at Children’s Hospital in Boston have grown bladders from skin cells and implanted them in sheep. They’re about to try the same process on a patient.		Reprogenesis, Curis.
Pancreas	Insulin-manufacturing cells are harvested from pigs, encapsulated in membranes, and injected into the abdomen. The method has been tested in animals and could be in human trials in two years.		BioHybrid Technologies, Neocrin, Circe Biomedical.
Urinary tract	Cartilage cells are taken from the patient, packed into a tiny matrix, and injected into the weakened ureter, where they bulk up the tissue walls to prevent urinary back-up and incontinence. The method is in late-phase clinical trials.		Reprogenesis, Integra LifeSciences.
Liver	A spongy membrane is built up and then seeded with liver cells. Organ in the size of a dime have been grown, but a ful-size liver could take 10 years due to its complexity.		Advanced Tissue Sciences, Human Organ Sciences, Organogenesis.

At the beginning of 2001, tissue engineering research and development was pursued by more than 3300 scientists and support staff in more than 70 start up companies or business units in USA with an expenditure of 600 million dollars. At least 16 European or Australian (22% of total) companies are also active. (Source of the information provided in table and the information underneath: ES 704; Garr, 2001)

Tissue Engineering and Biomaterials science;

Tissue Engineering-

Synthetic composites suitable for direct application to human tissue replacement must have the appropriate mechanical characteristics if the implant is to maintain overall physical function. If resorbable, these values must be maintained throughout subsequent processes. In loaded applications, interfacial strength should develop rapidly to allow the body to improve healing.

Unfortunately, limited properties continue to limit application of materials designed to fill both hard and soft tissue cavities. For example, ceramic-reinforced polymer matrices are widely regarded as the logical answer to how we can best match some of nature's feats of hard tissue engineering. The most significant means by which this can be achieved is via a poorly understood process known as ‘osseointegration.’ Such direct bone-implant bonding can form a strong interface between the body and the implant. When placed into a bony cavity, healing must occur in the space between the implant and the existing bone. New bone formation in such a gap is contingent upon chemical, mechanical and topographic influences that have not been clearly sorted out.

(Interdisciplinary Biomaterials Seed Grants; web source)

            Limitations exist for soft tissues; especially the interface between natural nerve tissue and synthetic analogs are searched to understand the thorough mechanism, control and achievement of nerve growth, a prospective approach in “restoring function to victims of paralysis” (Interdisciplinary Biomaterials Seed Grants; web source). By the study of Miller and coworkers (2000), Schwann cells were grown in an oriented manner, on micropatterned biodegradable polymey scaffolds. Also, “researchers at the University of Miami (Florida) achieved extensive remyelination of central nervous system axons in newborn rats with a genetic myelin deficiency”, by implanting Schwann cells; proving that “peripheral nerve cells could survive and function appropriately when implanted into the central nervous system” (Interdisciplinary Biomaterials Seed Grants; web source). Such works provide new treatment options for neurological disorders such as Parkinson’s and Huntington’s diseases; as Barker and Rosser (2001) proposed neural transplantation therapies for those diseases, which was proved to be succesfull to be translated into clinical studies.

Biomaterials science-

The emerging opportunities in this area are also driven by mutual expertises to develop, fabricate and test biomaterials targeted for clinical application. Collaborative studies came to be a must within the seeming direction of evolution of the area. “Engineering provides capabilities in modeling, sensing, mechanical property evaluation, corrosion, micro- and bulk fabrication” (Interdisciplinary Biomaterials Seed Grants; web source); with the contributions of computer aided tissue engineering, such as the studies of Sun and Lal (2002). “Health Sciences provides broad expertise in current biomaterials technology, biology-based experiments, drug design/delivery, tissue response and a much-needed clinical perspective. Mathematical and Physical Sciences provides new analytical techniques and a focus on specific chemical factors.” (Interdisciplinary Biomaterials Seed Grants; web source)

Microfabrication and genetic engineering provide us with new tools and approaches that were only dreamed of not long ago. Armani and Liu worked on microfabrication technology of a biodegradable polymer (Armani and Liu, 2000). Santini, with Langer and Cima, worked on a microfabricated controlled release device (Santini et. al., 1999). There were also groups working by the aid of recent genetic engineering techniques; on the subjects such as, genetic analysis systems for improvements, applications in tissue engineering and gene therapy, tissue engineering via local gene delivery (Anderson et. al., 1988; Madsen, S. and Mooney, 2000; Bonadio, 2000). Materials can now be designed to incorporate specific biochemical factors; surfaces can be engineered to precise levels of topography. These advances will trigger exponential growth in our understanding of biomaterial-tissue interactions and in their final clinical application.

Recent progress;     

 “Ultimately, any method for building new human organs will have to win approval from the national food and drug administration. And that means organ builders will need a standardized, reproducible manufacturing process. To achieve that goal, Griffith and her colleagues have turned to device invented by MIT engineer Emanuel Sachs and used for rapid prototyping and the manufacture of a variety of parts and tools: a three-dimensional powder printer, or 3DP machine.” (Sachs et. al., 1998)

“The machine builds up complex shapes layer by layer, based on a computer file capable of depicting the object as a series of horizontal slices. A roller pushes a thin layer of powder across a flat base plate resting on top of a piston. Next, an inkjet printer head distributes a glue, or binder, to solidify the powder only where the blueprint for that slice calls for solid material. The piston then ratchets the plate down by the thickness of the layer, and the process begins again. When all the layers have been printed, the new object can be removed from the machine, and the excess powder falls away.” (Mark et. al., web source)

“By adapting the powder to use polymer powders, multiple print heads and special binders, Griffith and her collaborators created a tool capable of mass producing polymer scaffolds for new tissue and organs. Not only does the printer allow the researchers to control a scaffold’s shape with great precision, it also allows them to build in chemical modifications to the structure’s surface that help tell different type of cells exactly where and how they should grow.” (Mark et. al., web source) Though Griffith focused on development of this technology mainly for the aim of tissue growth, technique seems to be applicable to be utilised by drug delivery system’s development as well.                                                                                                             

An area under development;

“Micro -medical   Devices-   The   application   of   microfabrication   techniques adapted  from  integrated  circuit  manufacturing  opens  up  new  pathways  for solving  traditional biomaterial  problems  and  developing  exciting methods of treating very old  diseases. For example, implanted electrochemical sensors can be used to monitor degradation products (e.g., lactic acid) being released by a resorbing composite. Such probes can be interrogated telemetrically.” (Interdisciplinary Biomaterials Seed Grants; web source)

A direct extension of this technology to tissue engineering involves drug release methods adapted to deliver specific biochemical growth factors. (Santini et. al., 1999, Vallbacka et. al., 2001) These allow diffusion of the desired compounds from synthetic reservoirs. These carriers could begin to release only after they are exposed to specific body fluids/cellular influences. Additionally, genetic engineering techniques also introduce new perspectives in growth factor delivery, or drug delivery via biodegradable scaffolds (Partridge et. al., 2002).

“Progress in this area has long been limited by a lack of knowledge regarding the optimal surface topography for such tissue-substrate interactions. The careful fabrication and testing of designed surface topographies is needed. Microscopy and chemical analysis ensures that design/fabrication goals were met. In vitro/in vivo molecular/cellular level interactions with such textured substrates are necessary to examine interlocking and tissue capsule development.” (Pins et. al., 2000)

Polymers in Controlled Drug Delivery (Brannon-Peppas, 1997)[1]:

Controlled drug delivery occurs when a polymer, whether natural or synthetic, is judiciously combined with a drug or other active agent in such a way that the active agent is released from the material in a predesigned manner. The release of the active agent may be constant over a long period, it may be cyclic over a long period, or it may be triggered by the environment or other external events. In any case, the purpose behind controlling the drug delivery is to achieve more effective therapies while eliminating the potential for both under- and overdosing.

        Other advantages: maintenance of drug levels within a desired range, the need for fewer administrations, optimal use of the drug in question, and increased patient compliance.

        The potential disadvantages: the possible toxicity or nonbiocompatibility of the materials used, undesirable by-products of degradation, any surgery required to implant or remove the system, the chance of patient discomfort from the delivery device, and the higher cost of controlled-release systems compared with traditional pharmaceutical formulations.

        The ideal drug delivery system should be: inert, biocompatible, mechanically strong, comfortable for the patient, capable of achieving high drug loading, safe from accidental release, simple to administer and remove, and easy to fabricate and sterilize.

        The earliest of these polymers were originally intended for other, nonbiological uses, and were selected because of their desirable physical properties, for example:

·            Poly(urethanes) for elasticity.

·            Poly(siloxanes) or silicones for insulating ability.

·            Poly(methyl methacrylate) for physical strength and transparency.

·            Poly(vinyl alcohol) for hydrophilicity and strength.

·            Poly(ethylene) for toughness and lack of swelling.

·            Poly(vinyl pyrrolidone) for suspension capabilities.

        Some of the materials that are being used or studied for controlled drug delivery include:

·            Poly(2-hydroxy ethyl methacrylate).

·            Poly(N-vinyl pyrrolidone).

·            Poly(methyl methacrylate).

·            Poly(vinyl alcohol).

·            Poly(acrylic acid).

·            Polyacrylamide.

·            Poly(ethylene-co-vinyl acetate).

·            Poly(ethylene glycol).

·            Poly(methacrylic acid).

        In recent years additional polymers designed primarily for medical applications have entered the arena of controlled release. Many of these materials are designed to degrade within the body, among them:

·            Polylactides (PLA).

·            Polyglycolides (PGA).

·            Poly(lactide-co-glycolides) (PLGA).

·            Polyanhydrides.

·            Polyorthoesters.

        Table 2. Environmental factors that induce change in polymer:

Stimulus	Hydrogel	Mechanism
pH	Acidic or basic hydrogel	Change in pH — swelling — release of drug
Ionic strength	Ionic hydrogel	Change in ionic strength — change in concentration of ions inside gel — change in swelling — release of drug
Chemical species	Hydrogel containing electron-accepting groups	Electron-donating compounds — formation of charge/transfer complex — change in swelling — release of drug
Enzyme-substrate	Hydrogel containing immobilized enzymes	Substrate present — enzymatic conversion — product changes swelling of gel — release of drug
Magnetic	Magnetic particles dispersed in alginate microshperes	Applied magnetic field — change in pores in gel — change in swelling — release of drug
Thermal	Thermoresponsive hrydrogel poly(N-isopro- pylacrylamide)	Change in temperature — change in polymer-polymer and water-polymer interactions — change in swelling — release of drug
Electrical	Polyelectrolyte hydrogel	Applied electric field — membrane charging — electrophoresis of charged drug — change in swelling — release of drug
Ultrasound irradiation	Ethylene-vinyl alcohol hydrogel	Ultrasound irradiation — temperature increase — release of drug

        Controlled release drug delivery can occur from a typical matrix drug delivery system (can be bulk-eroding and surface-eroding); or from from typical reservoir devices that are implantable or oral systems and transdermal systems.

        Prospective systems of future:

·            Copolymers with desirable hydrophilic/hydrophobic interactions.

·            Block or graft copolymers.

·            Complexation networks responding via hydrogen or ionic bonding.

·            Dendrimers or star polymers as nanoparticles for immobilization of enzymes, drugs, peptides, or other biological agents.

·            New biodegradable polymers.

·            New blends of hydrocolloids and carbohydrate-based polymers.

          Case:

Successful Delivery of Active BMP-2 Using Bone Osteoprogenitors on Porous Biodegradable Scaffold

Bone healing, replacement, and problems

“Fracture healing remains a fundamental problem in orthopaedic surgery. Although the majority of fractures heal well, difficulty in the form of delayed healing or non-union of fracture can be devastating. Fractures in anatomically compromised locations (e.g. talar neck or scaphoid), those with inadequate fixation or infection, those resulting from high-energy-type injuries with soft tissue stripping or segmental bone loss, or those occuring in poor bone-stock (i.e. osteoporosis) often result in delayed healing. … Treatment options for the orthopedic surgeon confronting these complex fractures, especially those that involve segmental bone loss, include bone autograft, vascularized bone-grafting, allograft supplemented with osteogenic proteins, bone transport or amputation. Unfortunately, patients generally endure a lengthy recovery with numerous procedures, potential donor-site morbidity and often an unsatisfactory end result. Similar difficulties occur with closure of the residual bony defects in craniofacial reconstruction, and oncology and trauma surgery.” (Wright, V., 2001)

       When the replacement of the bone is the case, a lack of sufficient material precludes the universal use of autogenous bone while the use of allogenic bone for transplantation carries potential risks of immune responses, pathogen transmission, and the necessary immunosuppression. (Partridge, K. et.al., 2002) Consequently, research directed towards improving fracture treatment and the development of optimal bone-subtitutes remains an area of intense interest.

Current approaches

A variety of bone grafts and implants has been used in orthopedic surgery; however, present bone substitutes do not behave physiologically or mechanically like true bone. For optimal bone healing, the milieu must contain osteoconductive and osteogenic elements. Scaffols provide the platform for bone in-growth and satisfy the osteoconductive requirement. Osteoinductive factors trigger osteoprecursors, which are stem cells in the microenvironment, to initiate the healing cascade of chemotaxis, mitosis and differentiation. Growth factors and other bioactive proteins usually play this role. Finally, osteogenic elements, such as the native osteoprecursors or muscle-derived stem cells, directly participate in bone formation after stimulation by the osteoinductive factors, by manufacturing osteoid in the osteoconductive scaffold. (Wright, V., 2001)

       To date, the most succesfull bone-grafting material is autogenous cancellous bone. This substitute has osteogenic, osteoconductive and osteoinductive properties, because it contains surviving osteogenic cells, bone collagen, bone mineral, bone morphogenic proteins (BMPs) and growth factors. However, the use of autogenous bone grafts has many shortcomings: patients often experience significant pain, infection and blood loss at the harvest site and a maximum failure rate of 15% is usually expected. Other approaches with synthetic materials, including metal prostheses, calcium-phosphate-based ceramics and pastes, methyl methacrylate constructs, and polymers, have been useful in a limited manner. The overwhelming shortcomings of these materials are their collective inability to integrate into surrounding tissues and their inability to become vascularized. Integration is important for long-term functional outcomes, as the structure of a patient’s tissue is dynamic and changes with time. (Wright, V., 2001)

Growth factors and gene teraphy

Growth factors are soluble proteins that promote cell division, maturation and differentiation. To coordinate the cellular response, cytokines and growth factors act locally through wound-specific signal transduction cascades. (Bonadio, J., 2000) Inductive growth factors (e.g. BMPs), such as other proteins in general, have a limited half-life and stability in vivo. Protein degradation, whether during incorporation or following release from polymer, can be caused by moisture, temperature, change in chemical environment (e.g. pH) or polymeric degradation products. (Madsen, S. and Mooney, D., 2000)

       Identifying the optimal growth factor for a particular application and determining the optimal method for delivering the growth factor to the surgical microenvironment is needed. Growth factor-induced vascularization is effective in supplying the oxygen and nutrients necessary for the survival of transplanted cells in organ substitution; however, growth factors have poor in vivo stability and so the biological effects are often unpredictable unless the delivery system is contrived. (Tabata, Y., 2000)

       The most obvious means of delivering growth factors in situ is direct application of the recombinant protein. In the past 36 years, since the discovery of the BMPs, researchers have demonstrated that one of the most important factors for in vivo healing is the presence of high and sustained levels of growth factor. Over 30 distinct forms of the BMPs exist although the most widely studied are BMP-1 through 7. The BMPs induce differentiation of multipotential mesenchymal cells, puliripotent murine stem cell cultures and rat bone marrow stromal cells as well as proliferation and maturation in osteoblast populations. (Partridge, K. et. al., 2002) Most growth factors are rapidly cleared and have half-lives of minutes. Even when the growth factor is bound to a collagen scaffold, its half-life is limited to several days. Additionally, although microgram doses are sufficient for the in vitro manipulation of cells, doses required for in vivo regional regeneration are typically in milligram quantities. Researchers have, therefore, turned to regional gene therapy for the sustained delivery of growth factor to a local site in vivo. (Wrigth, V. et. al., 2001)

       One gene therapy approach is the transfer of a gene encoding the desired growth factor into cells using viral or non-viral vectors; the transduced cells subsequently secrete the desired protein into the microenvironment. Gene therapy can take two forms: in vivo (direct) and ex vivo. The in vivo approach consists of directly injecting or implanting the gene–vector construct into the patient; this approach is attractive because of its simplicity. Viral vectors, such as retroviruses, composed of RNA can exhibit inefficient gene transfer, perhaps because of low transduction into non-dividing cells, but can retain a large portion of the viral genome. They are also expensive to manufacture. By contrast, the DNA-based adenoviral vectors composed of DNA can transduce many types efficiently but optimization of incorporated genes is controversial, possibly due to the immune response. There are also several concerns regarding the safety of patients with viral vectors. A number of studies in recent years have examined the potential of adenoviral delivery of BMP-2 in target cell populations. However, it is limited by the inability to perform in vitro safety testing on the transduced cells. A number of issues regarding the use of adenovirus remain, including potential immune response, clearance, and safety of the virus, quantification and duration of protein production. The issues of safety and ease of application have led to increasing efforts to develop non-viral transfection approaches. However, these approaches suffer from very low transfection efficiency. Particle-mediated gene therapy (e.g. the gene gun) is one approach used to address this limitation. An alternative approach is the incorporation of the plasmid DNA within biodegradable polymers for localized and sustained delivery to promote expression over the desired time frame. The ex vivo approach consists of isolating cells from a tissue biopsy. The cells are then grown in culture and transfected or transduced in vitro. The genetically altered cells can then be tested in vitro both for successful gene transfer and abnormal behaviour before their transduction into the patient. (Madsen, S. and Mooney, D., 2000; Wright, V. et.al., 2001; Partridge, K. et.al., 2002)

       To facilitate clinically applicable gene therapy, it is necessary not only to delineate the protein needed for proper tissue function and identify the gene, but also to be able to reproducibly deliver it in a durable manner to the diseased or injured tissue.  

Scaffolds

“Synthetic materials, such as poly(lactic acid) (PLA), poly(DL-lactic- co-glycolic acid) (PLGA), polyglycolic acid (PGA) have emerged as potential scaffolds for cell transplantation and tissue growth. These materials have the advantage of FDA approval and are currently used as suture materials and in drug delivery vehicles. Moreover, protocols for the generation of these materials with defined porosity using super critical fluid mixing and procedures for the surface modification of these materials with biological agents, have been developed.” (Partridge et.al., 2002) Tissue engineering has presented an alternative strategy based on transplantation of neo-organ constructs consisting of endogeneous stem/progenitor cells grown ex vivo within biodegradable scaffolds. The scaffold is resorbed over time, so that the neo-organ eventually will consist of transplanted cells and the stroma they produce. (Bonadio, J., 2000; Partridge, K., 2002)

       Mineralization factors (1,25 dihydroxy vitamin D3, the vitamin D receptor ligand, b-glycerophosphate, ascorbate, dexamethasone or another glucocorticoid receptor ligand, and the bone morphogenetic proteins) induce the bone marrow stromal cells to differentiate along the osteoblast pathway, characterized by mineralization of the extracellular matrix and expression of the osteoblast-associated gene marker osteocalcin. The formation of mineralized bone tissue within diffusion chambers in nude mice highlight the potential to tissue-engineer bone for orthopaedic use. (Halvorsen, Y., 2001; Partridge, K., 2002)

       Developed scaffolds generate attractive biomimetic scaffolds for cell growth and differentiation including isolated human osteoprogenitors. Thus, the selection and genetic modulation of primary human osteoprogenitor cells in combination with biodegradable polymer scaffolds, which interact and promote osteoblast differentiation and osteogenesis; is an alternative attractive approach for skeletal repair. (Partridge, K., 2002)

Study of Partridge and coworkers (2002) as a flow chart:

     Isolation and culture maintenance of osteoprogenitors

Þ Trasduction with adenoviral vector

Þ Checking secretion of active BMP-2 following adenoviral infection (characterization I)

Þ Calculation of concentration of BMP-2 in the media of infected cells (characterization II)

Þ Growth of adenoviral infected osteoprogenitors on PLGA scaffolds (cell seeding)

Þ Confirming cell adhesion, cell viability and cell differentiation

Þ In vivo studies (differentiation of adenovirally transduced human bone marrow cells in vivo)

Þ Histochemical and X-ray analysis

            Discussion and Conclusion:

Level of BMP which was used in the study can be hazardous, even fatal, especially when we consider prospectice clinical applications. BMP is aimed to be delivered in a controlled manner for this reason. Additionally, costs of the materials for pre-clinical research have to be considered, and BMP have quiet a high cost by this means. In order to lower this ‘cost effect’, BMP is produced in vivo, and this also provides other advances such as readiness of handling and manipulation, and lowering time that can be lost during transport processes (we also have to consider sysnthesis time in this case). The ability to infect a wide variety of cell types including dividing and nondividing cells combined with ease of manipulation, and high efficiency of gene transfer in the absence of integration into the host genome(remains as episomes), makes recombinant adenoviruses an attractive choice for gene therapy. In this study the high level of infection of the heterogeneous target cell population containing osteoprogenitor cells at different stages of differentiation confirms the selective advantage of adenoviral vectors for gene therapy.

            However adenoviralinfection has several key limitations

–         Immunogenicity in vivo is a significant issue,

–        long infection times are deleterious,

–        delivery of cells requires expansion in culture prior to viral infection and reimplantation

–        fate of adenoviral cells is unclear

            Though the adverse effect mentioned over is tried to be circumvented by “using a facile and reproducible technique to generate high titre of virus enabling infections of target cell populations at low MOI[2]” (Partridge et. al., 2002); those are still problematic even a number of them are said to be circumvented.

            In this study PLGA is used as the polymer scaffold, such as several other polymers PLGA is effected from several environmental factors, that are pH, ionic molecules, other molecules, electrical and magnetic fields, other forces being exerted, or expected to be exerted in physiological media such as osmotic pressures, enzymatic activities. Also random chemical events may take place within within polymer, or the occurrence rates of such events may change. As previously mentioned here, polymer to be utilised for controlled drug delivery is expected to be inert, biocompatible, mechanically strong, comfortable for the patient, capable of achieving high drug loading, safe from accidental release, simple to administer and remove, and easy to fabricate and sterilize. But those are idealized conditions, and further, we may not prefer such called advantages for possible specific purposes, for specific targets. There are other PLGAs having the same (75/25) molar ratio of lactic to glycolic acid but different molecular weight (Wang et. al., 2000), obviously expected to be having different properties in relation with the molecular weight differences. “The biodegradation of PLGA containing higher content of lactic acid moiety is lower than those containing a lower content of lactic acid moiety. PLGAs with a higher molecular weight degrade faster than those with a lower molecular weight, i.e. the molecular weight decreases more rapidly for higher molecular weight PLGAs than their lower molecular weight counterparts. Nature or properties of the hydrolysis / incubating media may have an effect on the biodegradation of PLGAs. A basic medium may slow down the biodegradation of PLGA in comparison with samples in an acidic medium. The rate of pH reduction for the incubating medium can be divided into three deferent phases, giving an inverted S-type pH profile for the non-buffered incubating media.” (Wu and Wang, 2001)

            In the end, further studies are recently carried on for further developing the DNA delivery scaffolds, that increases the both the rate and the efficiency of DNA release, wich was carried out by electrospinning of PLGA and PLA-PEG block copolymers.(Luu et. al., 2003) Obviously such studies should be developed and are need to be compared by experimental means and by the aid of these studies’ results that are published in several sources, journals and books, providing us the chance of comparing results from different available sources. We may here consider the human factor, some time in the future, also expect for clinical trials’ results for revealing several other complexities of the nature.

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Interdisciplinary Biomaterials Seed Grants; web source:                                            http://kcgl1.eng.ohio-state.edu/~lannuttj/ibsg.html

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