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Bone Generating Fibre Polyhydroxybutyrate (PHB)

adv-07

Ismat Zerin

Technical Editor, Textile Focus

Assistant Professor, National Institute of Textile Engineering and Research (NITER)

Abstract

Osteoporosis is a global public health problem. In osteoporosis, the mineral density of bone is reduced, the microarchitecture of bone is deteriorated, and the amount and variety of proteins in bone are altered. These can result in painful bone fractures. Bone tissue engineering is an attractive method to repair the bone defect. Biopolymer PHB (polyhydroxybutyrate) – possess a good biocompatibility and biodegradability, and it can be used for bone regenerations. It degrades slowly in body without an inflammatory reaction. These properties make it an interesting material as a cell carrier and implant for bone tissue engineering. This study gives an overview of the chemical structure of PHB, properties and application of PHB fibre and its utilization in the field of bone regeneration.

Day after day is increasing the human being in this limited earth. The environment pollution and the various diseases are also coming forwards with new faces. One of it, is Osteoporosis- means “porous bone“, i.e. bone deterioration, is a global public health problem that affects one in three women and one in 12 men over the age of 50. In osteoporosis, the mineral density of bone is reduced, the microarchitecture of bone is deteriorated, and the amount and variety of proteins in bone are altered. Which can result in painful fractures most likely to occur in the hip, spine and wrist, but other bones can break too. Bone tissue engineering scaffold is an attractive method to repair fragile bone [1]. Figure: 1 demonstrates an osteoporotic hip bone in compare to a healthy bone.

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To come round from such diseases and to have a healthy life cycle, life science i.e. medical science is improving rapidly. The advent of biomaterial has significantly influenced on the development and faster growth of various technologies in medical science. Several biopolymers, both natural and synthetic, have been investigated and find applications as sutures, scaffolds for tissue regeneration, tissue adhesives, hemostats and transient barriers for tissue adhesion, as well as drug delivery systems [2]. The biopolymer PHB (poly-hydroxybutyrate) – possess a good biocompatibility and biodegradability. It degrades slowly in body without an inflammatory reaction. These properties make it an interesting material as a cell carrier and implant for bone tissue engineering. Due to its biocompatibility, biodegradability and processability PHB have been investigated as matrices for drug delivery applications and tissue engineering.

Introduction of PHB-fibre

Polyhydroxybutyrate (PHB) is a thermoelastic polymer belongs to polyhydroxyalkanoates that serve many bacteria as intracellular storage molecules for carbon and energy [3]. Both prokaryotes and eukaryotes produce it although its accumulation is considered only in some prokaryotes. It accumulates as distinct inclusions in the cell and comprises up to 80% of cell dry weight for strains of Ralstonia eutropha, under conditions of nitrogen or phosphate limitation and excess of carbon source [1]. A poly-R(-) (3-hydroxybutyric acid) (abbreviated as PHB) is a biodegradable and biocompatible homopolyester and has prospects for various applications in which biodegradability or biocompatibility is required [4]. It is a linear polymer of R-3-hydroxybutyrate (R-3HB) and has a fundamental constituent of biological cells [4]. Transgenic plants provide a potential means of producing this polymer cost-effectively [3]. PHB is a stereoregular, isotactic, aliphatic and optically active polyester- like polyglycolide and is also degradable within the body. In 1925 PHB was invented by French microbiologists Maurice Lemoigne in the cytoplasm of bacterium Bacillus megaterium. Polyhydroxyalkanoates (PHAs) are commonly composed of ᵝ- hydroxy fatty acids where the R group changes from methyl to tridecyl and form poly -3 hydroxybutyrate (PHB, R= CH₃), The structure is shown in Figure: 2 [2]. The polymer is comprised predominantly of R (—)-3-hydroxybutyric acid and poly R(—)(3-hydroxybutyric acid) (PHB).

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Properties of PHB fibre

The PHBs are soluble in common organic solvents and can be processed into membranes, fibres, or microspeheres. The properties of poly hydroxybutyrate (PHB) which are shown in Table 3: Comparision of physical properties of PHB, polypropylene and copolymers of PHB with higher PHAs [5]. The physical properties of PHB are similar to polypropylene while strength parameters (tensile strength, Young’s modulus) and the most physical properties (crystallinity, melting temperature and glass transition temperature) are basically the same with others fibre. The difference consists in elongation at break, consequently toughness. While the ductile polypropylene breaks at elongation around 400%, PHB hardly exceeds 5% of it. PHB copolymers with higher PHAs have higher elongation at break due to much lower crystalinity.

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Polyhydroxybutyrate (PHB) is a naturally occurring ᵝ-hydroxy-fatty acid. Its ability to degrade and resorb in the human body environment makes it a suitable candidate as the matrix for bioactive and biodegradable composite implants that will guide tissue growth and be replaced eventually by newly formed tissue.

Manufacturing process of PHB fibre

PHB is manufactured mainly from plants in bacterial fermentation under conditions of nitrogen deficiency. It is synthesized in bacteria by the consecutive action of three genes: ketothiolase (phbA), acetoacetyl CoA reductase (phbB), and PHB synthase (phbC) [8]. The bacterial fermentation is however expensive and much more energy consuming technology. Australian Institute of Bioengineering and Nanotechnology focuses on producing the biodegradable plastic polyhydroxybutyrate in the leaves of sugarcane at commercially relevant levels. Figure: 4-a) shows the Storage PHB granules of an Azotobacter chrococcum cell [9]9]. Figure: 4-b) demonstrates the storage PHB granules in sugarcane leaf.[10].

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Figure 4: Basic PHB granules surrounded by a) membrane of Azotobacter chrococcum cell [9] b) PHB biopolymer in sugarcane leaf cells [10]

Pioneering work performed by Poirier (1992) in Arabidopsis, showed a dramatic increase in the amount of PHB on Arabidopsis plants. However in Arabidopsis, the formation of high levels of PHB is accompanied by severe negative effects on growth and development of the plant. The only exception is the work resulting in transgenic Brassica napus plants with seeds expressing up to 7% (w/v) PHB by weight. PHB of low molecular weight was also identified in yeast, plants and animals, however at a very low concentration [11]. Wróbel described to generate PHB from transgenic flax and displaying normal growth pattern. This was accomplished by transferring the bacterial pathway of PHB synthesis into flax by Agrobacterium-mediated transformation [13]. Figure: 5 demonstrates PHB synthesis process from plants.Figure 4: Basic PHB granules surrounded by a) membrane of Azotobacter chrococcum cell [9] b) PHB biopolymer in sugarcane leaf cells [10]

Figure 5: PHB extraction and synthesis process flowchart from plants (Adapted from [12])

Figure 5: PHB extraction and synthesis process flowchart from plants (Adapted from [12])

After synthesis of PHB polymer, fibre can be produced from it by melt spinning and gel spinning process. But because of rapid thermal degradation of PHB melt spinning is critical; results in low melt elasticity, slow crystallization after melt spinning, formation of large crystallites leading to extremely brittle material. By an addition of plasticizers and nucleating agents (like- boron nitride) brittleness can be improved. [5].

Again, PHB fibre spinning process is described by Gordeyev et al. [14]. It involves dissolving the PHB in 1,2-dichloromethane, as the solution gets highly concentrated to prepare a solid gel by evaporation. The gel was extrude at about 170°C. After extrusion and preconditioning stretch, hot drawing and annealing, the gel spun PHB fibre is produced [5]. Tensile strength of gel spun fibres is about double that of melt spun material of similar properties and the mechanical properties do not change significantly during storing a period of 120 days [14]. Besides fibre formation, orientated PHB can be prepared also in the form of films. A patented procedure [15] refers to PHB with Mw higher than 500,000 can be oriented at temperature 144-180°C. It exhibits tensile strength 80 MPa with 70% elongation. These reasonable elongations indicate that the material can form flexible foil which could be considered for packaging [5].

Degradability of PHB

PHB degrades by enzymatic and hydrolytic degradation. The enzymatic degradation begins on the surface of the materials, preferentially in less ordered regions between spherulitic crystals and proceeds to the core of the fibre. After four days of enzymatic degradation, the drawn fibre changed to aggregates of small fibrous fragments with a spongy structure and the diameter significantly decreased. Again, PHB is degraded by hydrolysis within the body. The degradation product is hydroxybutyric acid, which is like glycolic acid and lactic acid, a normal metabolite found in the body. Not only it degrades in the body but it can also undergo degradation in soil by soil bacteria. This property makes it attractive as a degradable packaging material.[6]

Application of PHB on bone tissue regeneration

Some 208 skeletal bones act as framework and 501 separate muscles provide the co-ordination that allows humans to walk upright, run and take charge of their environment [6]. PHB and PHB-based materials (copolymers and composites) are of particular interest for bone tissue application because of their biodegradability. Doyle et al. demonstrated that PHB based (Poly hydroxybutyrate and its composites reinforced with particulate hydroxyapatite-HA) electrospuned membrane has a consistent favorable bone tissue adaptation response with no evidence of an undesirable chronic inflammatory response after implantation periods of up to 12 months. Bone is formed close to the material and subsequently becomes highly organized, with up to 80% of the implant surface lying in direct apposition to new bone. The materials showed no evidence of extensive structural breakdown in vivo during the implantation period of the study [19]. It has been established that the strength and stiffness of these materials reduce on in-vitro environment exposure in phosphate-buffered saline at 37 °C for periods up to 4 months, and that the degradation rate is a function of composition and processing conditions [17]. Bone tissue engineering demonstrates in Figure: 6 which uses stem cells, growth factors and scaffolds- porous structures for supporting cell proliferation, differentiation and tissue formation- to generate new body tissues, is an attractive method for repairing fragile or fractured bones [1].

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Again, because of brittleness and hydrophobicity of PHB Xu and his co-workers made PHB ductile and hydrophilic by incorporating segments of polyethylene glycol (PEG) into the backbone of PHB. They produced weave PHB–PEG copolymer fibers into porous structures through electro spinning. Figure: 7 is demonstrated the porous copolymer scaffold [1].

Figure 7: Scanning electron microscopy image of a porous copolymer scaffold [1]

Figure 7: Scanning electron microscopy image of a porous copolymer scaffold [1]

Porous structures made from a copolymer of PHB and PEG are ideal scaffolds for bone regeneration as these are biodegradable, absorb water and calcium minerals well. Tensile measurements revealed that the developed copolymers could be strained up to 20 times its original length before failure. Laboratory tests showed that the flexible and porous copolymer scaffolds could absorb water much better than PHB scaffolds and retain their structural integrity throughout cell culture work. In tissue engineering, the use of scaffolds that mimic natural extracellular matrixes can greatly enhance the quality and success of bone tissue formation. [20]

Advantages and drawbacks of PHB fibre

Thermoelastic polymers, PHB have a number of advantages over polymers derived from oil: They are biodegradable, their production is essentially neutral with respect to carbon dioxide balance, and they are biologically renewable with the goal of sustainable development [3]. Due to their biocompatibility, processability, and degradability these polymers have been investigated as matrices for drug delivery applications and tissue engineering. The piezoelectric properties associated with these polymers make them attractive materials for orthopedic applications, such as bone plates [2].

Though PHB possess various merits several drawbacks hinder the wider application of it, including high susceptibility towards thermal degradation, difficult processing because of thermal instability as well as low melt elasticity. It is also a brittle material resulting in low toughness which increase further during storing and undergo physical ageing [5]. Currently the main problem, which limits the widespread use of PHB and its copolymers, is its relatively high cost compared with polypropylene. The fermentation process, substrates and product recovery are the major costs including the time-consuming extraction procedure from bacterial cultures. Therefore, the extraction process might be a challenge to a cost-effective industrial upscale production for large amounts of some PHA polymers [7]. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures and less for commercial packaging. The low number of applications seems to be the reason for the high price of this polymer where the applications are not developing because of too high a price, while the price is not decreasing due to a low volume of produced polymer [5].

Conclusion

The application of biomaterials in tissue repair started with biodegradability, which involved the development and application of bio-friendly materials. PHB and PHB-based materials (copolymers and composites) are of particular interest for bone tissue application because of biodegradability. It produce a consistent favorable bone tissue adaptation response with no evidence of an undesirable chronic inflammatory response after implantation periods [16]. It can be applied in most permanent bio-implants for today’s clinical use like, for example, hip–joint replacements. In this approach, It can be anticipated that engineered composite scaffolds made by biodegradable polymer PHB & PHB matrices with bioactive phases, is reviewed here, will play a vital role in regeneration of bone tissue. This review work on PHB biopolymer is recommended to lead further research activities which could be offer in future more advancement of PHB biopolymer on medicine.

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