Biomaterials for Tissue Engineering E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Frontiers in Biomaterials
- Sprache: Englisch
This volume reviews the published knowledge about bioactive composites, protein scaffolds and hydrogels. Chapters also detail the production parameters and clarify the evaluation protocol for analysis or testing and scaffolding biomaterials. The volume concludes with a summary of applications of porous scaffold in medicine. Each chapter links basic scientific and engineering concepts to practical applications for the benefit of the reader.
The text offers a wealth of information that will be of use to all students, bioengineers, materials scientists, chemists and clinicians concerned with the properties, performance, and use of tissue engineering scaffolds — from research engineers faced with designing the biomaterials and techniques to physicians and surgeons charged with shepherding the use of the scaffolds into the applied clinical settings.
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Seitenzahl: 543
Veröffentlichungsjahr: 2017
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Frontiers in Biomaterials
(Volume 4)
(Biomaterials for Tissue Engineering)
Mehdi Razavi
BENTHAM SCIENCE PUBLISHERS LTD.
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FOREWORD
Biomaterials have come a long way since the first total joint replacements, which were introduced at a time when biomaterials were selected for their corrosion resistance. Orthopaedic surgeons initially selected materials which would stimulate the least reaction from the body. Materials used were “nearly inert” metal alloys and polymers. Total joint replacements revolutionised surgery and were life changing for patients. However, such materials are eventually rejected by the body, not in the same way as transplants, but because a thin layer of scar tissue forms around them, isolating them from the body, eventually causing the implant to be forced out of position. This became more problematic when clinicians attempted to repair or restore other parts of the skeleton or other tissues.
In 1969 (published in 1971), the invention of Bioglass® by Larry Hench, then at the University of Florida in Gainesville (USA), changed the face of orthopaedics. Bioglass was the first synthetic material that was found to bond with bone (no scar tissue). It is also biodegradable. However, it was not until the mid-1990s when the first Bioglass synthetic bone graft for bone regeneration reached the market. Now, it has been used in more than 1.5 million patients. Between the concept and clinical use of Bioglass, other bioactive ceramics reached clinicians first, such as synthetic hydroxyapatite, which is similar to bone mineral and also bonds with bone, albeit slower than Bioglass. This triggered the use of other calcium phosphate variants, such as tricalcium phosphate.
I mentioned that bioceramics can be biodegradable. This is possible by dissolution (also happens in water) or by cellular action (e.g. macrophages or osteoclasts). Hench termed the combination of biodegradation and bioactivity as 3rd Generation Biomaterials in a Science review in 2002.
Biomaterials are now being designed to deal with the body’s own healing for many different clinical indications. To work well they must be used as temporary templates or scaffolding, specifically designed for the tissue that is being repaired. Scaffolds made of bioactive and biodegradable materials could present a 4th Generation if they are able to stimulate another course of action, e.g. blood vessel growth or bearing load. They can be labelled as 5th Generation, if they do both.
Remarkably, biomaterials have now gone beyond bone and orthopaedics. Almost every tissue in the body has received research attention, with clinical products at various stages of development. In this book, scaffolds for nerves, cardiovascular system, liver, kidney and skin applications are described in addition to bone, cartilage and dental. The translation of new devices from concept to clinic is a great challenge for biomaterials researchers, one that is certainly not lost on the authors of this book.
This book begins with important, and perhaps more conventional biodegradable materials, which are the biodegradable polymers that are used in sutures. Bioceramics are usually too brittle for load bearing structures that must take cyclic load, therefore, in this book they have been included within composites with polymers as the matrix. Metals are now also being made to be biodegradable.
Scaffolds are often designed to mimic the macrostructure of the host tissue, with blood vessels growing through the pore networks to feed the new tissue. Hydrogels are another important type of polymers which mimic the extracellular matrix of tissues. Hydrogels are particularly beneficial for cell types that exist in a 3D gel-like environment. Their unique property is their ability to transport nutrients through their watery networks to cells.
Scaffolds can be employed as an implant on their own, or can be seeded with cells (e.g. stem cells) in vitro prior to implantation, which is termed as tissue engineering.
The concept of bioactive, biodegradable and strong scaffolds is an important area in healthcare. The UK Government highlighted Eight Great Technologies in 2013, suggesting great need and opportunity for growth. Two of those are Advanced Materials and Regenerative Medicine. When new medical devices are created in the laboratory, they must be translated to clinic. In order to deliver these scaffolds, new manufacturing methods are also needed, such as Additive Manufacturing and 3D printing, which can create the required architectures and also promote reproducibility in large numbers.
Other aspects of technology transfer are the need to pass tests prescribed by regulatory bodies. The devices often highlight ambiguities in the tests, so new tests have to be developed. There is a large area of research in tests that can more closely assess the in vivo situation. While researchers often study how tissue specific cells respond to scaffolds, an important area often neglected is that how immune cells respond.
This new book provides a basic level of understanding of all of the above topics, starting from scaffold design; some key biomaterials; manufacturing techniques; to technology transfer aspects that include testing scaffolds both in vivo and in vitro. It provides the necessary foundation of science and technology. For the experienced researcher the book provides a comprehensive overview of the important current topics in the field. Happy reading.
Julian R. Jones Department of Materials, Imperial College London, South Kensington Campus, London SW7 2AZ, UK, E-mail: [email protected]PREFACE
Tissue engineering aims to regenerate damaged tissues by using a 3-dimensional bioscaffold, cells, biomolecules and growth factors. The success of this strategy depends on the biomaterials selection, design and development techniques of bioscaffolds and evaluation methods. Although, this field is new, upward advances are being made in order to be translated to patients. Therefore, it was found that an appropriate book which discusses the novel 3D bioscaffold designs and experimental procedures could be helpful for students and researchers. This book reviews the published resources and discusses the bioscaffolding materials, used techniques and presents the production parameters to clarify the evaluation protocols for analysis or testing. This book covers the chapters by leading bioengineers, biologists, dentists and clinicians. Therefore, the text has basic information that will be of use to bioengineers, clinicians and surgeons who deal with the tissue engineering strategies. This is a reference book for undergraduate and graduate courses and clinical laboratories. Finally, the editor thanks for the support of all the contributors and the publisher who made the publication of this book possible.
Mehdi Razavi Department of Radiology, School of Medicine Stanford University, Palo Alto, California 94304 USA E-mail: [email protected]List of Contributors
Synthetic Biopolymers
Mahdis Hesami*Abstract
The striking role of tissue engineering in saving people’s life is inevitable. The number of patients waiting for an organ donor is increasing every minute. On the other hand, life expectancy has increased which results in a growing demand of scaffold production. The developed technology seeks for materials which target specific cells, proliferate, regenerate the targeted tissue and restores its function. To fulfill this, not only the material type but also the fabrication techniques should be engineered carefully. Among various materials, synthetic polymers have attracted more attention due to their tailorable properties. In this chapter an overview of various synthetic polymers, their degradation, application and their various blends is discussed. The polymeric nanocomposites used as scaffold have been introduced briefly and the future research in the material selection for porous scaffolds has been reviewed.
INTRODUCTION
Saving people’s lives is an admirable goal throughout history. Various materials including metals, glasses and woods have been used in human body more than 2000 years ago. Romans, Chinese and Aztecs were the first who used gold in dentistry. During centuries, scientists and surgeons made lots of effort to treat patients who lost one of their organs. The first autografting was done in 17 century by a Dutch doctor with a piece of dog bone. Although the patient was excommunicated, the therapy worked well [1]. The next big jump toward implantations happened during world war II when the injured airplane pilots by shards of polymethyl methacrylate (PMMA) felt much better than those injured by standard glass [1]. Nowadays population is growing much faster than before while the life expectancy is growing even faster. According to world health organization (WHO) report, about 8.5 million people die every year as a result of
injury. This number is 32% more than fatalities due to malaria, HIV and tuberculosis and highlights the necessity for a solution [2]. Organ transplantations including autografts and allografts have been regarded as an efficient therapy although they have disadvantages. In some cases the transplanted organ is rejected by the patient’s body because of immunogenic responses. In many cases the shortage of donor organ leads to patient’s death. Every 12 min, one name is added to the transplant waiting list while an average of 22 people die each day waiting for transplants [3]. Tissue engineering is a promising alternative which provides the potential for regenerating tissues and organs of the human body. Various materials have been employed for this purpose including: metals, ceramics, natural and synthetic polymers. Among them, synthetic polymers have attracted more attention due to their superior characteristics. Their Excellency is because of their tailorable properties, biodegradability, biocompatibility and mass production. Metals used as clinical sides are not bio-degradable which means they require a second surgery for removal [4]. On the other hand, bio activated ceramics confront similar problems. They may react with physiological fluids in the body and form unwanted bonds to hard or soft tissues. Consequently, their biocompatibility is not well guaranteed [5].
For tissue engineering purposes, the synthetic bio polymer must have tailorable chemical and mechanical properties as well as proper architecture and morphology [6]. This is specifically important in porous scaffolds where interconnected pore networks provide place for cell attachment and growth. Furthermore, synthetic bio polymers can be mass produced and have a long shelf time. Synthetic bio polymers offer distinct advantages of biological properties and versatility of chemistry which leads to produce scaffolds with predictable uniformity and free from immunogenicity concerns. However in order to achieve these goals some criteria should be considered for selecting the synthetic bio polymer used for scaffold applications. These include material chemistry, molecular weight, polymer architecture, solubility, hydrophilicity/hydrophobicity, lubricity, surface energy, water absorption, and erosion mechanism (bio-degradation mechanism where necessary) [5]. In this section, the effect of mentioned parameters will be explained. In section 2, different synthetic biopolymers used in porous scaffolds will be introduced. In section 3, the application of polymer nanocomposites in scaffolds will be discussed briefly and finally the future research trends in this field will be reviewed.
REQUIRED CHARACTERISTICS FOR A SCAFFOLD
Biocompatibility: The scaffold applied inside the human body should be fabricated from biocompatible material in order not to cause an immunological reaction or foreign body attack [7]. This includes the blood compatibility as well. It means that the material should perform an appropriate host response and not cause toxic or injurious effects. Furthermore it should support the appropriate cellular activity such as facilitation of molecular signals [8].Biodegradation: The planted scaffold should be bio-adsorbed by the surrounding tissues in order to omit the necessity of a surgical removal. However to achieve this, two important issues should be considered. First, the degradation should not produce toxic and harmful byproducts. Second, the kinetic of degradation should be designed appropriately according to the final application. For example, in skeletal tissue engineering, bio-adsorption of the scaffold should be relatively slow, since the mechanical strength should be adequate until tissue regeneration is almost completed. However, for the skin scaffold, bio degradation rate should be relatively high. If the scaffold remains for a long time, the remaining material may retard the tissue regeneration rather than healing effect [9].Processability: In contrast to metals and ceramics which have poor processability, polymers can be processed easily [10]. There are plenty of methods for producing porous scaffolds based on synthetic biopolymers. The method selection depends on many parameters and should be engineered carefully. This issue will be discussed in another chapter.Pore Density: An important factor for a scaffold is porosity and adequate pore size. The mission of scaffolds is to provide an appropriate environment for attaching the cells and growing them. In order to fulfill this goal, interconnected micro pores with sufficient pore size are necessary. The cells can be seeded and grow on the micro pores which provide vascular formation as well as waste transport. For bone tissue engineering a minimum pore diameter of 150 μm is suggested while for soft tissue engineering a range of 200-250 μm is recommended [11].Mechanical Properties: Generally the mechanical properties of scaffold should be sufficient to shield cells from damaging under compressive or tensile forces [12]. However, in some applications mechanical properties play a striking role, e.g. in skeletal systems. Mechanical environments markedly affect the bone tissue scaffolds. The amount of load on the bone tissue engineering affects its reconstruction.BIOPOLYMERS
Bio polymers can be divided into various classes regarding to synthesis, properties, biodegradability and application. The designed scaffold may be used as a ‘soft’ or ‘hard’ tissue; therefore the kind of material used differs. For soft tissues e.g. cardiovascular substitutes or skeletal muscle, generally a wide variety of polymers are employed. Hard tissues, e.g. bone substitutes, are usually fabricated from rigid polymers. In this section the synthetic polymers used as scaffold have been reviewed regardless their ways of categorizing. Their application, properties, synthesis and biodegradation mechanism have been scrutinized as well.
Polyesters (Poly (α-Hydroxy-Acid))
Among various synthetic bio polymers, poly (α-hydroxy-acid)s are very popular due to their distinct properties. The advantage of these poly esters is that their degradation rate is tailorable. Depending on the final application, their degradation period can be controlled from months to years [12]. This is the reason why these biopolymers are most common polymers for scaffold application. However just like a double edged sword, they also have disadvantages. Within degradation, poly (hydroxy acids) produce acidic species which may decrease the local pH and accelerate the degradation rate. This may cause inflammation for surrounding implant [13]. They also suffer from their hydrophobicity which hinders smooth cell seeding. To overcome these drawbacks, copolymers or blends can be used. The most applicable poly (hydroxy acids) include PLA, PCL and PGA.
Polycaprolactone
Polycaprolactone (PCL) is a semi-crystalline polymer with a glass transition temperature (Tg) of approximately –65ºC and a melting point (Tm) of approximately 60 ºC which is prepared by ring opening polymerization of ε-caprolactone. PCL imparts good biocompatibility, biodegradability and permeability and shows excellent cell attachment and growth. Scheme (1) shows its structure.
Scheme 1) Structure of polycaprolactone.Biodegradation
The biodegradation of PCL takes place because of the ester linkages in the linear polyester which are susceptible to hydrolysis. In comparison to other aliphatic polyesters, degradation kinetics of PCL is slower because of its hydrophobic nature and high crystallinity. The products generated by biodegradation are either metabolized via the tricarboxylic acid (TCA) cycle or eliminated by direct renal secretion [14] Generally, degradation kinetics of poly(hydroxy acid) is affected by various factors including: degree of crystallinity, site of implantation [15] and molecular weight [16].
Scaffold Preparation Techniques
Polycaprolactone scaffolds can been constructed with a variety of solid free-form (SFF) techniques including fused deposition modeling (FDM) [17, 18], photo polymerization of a synthesized PCL macromere [14], shape deposition modeling [19], precision extruding deposition [20], low-temperature deposition [21] multi-nozzle free-form deposition [22] and selective laser sintering (SLS) [23]. The maximum porosity obtained by FDM technique was 80% while the non-woven method provides irregular porous structures with a pore size of 20-100 μm [23].
Direct solid free-form techniques provide porous scaffold with an appropriate control over porosity, pore size and interconnectivity. However they offer limited micro scale resolution and also require multifaceted manufacturing control. To overcome these problems and benefit the SFF technique, indirect SFF method has been employed [24]. Porogen-based SFF, a method of indirect SFF technique, has been recently applied for fabrication of PCL scaffolds as an alternative for bone tissue engineering. Employing this technique has resulted in scaffolds with a pore size of 200μm. Furthermore, the resolution of the 3D-SSF system was at least 2-fold as compared to directly built scaffolds [25].
In order to increase the surface area of PCL scaffolds, various methods have been employed e.g. fused deposition modeling [26]. Li et al. used needleless electro spinning for the first time to produce PCL 3D fibrous tissue engineering scaffolds. In comparison to the solvent casting method, needleless electrospun scaffolds showed higher porosity with higher surface-to-volume ratio which results in the enhancement of protein adsorption, cell attachment and proliferation. These scaffolds showed great potential in soft tissue engineering. With a 3D structure and larger pore size, fibroblasts were able to migrate up to 800 μm into the scaffolds [27].
Application
PCL is often used in bone tissue engineering and cartilage. In contrast to autogenic and allogenic grafts which suffer from various problems, bone tissues based on PCL have shown successful results. Disadvantages of grafting include organ rejection and donor site morbidity. On the other hand, metal implants used as bone tissues, can’t perform healthy bone either. They are not bio-degradable so they require a second surgery for removal. Furthermore, they can’t remodel during time and they may cause stress shielding. Another choice for bone tissue organs is bioceramic especially hydroxyapatite (HA) which is osteogenic. However, HA is brittle and can’t be processed into complex shapes [28]. PCL based scaffolds overcome these problems and recently they attracted much attention for bone repairment.
PCL-based scaffolds are also proper for skin replacement [29, 30] and nerve tissue engineering [31]. The application of PCL in tissue-engineered skin is because of its good mechanical properties as well as biocompatibility. Natural scaffolds such as collagen lattice used in skin tissue engineering suffer from shrinkage and interspecies pathogen transfer. Synthetic polymers also have shortcomings which must be considered. One of these shortcomings is low mechanical strength which can be compensated by producing biaxially stretched PCL. The results showed that cell attachment and proliferation of these stretched PCL was 20-40% better than that on polyurethane [29].
Another application of PCL-based scaffold is in nerve tissue engineering. In the case of nerve tissue engineering, electrical conductivity is vital. Consequently, applied scaffold should be prepared from conductive polymer or its blends. Since PCL is not conductive, its blends with a conductive polymer have been used for nerve tissue engineering which is explained in the following paragraphs.
Polycaprolactone Blends: PCL/ Hydrogels
Recently, the blends of PCL with natural hydrogels (gelatin, collagen, chitosan and alginate) have attracted much attention. Chitosan is a natural bio polymer which is extensively used for wound healing applications and for skin tissue engineering in combination with other biopolymers. Blending of two polymers is an appropriate method to bring desired physico-chemical characteristics of both polymers. Electrospun mats of chitosan/ PCL with a composition ratio of 1:5 were used for skin tissue engineering. In comparison to PCL alone, the blend showed enhanced cell adhesion, cell spreading and proliferation [32].
Introduction alginate to PCL can overcome the PCL deficiencies as well as alginate ones. PCL can produce acidic by-products during degradation, causing an inflammatory response. On the other hand, alginates have poor mechanical properties and limited 3D shape-ability due to their hydrophilic nature. Blending of these polymers can improve low mechanical strength and biocompatibility of alginate and PCL respectively. These nano fibrous scaffolds could be suitable for the regeneration of various hard tissues [33].
PCL/collagen composite scaffolds prepared by electrospinning have good mechanical properties as well as good cell adhesion and proliferation. They have been studied in various tissue engineering fields such as skin [34] and vascularization vessels [35].
PCL/ gelatin nano fibers were extensively studied by Ramakrishna et al. they used these scaffolds for dermal reconstitution [36] as well as neural tissue engineering. For this purpose C17.2 nerve stem cells were carried out. These cells can be used as neuron precursors since they are involved in the normal development of cerebellum, embryonic neocortex and other structures. In comparison to PCL, PCL/ gelatin nanofibers have promoted neurite extension [31].
Polycaprolactone Copolymers
In order to balance hydrophobicity and crystallinity of caprolactone, its copolymers with hydrophilic monomers has been prepared. Polyethylene glycols (PEGs) have been most commonly copolymerized with caprolactone to prepare hydrophilic, non-toxic, non-immunogenic and non-antigenic copolymers. Davaran et al. [37] synthesized triblock PCL-PEG-PCL copolymers by ring-opening polymerization of caprolactone in the presence of PEG and employed electrospinning to produce nano fibrous scaffolds. They studied the effect of composition and PEG molecular weight on morphology of nanofibers and concluded that, by increasing molecular weight of PEG, the fiber diameter decreased and the fiber morphology became finer. These results are of significant importance for design of scaffolds. By applying this method the favorable properties of PEG retains and the spin ability improves.
Polyglycolic Acid
Poly (glycolic acid) (PGA) -also called polyglycolide- is a rigid thermoplastic material with high crystallinity (46-50%). It can be prepared by polycondensation or ring-opening polymerization of glycolic acid. The glass transition temperature and melting point temperatures of PGA are 36ºC and 225ºC, respectively. According to some reports, PGA is not soluble in most organic solvents due to its high crystallinity; the exceptions are highly fluorinated organic solvents such as hexafluoro isopropanol [6]. Scheme (2) depicts the structure of PGA.
Scheme 2) structure of polyglycolic acid.Biodegradation
Polyglycolic acid is a bulk degrading polymer, degrades by the non-specific scission of the ester backbone. The degradation process happens in two steps, first, diffusion of water molecules into the amorphous regions of the matrix and second, hydrolytic chain scission of the ester groups. Finally, the crystalline regions of the polymer will be triggered to degrade the polymer completely [6].
Application
Polyglycolic acid scaffolds have various applications including heart valve, myocardial tissue, esophagus, trachea [38] and wound dressing [39]. They are also served as the template for neocartilage formation. The grown neocartilage has stained positively for extracellular matrix structures found in native cartilage, such as sulfated glycosaminoglycan, collagen type II, and elastin [40].
Disadvantages of PCL include: high rate of degradation, acidic degradation products and low solubility. Therefore, several copolymers containing glycoside units are being developed to compensate the inherent shortages of polyglycolide [41]. The most common copolymer is poly (lactide-co-glycolide) which will be explained in the following paragraphs.
Polylactic Acid
Poly (lactic acid) –also called polylactide- is aliphatic polyester derived from renewable resources, such as corn starch, tapioca roots, chips or starch. It exist in three isomeric forms due to its monomer chirality: semi crystalline d(-), semi crystalline l(+) and racemic (d,l) which is amorphous [6]. Poly (L-lactide) (PLLA) is a semi crystalline polymer with 37% crystallinity. The degree of crystallinity depends on the molecular weight and polymer processing parameters. It has a glass transition temperature of 60–65°C and a melting temperature of approximately 175°C. PLLA has high tensile strength and modulus and therefore it is an ideal biomaterial for load bearing structures.
Poly (DL-lactide) (PDLLA) is an amorphous polymer due to the random distribution of L- and D-lactide units. It has a glass transition temperature of 55–60°C and a melting point of approximately 260ºC. In comparison to PLLA, PDLLA shows much lower strength (1.9 GPa) which can be attributed to its amorphous nature. This polymer loses its strength within 1–2 months when hydrolyzed and undergoes a loss in mass within 12–16 months [41]. In Scheme (3) structure of PLA is shown.
Biodegradation
PLA degrades by bulk hydrolysis and produces lactic acid which exists in the human body. The body transports the produced L(+) lactic acid to the liver, converts it into pyruvic acid and upon entering the tricarboxylic acid cycle, secreting it as water and carbon dioxide and finally the carbon dioxide will excrete by the lungs [42]. However, some inflammatory responses have been reported since the produced lactic acid can lower the pH locally.
Scheme 3) Structure of polylactide.The hydrolytic degradation of PLA is influenced by various factors including [43]:
Water permeability, solubility (hydrophilicity/hydrophobicity).Additives presenting in the final scaffold (acidic, basic, monomers, solvents or even drugs): Basic compounds can catalyze ester linkage scission and thus accelerate polymer degradation on one hand and decelerate the degradation by absorbing the carboxylic acid sites on the other hand. The balancing between these two mechanisms determines the rate of degradation.Morphology of the PLA: (crystalline, amorphous and the degree of crystallinity), the higher the degree of crystallinity the lower rate of degradation.Porosity: by increasing the amount of porosity, the bio-degradation accelerates since the oligomers and low molecular weight produced during degradation can diffuse faster and expedite the degradation. These oligomeric chains are ended by carboxylic acid groups which may facilitate the autocatalytic degradation of the polymer.Glass transition temperature.Molecular weight and molecular weight distribution: wide molecular weight distribution means that the numbers of carboxylic end groups are higher compared to narrow molecular weight distribution which results in faster degradation rate.Sterilization: the types of materials used for sterilizations or the employed technique will affect the degradation.Site of implantation.Scaffold Preparation Techniques
Porous scaffolds of PLA can be fabricated by different techniques including solvent casting [44], ice particle leaching method, gas foaming/ salt leaching method [45], freeze drying [46] and thermally induced phase separation. For producing fibrous scaffolds, electrospinning [47] or wet spinning can be also employed.
Application
Polylactic acid (PLA), and their copolymers have different applications in three dimensional culture and transplantation of articular, auricular, nasal, costochondral, tracheal, chondrocytes and intervertebral disk [48]. several cell types were successfully grown on the surface of PLA scaffolds including MC3T3-E1 osteoprogenitor cells [49], osteoblasts [50] chondrocyte [51], keratinocytes [52], hepatocytes [53], Bone marrow-derived mesenchymal stem cells (BMSCs) (cardiovascular applications such as tissue-engineered heart valves) [54], pneumocytes [55], Hep G2 cells [56] and neural stem cells [57].
Polylactide Copolymers and Composites; Poly(lactic-glycolic Acid)
PLGA is a linear copolymer with a Tg around 37°C that can be prepared at different ratios between its constituent monomers, lactic (LA) and glycolic acid (GA). It is preferred for the fabrication of bone substitute because of its superior control over degradation rates, crystallization and physico-chemical properties such as hydrophobicity/hydrophilicity balance. PLGA can be synthesized via different synthesis processes including solution poly-condensation, melt/solid polycondensation process and ring opening polymerization. In spite of the molecular weight of the monomers and their ratio, tacticity (stereochemistry) of the copolymer also influences dramatically the ultimate properties. By increasing the molecular weight, the degradation rate decreases. Also if the ratio of LA to GA enhances the degradation rate decreases because LA component is less hydrophilic compared to GA. PLGA degrades by hydrolysis of its ester linkages and converts into LA and GA byproducts [58].
PLGA materials have been used extensively to fabricate scaffolds for engineering musculoskeletal tissue. These include scaffolds for bone [59] cartilage and meniscus. PLGA scaffolds showed acceptable results by culturing various cells including 3T3 fibroblasts [60], osteoblasts [50, 61, 62], chondrocytes [63], keratinocytes [52], Bone marrow-derived mesenchymal stem cells (BMSCs) [54, 64], Primary neonatal Schwann cells [65] and Adipose tissue [66].
In order to produce porous scaffold out of PLGA, various techniques have been employed including phase separation [67], gas forming [68], porogen leaching [44] and solid free form techniques [69]. Porogen leaching method has been widely used by different researchers however this technique suffers from some serious problems such as incomplete solvent removal and restricted thickness. Some improvements are exerted by combining two methods e.g. gas forming and particulate leaching which omits the need of solvent and combination of the porogen leaching and melt-molding. Nowadays, selective laser sintering is used as a useful technique for producing scaffolds of PLGA [58]. Scheme (4) depicts the structure of PLGA.
Scheme 4) Structure of poly (lactide-co-glycolide).Polylactide/ Poly(lactic-glycolic Acid) (PLA/PLGA) Blends
An important issue in polymer blends is their miscibility which determines the physicochemical and final properties of the blend. In tissue engineering field, this aspect is of great importance because tailoring the ultimate properties is influenced by the blend characteristics. The miscibility of blends can be investigated by various method such as thermal (detecting the Tg), rheological methods and microscopic methods.
The researches have shown that blends of PLLA and PLGA (LA: GA=50:50) are immiscible and create a biphasic morphology (matrix-droplet). However in some compositions (75/25 PLA/PLGA) a certain degree of compatibility has been observed. In addition to blend composition, other factors also affect the miscibility including lactic to glycolic ratio in PLGA, blending method and molecular weight of each component [70, 71].
Addition of PLGA to PLLA has some benefits: the in vitro degradation of the blend has accelerated compared to its component. Furthermore, presence of PLGA can reduce the inflammatory reaction at the site of implantation.
Polylactide/ Polycaprolactone (PLA/PCL)
In order to overcome the brittleness of PLA, blending with a tough bio-polymer has been suggested. PCL is a bio degradable polymer which also improves the fracture toughness of PLA. Blends of PLLA and PCL form immiscible phases, with the PCL phases finely dispersed in the PLLA-rich matrix phase [72]. The presence of PCL in PLLA/PCL blends reduces the crystallinity of PLA. As a consequence, elongation at break increases and ductility improves. These blends have been used for bone regeneration and their efficacy tested using bovine periosteal tissues. The addition of PCL decreased the brittleness of PLLA membranes and at 50% PCL content, the elongation was also improved without decreasing the porosity on the membrane surface which is important for tissue adherence [73].
Polylactide/ Polyethylene Glycole (PLA/PEG)
The high hydrophobicity of PLA restricts its applications in some certain fields of tissue engineering. Therefore copolymerization with a hydrophilic bio-polymer such as PEG has been considered. Another way is blending of PLA with PEG which results in a partial miscible blend. Introduction of PEG to PLA also increases the biodegradation rate due to the enhancement of the surface hydrophilicity. These scaffolds can be used both in bone and skin tissue engineering. The result of dermal fibroblast cell culturing has proved the cellular interaction with nanofibrous mats. Cultivation of osteoblasts-like MG-63 cells also showed the adherence and proliferation of cells in the scaffold [74, 75].
In addition to electrospinning and freeze drying methods, gas forming technique has also been employed for preparation of PLA/PEG scaffolds. In this technique porosity is created by dispersing gas bubbles inside a viscous polymer solution. The gas bubbles can be formed by a blowing agent via chemical reactions. Dehghani et al. used this method for preparation of PLA/PEG scaffolds by tailoring the porosity. According to their results, mechanical properties of PLA/PEG blends with less than 30wt% PEG were suitable for the fabrication of porous scaffolds. However, increasing the concentration of PEG to above 50% resulted in blends that were brittle and had low mechanical integrity [76].
Introduction of PEG to PLA not only improves the hydrophilicity of PLA but also increases the porosity. In comparison to PLA, blends of PLA/PEG have a higher porosity. The porous films of PLA/PEG can be used for wound dressing. The enhanced porosity and hydrophilicity result in a higher water vapor transmission rate and higher oxygen permeability which are essential parameters for healing a wound. In order to prepare antimicrobial wound dressings, antibiotic drugs can be loaded on these porous matrix films. The results published by Phaechamud et al. confirm that these drug-loaded porous films, will efficiently inhibit the bacterial growth so they can be used as an effective dressing for treatment or prevention of bacterial wound infection [77].
Polylactide/ Polyhydroxybutyratevalerate (PLA/ PHBV)
Despite of suitable properties of PLA, it exhibits some drawbacks such as high brittleness and low heat resistance. These drawbacks need to be addressed in order to widen its range of applications in tissue engineering. Polyhydroxy-butyratevalerate (PHBV) is a copolymer of PHB with randomly arranged 3-hydroxybutyrate (HB) groups and 3-hydroxyvalarate (HV) groups which exhibits high stiffness and crystallinity. This polymer is a good candidate for blending with PLA to improve its brittleness. Peng et al. [78] studied the effect of the PLA/PHBV blend composition on the morphology as well as thermal and mechanical properties of microcellular PLA/PHBV injection molded component. According to their results, PLA/PHBV blends containing less than 30% PHBV were miscible. It was proved that addition of PHBV to PLA improves the strain-at-break significantly.
The capability of cell adhesion and growth on PLA/PHBV substrates was studied by Stantos et al. They used Vero cells (a fibroblastic cell line established from the kidney of the African green monkey) for this case and studied various blend compositions. They concluded that (60/40) and (50/50) PLA/PHBV blends had the best cell adhesion among other compositions [79].
Hybrid of Poly (Hydroxyl Acids) with Collagen
As mentioned before, poly (hydroxy acids) suffer from their hydrophobic property which results in lack cell-recognition signals and hinders smooth cell seeding. In contrast, naturally derived collagen has the potential advantages of specific cell interactions and hydrophilicity, but collagen based scaffolds have poor mechanical strength. In order to benefit the properties of each polymer, hybridization of collagen and polyesters have been employed. Chen et al. produced PLGA-collagen hybrids by combining porogen leaching and freeze-drying techniques. By applying this method pore structures of the polymer sponges could be manipulated. These hybrids benefit both the synthetic polymers and collagens [80].
Polyhydroxyalkonates
Polyhydroxyalkonates (PHA) are aliphatic polyesters which are produced by microorganisms under unbalanced growth conditions. PHAs are generally biodegradable and have good biocompatibility therefore, they have been regarded as good candidates for tissue engineering biomaterials. Today, there are more than 100 known PHAs which have different structures and various physical- mechanical properties [81]. The most applicable PHA in tissue engineering include poly 3-hydroxybutyrate (PHB), 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO). PHB is the most common member of the PHA family which has good biocompatibility for adrenocortical cells, osteoblasts, epithelial cells, fibroblasts, endothelium cells, and isolated hepatocytes [82]. Scheme (5) shows the general structure of polyhydroxyalkonates.
Scheme 5) Structure of polyhydroxyalkonate: m=1, R=CH3, the monomer structure is 3-hydroxybutyrate; m=1, R=C3H7 monomer is hydroxyhexanoate.Biodegradation
Another superiority of PHB among various PHAs is its existence in blood. Low molecular weight PHB is widely distributed in biological cells, being found in representative organism of nearly all phyla [83]. However the toxicological properties of other PHAs should be checked before use. For example hydroxyvalerate may be cytotoxic and may cause inflammatory effects. In general, various parameters affect biodegradation of PHAs including: composition of the material, methods of processing, crystallinity and molecular weight [81].
Scaffold Preparation Techniques
There are currently several methods available for fabrication of PHA scaffolds, including solution casting, electrospinning, phase separation, solvent casting, fiber bonding and particulate leaching, solid freeform fabrication, and particle sintering [84].
Application
The main application of PHAs is in the bone tissue engineering. Application of PHB as bone tissue showed no evidence of an undesirable chronic inflammatory [85]. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBHHx scaffolds have been applied for tendon repairing. In order to evaluate their facilitation of tendon movement recovery and complete restoration of load bearing and function, rats marrow recipient were employed [86].
Another application of PHAs is in neuro tissue engineering. PHA scaffolds can be used for cultivation of neural stem cells which may be useful for repairing central nervous system injury [84]. An example is PHBHHx which promotes neural stem cells differentiation into neurons. Nanofiber scaffolds prepared from PHBHHx, are good candidates for treating central nervous system defects [84].
Blends of (Polyhydroxy Alkonate) PHAs: Poly 3-hydroxybutyrate/ Polyethylene Glycol (PHB/PEG)
A drawback of PHB is its poor hydrophilicity. In order to improve the hydrophilicity, it would be practical to blend it with a hydrophilic polymer such as PEG. Cheng et al. [87] produced PHB/PEG blends for blood vessel. Their results showed that PEG played an important role in resisting platelet adhesion. Furthermore, by increasing the PEG loading, the number of live cells increased which is an evidence for the suitability of the blend for blood vessel.
Recently, Karahaliloglu et al. [88] used plasma polymerization to graft PEG on the surface of PHB nanofibers. In order to improve the drawbacks of PHB, they used surface modification. At first nanofibers of PHB were produced by electrospinning then PEG was grafted on its surface. L-929 cells were used to test the proliferation. The results proved that surface modification has improved the proliferation.
Poly 3-hydroxybutyrate/Poly (lactic-co-caprolactone)(PHB/PLCL)
Punyodom et al. [89] produced a blend of PLCL, PHB by electrospinning. They observed that by increasing PLCL loading in PHB blends, the average fiber diameters and the distribution of diameters in the electrospun scaffolds decreases significantly. Furthermore, PLCL has improved the brittleness of PHB. On the other hand presence of PHB improved cell adhesion and growth of PLCL.
Poly 3-hydroxybutyrate /Poly (3-hydroxyvalerate) (PHB/PHBV)
PHB is known as a rigid and highly crystalline polymer with slow degradation rate that results in a poor processing window. In contrast, PHBV has lower glass transition and melting temperatures and therefore it is more flexible and easier to process. Blending of PHB with PHBV will decrease the melting temperature, leading to the possibility to process the materials at lower temperature. Blends of PHB/PHBV have been studied in various compositions. PHB/PHBV 50/50 scaffolds have been used in bone tissue engineering [90] and PHB/PHBV 30/70 have been used as scaffold for human adipose tissue-derived stem cells [91]. Recently, Morshed et al. [92] prepared random and aligned PHB/PHBV nanofibers by electrospinning and evaluated the Schwann cells (SCs) effect on them. They observed that aligned nanofibrous scaffolds showed higher SCs proliferation after 14 days compared to random nanofibers. These results confirm the suitability of the scaffolds for using in nerve tissue engineering.
Poly (Propylene Fumarate)
Poly (propylene fumarate) (PPF) is a linear polyester polymerized of fumaric acid. The fumarate double bonds are reactive and crosslink at low temperatures, making it valuable as an in situ polymerizable biomaterial especially for orthopedic applications. Although crosslinked networks may be formed from PPF alone, a variety of crosslinking agents have been explored in combination with PPF for the formation of crosslinked, degradable polymer networks with tunable material properties. For example, crosslinked networks of PPF with N-vinyl pyrrolidinone, poly(ethylene glycol)-dimethacrylate, PPF-diacrylate and diethyl fumarate have been developed [93]. In Scheme (6), the structure of poly (propylene fumarate) has been shown.
Scheme 6) Structure of poly (propylene fumarate).Biodegradation
Degradation of PPF starts by hydrolysis of the ester bonds (as shown in Scheme 6) and results into fumaric acid and propylene glycol which are biocompatible and insoluble in water [94].
Application
The double bonds in PPF allow the linear polymer to be crosslinked into a solid, polymeric network which makes it appropriate for bone tissue engineering scaffolds. This polymer was either used as an injectable in situ curing material or as preformed scaffold [95]. The PPF scaffolds were used for large cranial defects in a rabbit model. The scaffolds were photo crosslinked by UV laser and produced with various porosities. The results showed that bone ingrowth in PPF scaffolds implanted into cranial defects was <3% of the defect area which confirms their suitability as platform for bone tissue engineering [96].
Although PPF has good mechanical properties, for bone tissue application it must mimic the bone extracellular matrix. Bone is a structure composed of hydroxyapatite crystals deposited within an organic matrix consisting of ∼95% type I collagen. Therefore, hydroxyapatite and other calcium phosphate have been introduced to PPF to improve bone regeneration capacity. The results revealed that scaffolds with each of the calcium phosphate coatings were capable of sustaining recombinant human bone morphogenetic protein-2 (rhBMP-2) release and retained an open porous structure [97].
Polyanhydride
Polyanhydrides are a class of biodegradable and biocompatible polymers which degrade by surface erosion. Generally, they are polymerized by polycondensation method through dehydration of the diacid or a mixture of diacids [98].The dicarboxylic acid monomers are converted to the mixed anhydride of acetic acid by reflux in excess acetic anhydride. High molecular weight polymers are prepared by melt-polycondensation of pre polymer in vacuum under nitrogen sweep [6].
Polyanhydrides are chemically reactive which has both advantage and disadvantage. Their advantage is their degradation by surface erosion without the need of incorporation various catalysts or excipients. However because of high reactivity, polyanhydrides may react with compounds containing free amino groups or other nucleo polyanhydrides [99]. Another drawback of polyanhydrides is their limited mechanical properties which restrict their use in load–bearing applications such as orthopedics. For orthopedic means they are blended or copolymerized with other polymers [6].
Scheme 7) Structure of polyanhydride.Biodegradation
Polyanhydrides are surface eroding polymers which do not allow water to penetrate into the material and erode layer by layer. Their degradation proceeds by hydrolysis of the anhydride linkage. By choosing appropriate diacid monomers, the hydrolytic degradation rates can be manipulated. Poly (sebacic acid) degrades quickly (about 54 days in saline), while poly (1,6-bis(-p-carboxyphenoxy) hexane degrade much more slowly (estimated 1 year). In order to control degradation rate for a specific application, different amounts of these monomers can be combined in polymer [100].
Application
Polyanhydrides may have an orthopedic application but their Young’s Modulus should be improved by formation of cross-linked networks. Two various strategies have been employed to improve mechanical properties of polyanhydrides: copolymerization and photo polymerization.
Incorporation of aromatic imide groups to anhydride monomers can increase the thermal and mechanical properties of the copolymers. Langer et al. [101] first, synthesized a series of poly(anhydride- co-imides) containing pyromellitic acid derivatives for potential use as degradable, high compressive strength materials. Their results proved that the mechanical and thermal stability of the materials are increased by the incorporation of imide groups in the polymer backbone.
To produce photopolymerizable polyanhydride, cross linkable methacrylate groups were introduced to polyanhydride chains which results in controlled degradation and improves mechanical properties. In general, the core of the molecule consists of hydrophobic repeating units, such as sebacic acid, carboxyphenoxy propane, or carboxyphenoxy hexane but other anhydride monomers, including methacrylated tricarballylic acid (MTCA, trimethacrylated) and methacrylated pyromellitylimidoalanine (MPMA-ala, amino-acid containing) have been synthesized to impart greater crosslinking density and a biologically recognized component, respectively [102].
Photo crosslinkable polyanhydrides have been employed for several applications. Because they are injectable, they can be formed directly in a bone defect through a photo initiated polymerization. When new bone fills the defect, the injected polymer will degrade [103].
Polyurethane
Polyurethanes can be tailored to have a broad range of properties, from soft to hard tissue applications [6, 104] In contrast to aliphatic linear polyesters that are more appropriate for hard tissue engineering due to their high glass transition temperature and high modulus, polyurethanes can be used as soft tissue scaffolds as well. They exhibit a wide range of properties through variability of the hard segment (diisocyanate), the soft segment (polyethers or polyesters), the chain extenders, and the ratios in which they are reacted [105]. Scheme (8) shows a general structure of polyurethanes.
Scheme 8) Structure of polyurethane.Biodegradation and Biocompatibility
An important issue in polyurethanes is the toxicity of their degradation product which limits their usage as bio materials. Recently polyurethanes with non-toxic degradation products have been developed. Guan et al. [106] synthesized polyurethanes from polycaprolactone (PCL) and 1,4-diisocyanatobutane (BDI) with putrescine used as a chain extender. The hard segments of polyurethane were built by BDI and the soft segments were prepared by polyethylene glycol (PEG) which also increases the hydrophilicity of the scaffold. Putrescine, as a polyamine can improve cell growth and differentiation, following complete degradation. This group also studied the degradation of the polyurethanes. According to their results, there was no evidence of an autocatalytic effect during the degradation process. Furthermore, degradation behavior is different from that of poly (α-hydroxyester) such as poly (L-lactide) and poly (lactide-co-glycolide). In contrast to polyhydroxy esters, the pH will not decrease during degradation. In addition, the use of BDI as a hard segment with chain extension by putrescine would be expected to ultimately yield a hard segment degradation product of putrescine, which is already present in the body and has been implicated as an important mediator of cellular growth and differentiation in response to growth factors [107, 108].
Scaffold Preparation Techniques
Methods used for preparing polyurethane porous scaffolds include electrospinning [109, 110], solvent casting/salt leaching [111], phase inversion [112], laser excimer [113] and thermally induced phase separation [114]. Electrospun polyurethanes are elastomeric, have small diameter, high porosity and controlled degradation rate. Although these properties aid the development of soft tissue scaffolds, it is difficult to make a scaffold with large pore sizes [110]. In the case of solvent casting/salt leaching method, pore size can be controlled by manipulating the size of the salt particulate. However, the resulting scaffold may have limited interconnectivity, which would adversely influence cell seeding and ingrowth [111]. Phase inversion method results in scaffolds with low interconnectivity and week control over pore size [112]. Scaffolds prepared by laser excimer method exhibit straight pores but achieving connectivity is still a challenge [113]. In the case of thermally induced phase separation method, pore size and pore structure can be controlled by varying the preparation conditions [114].
Application
Polyurethanes have a vast majority of applications including hard and soft tissues. However, the diversity of soft-tissue applications is far greater. These include endothelium, cardiovascular [107, 111], epithelial, cartilage skin related tissue [114] and bladder muscle reconstruction.
For endothelium reconstruction, the surface of polyurethane is usually amino modified. The introduced amino groups provide the opportunity to immobilize bio macromolecules such as gelatin, chitosan or collagen onto PU scaffold surface which in return enhance cell–material interaction and accelerate the endothelium regeneration [115].
