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Laboratory of Polymers & Biomaterials

Institute of Fundamental Technological Research

  • 1 Oliwia Jeznach as a laureate of the Kosciuszko Foundation scholarship

    It is our pleasure to inform you that our team member, Mrs. Oliwia Jeznach has become one of the laureate of scholarship granted by the Kosciuszko Foundation. Congratulations Oliwia!
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  • 2 Professor dr hab. Andrzej Ziabicki

    We announce with very deep regret that on March 14th, 2019 left us our great Master and Teacher - Professor dr hab. Andrzej Ziabicki.
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  • 3 New internschip students in our laboratory!

    As every year new internship students will join our research team! To get know who will join us this summer please see in the link below...
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  • 4 Polish-Israeli Conference on Electrospinning and Tissue Engineering

    It is our great pleasure to announce the 1st Polish-Israeli Conference on Electrospinning and Tissue Engineering (PICETE 2018), which will be held on 4-5th October 2018 in Warsaw, Poland, at the Institute of Fundamental Technological Research, Polish Academy of Sciences.Kindly visit the PICETE2018 conference website.
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  • 5 New OPUS and PRELUDIUM grant in our laboratory!

    We would like to inform that National Centre of Science granted the project of our laboratory leader, prof. Paweł Sajkiewicz and our colleague, Piotr Denis, M.Sc., in OPUS and PRELUDIUM program, respectively. Both, projects were subbmited in the ST8 category - Process and production engineering. Congratulations for the project leaders!
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Polymers have many different functions in the area of tissue engineering and can be processed into various forms. One of them is hydrogel. Hydrogels are usually recommended for medical applications in which 3D structure is required with combination of highly hydrated enviroment similar to native extracellular matrix (ECM)Moreover, this type of polymer scaffold can often be processed under relatively mild conditions, and may be delivered in a minimally invasive manner. Consequently, hydrogels have been analyzed as scaffold materials for drug and growth factor delivery or engineering tissue replacements.

 


 

Hydrogels are three-dimensional, hydrophilic, polymeric networks capable of absorbing large amounts of water or biological fluids. Due to their ability to absorb and retain large amount of water, porosity and relatively low stiffness, they mimic natural living tissue. They can be chemically stable or they may degrade and eventually disintegrate and dissolve.Hydrogels are able to fill a wound and also can act as a scaffolds promoting cells growth and differentiation. Hence, they can be put into internal or external injury areas as a wound dressing or implant inside the body in different way. The very interesting class is s.c stimuli-responsive hydrogels,  The response of such gels relies on initiation of gelation at certain pH, electric field, light or temperature conditions. Particularly interesting from our perspective are thermogels, in which gelation occurs at body temperature. Such  materials can be injected as solutions followed by formation of gels at body temperature at the place of tissue regeneration. Thus, materials in this form have a  huge potential in tissue engineering as a surgery substitute. Thinking about physical crosslinking, most of important hydrogels belong to polysaccharides. One of the examples of polymers which are able to crosslink at body temperature is  hyaluronan and methyl cellulose.

Classification of hydrogels:

The main classification is related to gelation (crosslinking) mechanism. There are two possible ways that gelation can take place:

- by physical linking,

-by chemical linking.

How to obtain hydrogel?

Hydrogels obtained by physical interactions are divided into materials with relatively strong physical bonds (Lamellar microcrystals, double or triple helices)  and with rather weak physical bonds (hydrogen bonds, block copolymer micelles and ionic interactions). This type of interactions is reversible. Hydrogels formed chemically consist of covalent bonds, generated by condensation, addition  or polymerization process. Hence, this type of cross-linking is permanent. Although, chemical bonds are much stronger than physical interactions, there is a possibility that chemical cross-linking agents may be toxic to living cells. Hence, it has been reported that physical cross-linking is safer and easier to produce  because of redundant additional cross-linkers [3].

Advantages

-significant water absorption ability,

-flexibility and softness similar to living tissues,

-easy formation,

-can be injectable (alternative for invasive surgery).

The main disadvantage of hydrogels are relatively poor mechanical properties. There are some methods which enable to overcome hydrogels’ mechanical problems. One of them is  structure modification by involving reinforcing of nanoparticles. One of the examples is inclusion of  electrospun nanofibers into hydrogel matrix. For that purpose layering, mixing with short fibers  or combination of electrospinning and electrospraying are  used.

It was demonstrated that electrospun nanofibers can improve additionally biological activity of hydrogels. The best  cells proliferation and differentiation has been reported for lower nanofiber diameter [1].

Materials

Hydrogels may be produced from various polymers which can be natural, synthetic or mixed. They are presented in Table1.

Natural

Synthetic

Natural-Synthetic

alginate

Polylactic acid (PLA)

PEG-fibrinogen

chitosan

Polyacrylic acid (PAA)

PEG-heparin

hyaluronan

Polyethylene glycol (PEG)

PEG–chitosan

collagen

Polyethylene oxide (PEO)

PAA–alginate

methylcellulose

Polycaprolactone (PCL)

Chitin–PLGA

fibrinogen

Poly(lactic-co-glycolic acid) (PLGA)

 PAA–chitosan

gelatin

Polyvinylalcohol (PVA)

PMAA–alginate

heparin

Poly(N-isopropylacrylamide) (PNIPAAm)

Methacrylated gelatin

laminine

2-metoxyloxirane (Pluronic F127)

 

Table 1. Materials commonly used as hydrogel composites [1],[2],[3],[4].

 

Application

Because of many benefits, hydrogel scaffolds  are great candidates for tissue engineering applications and technical products, e.g.:

wound care (PEG, methyl cellulose, alginate, hyaluronan), 

drug delivery system (PAA,PVA, chitosan),

injectable biomaterial (polyesters, polysaccharides),

cosmetic products (alginate, heparin, chitosan),

CNS implants (methylcellulose, hyaluronan, laminine),

Cardiac construction (methacrylated gelatin linked with carbon nano tubes),

Cartilage and bone regeneration system (PCL-fibrin, PCL-alginate and PLA-Poly(lactide-coethylene oxide fumarate).

 

The water absorption mechanism leads to generation of 4 types of water bounds:

  1. The primary water bound – obtained by linking polymer hydrophilic groups with water.
  2. The secondary water bound – obtained by linking polymer hydrophobic groups with water.
  3. The total bound water – consist primary and secondary water bound.
  4. The bulk water is a water additionally by hydrogel [3].

Literature:

  1. Annabel L. Butcher, Giovanni S. Offeddu, and Michelle L. Oyen, “Nanofibrous hydrogel composites as mechanically robust tissue engineering scaffolds”, Trends in Biotechnology November 2014, Vol. 32, No. 11.
  2. Malgosia M.Pakulska, Brian G. Balliosand Molly S.Shoichet,Injectable hydrogels for central nervous system therapy”
  3. Syed K. H. Gulrez, Saphwan Al-Assaf and Glyn O Phillips, “Hydrogels: Methods of Preparation, Characterisation and Applications”Progress in Molecular and Environmental Bioengineering - From Analysis and Modeling to Technology Applications, Prof. Angelo Carpi (Ed.), ISBN: 978-953-307-268-5, InTech,
  4. Anwarul Hasan , Ahmad Khattab , Mohammad Ariful Islam , Khaled AbouHweij, JoyaZeitouny , Renae Waters , MalekSayegh , MdMonowar Hossain , and Arghya Paul,“Injectable Hydrogels for Cardiac Tissue Repair after Myocardial Infarction”Adv. Sci. 2015, 2, 1500122.

Crystallization of polymers from solution occurs usually during electrospinning process. At the moment we will discuss some very general aspects of solution crystallization. Crystallization is in general an example of a separation process in which mass is transferred from a liquid phase (solution or melt) to a solid crystal. The thermodynamic driving force for crystal nucleation and growth is the concentration excess (supersaturation) of the solution above the equilibrium (saturation). Once crystallization is occurred, equilibrium is set between the crystals and the residual mother liquid, the balance being determined by the solubility (concentration) and the temperature. The resistances to growth are related to mass transfer within the solution and the energy needed at the crystal surface for incoming molecules to orient themselves to the crystal lattice.

Solution crystallization is a complex process being governed by various parameters. The first one is a solubility which is a function of temperature and related saturation. For most materials solubility increases with temperature (Fig. 1).

solubility abd saturation curvesFigure 1. Solubility and saturation curves

 

Considering solution crystallization at constant temperature, it starts after solvent evaporation being sufficient to cross the saturation curve. Immediately after crossing saturation curve, there is metastable region in which spontaneous nucleation is immposible because of the role of interfacial energy being in opposition to the driving volume free energy. In the metastable region, there is an equilibrium between the processes of crystal formation and dispersion. Above the next saturation line, called supersaturation, crystals can form spontaneously (homogeneously. During crystallization, the crystals are grown from solutions with concentrations higher than the saturation level in the solubility curves. Once nuclei are formed the crystals will continue to grow so long as supersaturation exists. There are several factors controlling the rates of crystal nucleation (primary nucleation) and growth (secondary nucleation). The most important is the degree of supersaturation, viscosity, interfacial tension between the solute and the solvent, boiling temperature of a solvent as well as strength of a solvent which affects position of the saturation line.

Supersaturation is critical because it is the driving force for crystal nucleation – either spontaneously (primary nucleation) or in the presence of existing crystals (secondary nucleation - growth). Crystal growth is the increase in size of crystals as solute is deposited from solution. These often competing mechanisms ultimately determine the final crystal size distribution. The relationship between supersaturation, ΔC, and rate of nucleation, N, and growth, G, can be defined by simplified equations:

wzr 1 supersaturation vs nucleation rate

where kg is growth constant, g - growth order, kn - nucleation constant, n - nucleation order. For organic crystallization systems, the value of the growth order is typically between 1 and 2 and the value of the nucleation order is typically between 5 and 10, leading to the scheme drawn in Fig. 2.

 

scheme of nucleation growth rate and crystal size vs supersaturation

Fig. 2. Scheme of nucleation, growth rate, and crystal size vs. supersaturation.

 

At low supersaturation, crystals can grow faster than they nucleate resulting in a larger crystal size distribution. However, at higher supersaturation, crystal nucleation dominates crystal growth, ultimately resulting in smaller crystals. This is a problem of relatively low total thermodynamic driving force (high positive input of surface free energy being in opposition to the negative volume free energy) at low supersaturation which can be enhanced by nucleation on earlier formed crystals (growth). If supersaturation is high, there may be further effective nucleation and the growth will not be so great. In practice, slow evaporation rate maintaining a low level of supersaturation produces large crystals and fast evaporation results in formation rather small/defective crystals.

          Additional parameter involved in thermodynamics of solvent crystallization is interfacial tension between the solute and the solvent. In the case of solvents providing high interfacial tension between the solute and the solvent, there will be higher surface barrier against crystallization. It will lead to retardation of crystallization, particularly evident for the stage of primary nucleation.

          Viscosity of solution is crucial for molecular transport which is needed for crystal growth. The rate of crystal growth is controlled by the diffusion of the solute through the solvent to the surface of the crystal and by the rate of the reaction at the crystal face when the solute molecules rearrange themselves into the crystal lattice. These rates of crystal growth can be represented by the equations

                   wzr 2 crystal growth

where dw is the increase in weight of crystals in time dt, A is the surface area of the crystals, c is the solute concentration of the bulk solution, ci is the solute local concentration at the crystal/solution interface, cs is the concentration of the saturated solution, Kd is the mass transfer coefficient to the interface and Ks is the rate constant for the surface reaction. The equations are usually combined into more simple final form:

wzr 3a

   where

wrr 3b

          It is evident that the growth is faster for higher values of K, which is inversely proportional to the viscosity.

          Additionally discussing solution viscosity, one should be aware that it reflects the strength of solvent for particular polymer. The higher is the viscosity for given polymer the better is the solvent. This fact can be seen from Mark-Houwink equation, providing relation between solution intrinsic visocity [h] and polymer molecular weight, M:

wzr 4 mark houwink

in which the exponent “a” depends strongly on a polymer configuration. In a “poor” solvent, the segments of a polymer molecule attract each other in solution more strongly than they attract the surrounding solvent molecules. The polymer molecule assumes then a tighter configuration, and the solution has a lower intrinsic viscosity resulting in Mark-Houwink “a” constant close to 0.5. For better solvents, the molecules tend to adopt more extended configuration, resulting in higher “a” constant values (higher viscosity).

Furthermore, the better is solvent the higher is saturation concentration (higher positions of saturation lines on Fig. 1). This fact can be important for the final crystal structure because of shorter time accessible for crystallization in the case of better solvents. Similar effects can be expected for solvents with low boiling points.

Methods  for nanofibers producing are known from about century. However, only about 20 years ago appeared appropriate analytical methods, allowing for the precise study of theis structure and morphology. Materials with nanometer scale offer many new opportunities to support the tissues and organs regeneration. The main advantage of nanofibers is their similarity to the extracellular matrix of collagen, whereby the cells treat them as their native environment. 


 

                     publications about nanofibres

               Increasing number of publications about nanofibers in the years 1999-2012

Source: www.nafigate.com/en/section/portal/app/theme/detail/30-a-dramatic-rise-of-interest-in-nanofibers-has-been-confirmed

 

Methods 

Methods of producing nanofibers include: 

- drawing, 

- template synthesis, 

- phase separation,

- molecular self-assembly, 

- electrospininng. 

 

In drawing sodium citrate is used. Pipette with a diameter of several micrometers is immersed in a drop of a solution of sodium citrate in chlorine- gold acetic close contact surface. Drawing the fiber occurs when removing the pipette from the solution at a suitable speed (about 10-4 m / s). Then the nanofibers is deposited onto a suitable surface by contacting it with the end of the pipette. For one drop of these steps are repeated several times, because the nanofibers have a small volume. The drawing is required nanofibers of high viscosity material, since it can be subjected to large deformations without losing consistency.

 

 
Template synthesis is characterized by using a specific template in order to obtain a nanofiber with a desired shape and size . Nanoporous membrane template is made ​​of alumina . The polymer is the pressure exerted pressures to which the material is squeezed through the pores of the template into a chamber containing a solution solidifying in contact with the nanofibers solidify. The resulting nanofibers are dimensioned by the membrane pore size.
 
The phase separation method is based on the solution of the polymer and the solvent is subjected to gelation . The solvent is then removed . The remaining phase is a polymer fiber. Fibrous structure is left at a relatively low temperature ( freezing of the structure). The frozen gel is placed in a freeze dryer and lyophilized.
 
Molecular self-assembly  uses the phenomenon of order, that is, the formation of organized spatial structures in the form of nanofibers. The mechanism of self-assembly based on intermolecular forces leading to connect individual particles in chains. The shape of the resulting nanofibres depends on the shape of the constituent particles.
 
Electrospinning nanofibers  involves preparation of nanofibres from polymer melts ( electro -alloy ) or a solution thereof ( from the electrospinning solution) by the electric field .

 

Types of nanofibres 

Changing different parameters of electrospinning process like polymer concentration, humidity, evaporation rate or by selecting polymer with different molecular weight we can create fibres with different morphology. We can distinguish many types of nanofibres:  porous, flattened ,  ribbon like, branched, helical or hollow.  

 

                         porous nanofibres

                                                                     Porous nanofibres

Source: Nishath Khan,  ,,Applications of Electrospun Nanofibres in the Biomedical Field. A Review'', Surg Vol 5, No 2 (2012) 

 

Porous nanofibres can be produced in high humidity conditions during the electrospinning nanofibers. The pore size ranges from several tens nm to 1 micron, and depends on the type of polymer and solvent, as well as process conditions

   

                                 Plat-nano-ribbons

                                                                    Ribbon like nanofibres

Source: Aravind Dasari, Berta Herrero, ,,Nano-manufacturing multifunctional ultrathin fibres'', Multifunctional Nanocomposites Group of IMDEA-Materials

 

The way how morphology of the flatted or ribbon like nanofibers appear can be explained by evaporating the solvent during the electrospinning process. Flat shaped fibers can be achieved using a solution of polyvinyl alcohol (PVA) with high molecular weight and increased concentration of polymer in solution. Evaporation of water solvent decreases with increasing viscosity of the solution. Wet fibers become flatten during impact with the collector.

  

                                 branched nanofibres 

                                                                        Branched nanofibres

Source: http://cheed.nus.edu.sg/stf/cheleejy/Gallery.html

 

Branched nanofibres can be obtained by the detachment of the small stream from the surface of the main stream. This happens when there is an imbalance between the forces of surface tension, electrical and which leads to instability of the shape of the stream. This instability can be reduced as a result of tearing or rupture of the original jet stream into two smaller ones.

 

Hollow fibres can be obtained using a sequence of processes of chemical and electro deposition from vapor deposition (CVD) and oriented coaxial spinning. When fibers are produced by electrospinning constituting the core, and then when CVD is applied to the core layer. The inner core is removed by annealing. Hollow nanofibers can be obtained in a single stage process using a coaxial spinning direction.

 

Characteristic 

The basic characteristics of the nanofibers is their morphology (fiber diameter) and mechanical properties. The nanofibers may be characterized using SEM (morphology, fiber diameter, chemical composition - EDS detector), AFM, TEM or nanoCT. In order to investigate the mechanical properties by standard methods we can use static tensile test. The deformation of nonwoven in the direction transverse to the rotation and the collector is investigated by this method.  One of the most important properties for medical application is surface contact angle which also usually is characterised.  Hydrophilicity of the material  influence on the cell repopulation of its surface. Of course also materials structure should be studied. For this reasone DSC, XRD or spectroscopy like FT-IR can be used. 

 

Materials 

For medical application mainly polymer fibres are used. From this group of materials, both, synthetic and natural polymers are used. Natural polymers used for this applications are hyauronic acid, collagen, gelatin, chitosan, elastin, wheat protein or silk. From this polymers one of the best  studied is collagen and gelatin. Collagen is compatible with number of cell types and create a suitable enviroment for cell growth.  The same like collagen, hyauronic acid,, is natural component of ECM (Extracellular Matrix). To synthetic polymer group used for nanofibres includes PLA, PET, PCL or PLGA.  

 

Funcionalisation 

In general, for medical applications special surface properties with regard to chemical composition, hydrophilicity, roughness, crystallinity, conductivity, lubricity or density are required for the success of these applications. To increase the range of applications nanofibers surface functionalization is needed. For this purpose, the surface of nanofibers is applied to the respective proteins. 

 

Applications in Tissue Engineering 

Nanofibres can be succesfuly use in muscoleskeletal tissue engineering. Attempts are made to regenerate bone tissue, cartilage, ligament or skeletal muscle. In case of bone tissue it is the most important to recreate 3D structure and approperiate physical and mechanical properties like mechanical strenght, pore size, porosity and hardness.  Cartilage is more problematic then bone tissue because of its specific contruction. Morover nanofibres can be used to build structures for skin or blood vessels regeneration. nanofibres also can be used as a drug delivery system to improve the terapeutic efficiency and safty of drugs. 

 

Literature: 

1. Ramakrishna S., Fujihara K., Teo W. E., Lim T.C., Ma Z.,  ,,An Introduction to Electrospinning and Nanofibres'', World Scientific, 

2. www.zasoby.open.agh.edu.pl/~11sashot/strona.php?t=pm&h=nw&v=

3. www.aksolotl.org/index.php/prezentacje/98-nanowlokna-nowa-nadzieja-medycyny-regeneracyjnej

Synthetic and natural polymers (biopolymers) are commonly used in tissue engineering because of valuable properties eg. biocompatibility, biodegradability, good mechanical properties etc. For this reasons, a lot of current research studies for medicine is focused on this group of materials. Polymers, give the great opportunity to fulfill one of the main assumption of regenerative medicine and tissue engineering, which is formation of wholsome tissue in in vivo conditions.  

 


 

       Recent years polymers have become the most used and investigated materials for applications in medicine. In the case of tissue engineering the most important features of polymers are ability to be easily reproduced, their versatility, tuneable properties and biodegradability. Biomedical polymers can be divided into two main groups: naturally-occuring polymers and synthetic polymers.

 

Natural polymers

 Among natural polymers we can distinguish: proteins (e.g. silk, collagen, fibrin), polysaccharides (e.g. alginate, hyaluronic acid, chitosan) and polynucleotides (e.g. DNA, RNA). Proteins and polysaccharides are very often used in tissue engineering. The most common natural polymers are presented below.

 Collagen is ECM protein. We know 27 types of collagen, wherein type I collagen is the most abundant. It is fibrillar, rod-shaped molecule, which can be found for example in tendon, ligament, bone, skin and cornea. Collagen fibers are the tissue structural framework and are responsible for suitable tensile strength. Great importance of collagen in tissue structure causes that this polymer is very often used as material for tissue engineering scaffolds.

 

aligned fibresrandom

 Figure 1. Aligned (A) and randomly oriented (B) nanometer scaffolds.

 

Silks are proteins produced in fiber form by silkworms and spiders. They are generally composed of β-sheet structures. These structures allow to tight packing of stacked sheets of hydrogen bonded chains. The assembly of silk and the strength and resiliency of silk fibers are the result of large hydrophobic domains interspaced with smaller hydrophilic domains. Silks are used as biomaterials because of their biocompatibility, controlled proteolytic biodegradability, impressive mechanical properties, morphologic flexibility and the ability to immobilize growth factor.

 Fibrin is a fibrous protein involved in the clotting of blood. It is formed by polymerization process with the action of thrombin and activated factor XIII, which convert fibrinogen into fibrin. Cross-linking is one method in which diverse microstructural and mechanical properties of fibrin networks can be achieved. What is interesting, by using fibrin made from a patient’s own blood, autologous scaffolds can be manufactured. The fibrin disadvantages are morphological deconfiguration and rapid degradation in physiological conditions, and thus combinations of fibrin with strength enhancers such as PCL/polyurethane, hyaluronic acid and PLGA are used.

 Hyaluronic acid (HA) is a natural linear glycosaminoglycan, co-polymer of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is present in connective, epithelial and neural tissue and synovial fluid. What important, HA is biocompatible, biodegradable and has important tissue healing properties, such as induction of angiogenesis, and the promotion of cell migration, adhesion, and proliferation. It has been suggested that this polymer display also anti-inflammatory and bacteriostatic action.

 Alginates are a low-cost marine materials extracted from the cell walls of brown seaweed. Alginates are salts of alginic acid. Naturally, alginates occur as calcium, magnesium and sodium salts. The very important feature of alginates is that they can form hydrogel thanks to ion-crosslinking, for example Ca2+-crosslinking. Chemically, alginates are linear copolymers containing blocks of (1,4)-linked β-D-mannuronate (M) and α-L-guluronate (G) residues. The M/G ratio, G-block length and molecular weight affect the physical and mechanical properties of alginate and resultant hydrogel.

Chitosan is cationic polysaccharide obtained from deacetylation of chitin built from (beta-1,4-linked N-acetylglucosamine units. Deacetylation is used due to  insolubility of chitin in common solvents and difficult processing. Degree of the deacetylation has impact on crystallinity and molecular weight of the chitosan. This cationic polymer can form electrostatic interactions with negatively charged cell surfaces and also display antimicrobial activity. Thanks to ability of chitosan to interact with glycosaminoglycans (GAGs) scaffolds based on chitosan can have a direct impact on the modulation of cytokines and growth factors and thus local tissue regeneration.

 

Synthetic polymers

 Synthetic polymers are also common used in tissue engineering. They are  much more reproducible and have better mechanical properties in comparison to natural polymers. Examples of the most frequently studied synthetic biomedical polymers are presented below.

 Poly(caprolactone) (PCL) is thesemi-crystalline aliphatic polyester. Properties such as great organic solvent solubility, a melting temperature of about 55–60C and glass transition temperature of  −54C causes that this polymer is common used as tissue regeneration support structures. Due to relatively long degradation profile PCL is suitable for use in tissues with longer regeneration process.

 

Figure 2.Electrospun nanofibrous membrane made from PCL and collagen.

 

Poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their copolymer poly(lactic-co-glycolic acid) (PLGA) are also polyesters. Furthermore, these polymers are poly(caprolactone) (PCL) is thesemi-crystalline aliphatic polyester. Properties such as great organic solvent solubility, a melting temperature of about 55–60C and glass transition temperature of  −54C causes that this polymer is common used as tissue regeneration support structures. Due to relatively long degradation profile PCL is suitable for use in tissues with longer regeneration process.

Poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their copolymer poly(lactic-co-glycolic acid) (PLGA) are also polyesters. Furthermore, these polymers are poly(hydroxy acids). Hydrolysis of the ester bonds in the backbone of their chains causes breaking down of PLA, PGA and PLGA to their monomeric units - lactic acid and glycolic acid respectively. These breakdown products can be then simply cleared by natural metabolic pathways.

Poly(ethylene glycol) (PEG) is polyether. Its polymerization involves ethylene oxide condensation. Chains of PEG which are greater than 10 kDa are defined as poly(ethylene oxide) (PEO). Molecular weight, cross-linking and polymer concentration affect mechanical properties of PEG scaffolds in tissue engineering. These polymers are biocompatible, biodegradable, non-toxic, low-immunogenic. Despite the lack of natural ability of PEG
to binding proteins or cells, cell adhesion is possible thanks to incorporation of RGD peptides.

Poly(urethanes) are traditionally and most commonly formed by reacting a di- or polyisocyanate with a polyol. Therefore  fundamental constituents of polyurethanes are:  hard segment - diisocyanate, the soft segment - polyethers or polyesters and chain extenders. Properties of resultant polyurethane depend on the ratios of these components. Due to unfavorable degradation profiles combinations of polyurethanes with other biomaterials are made to improve degradation rate of polyurethane-based scaffolds.

 

 

Literature:

 

  1. Ozdil, D., & Aydin, H. M. (2014). Polymers for medical and tissue engineering applications.Journal of Chemical Technology and Biotechnology,89(12), 1793-1810.
  2. Silva, E. C., Omonte, S. V., Martins, A. G. V., de Castro, H. H. O., Gomes, H. E., Zenóbio, É. G., ... & Souza, P. E. A. (2017). Hyaluronic acid on collagen membranes: An experimental study in rats.Archives of Oral Biology,73, 214-222.
  3. Lee, K. Y., & Mooney, D. J. (2012). Alginate: properties and biomedical applications.Progress in polymer science,37(1), 106-126.
  4. Axpe, E., & Oyen, M. L. (2016). Applications of alginate-based bioinks in 3D bioprinting.International Journal of Molecular Sciences,17(12), 1976.
  5. Vepari, C., & Kaplan, D. L. (2007). Silk as a biomaterial.Progress in polymer science,32(8), 991-1007.

 

There are various methods of nanofibers production. One of the most flexible and cost-effective is electrospinning, allowing formation under the influence of high electrical voltage of long, continuous fibres with diameter ranging from  few nanometers to several micrometers. Electrospun nanofibers are created from electrically charged jets of polymer solution or polymer melt. Unusual properties of resulting fibers predispose them for plenty of applications with the most promising being nowadays tissue engineering.

 


 

 

History of Electrospinning

 

Observation of electrospinning process started at the end of XIX century and continues during XX century (Reyleigh, Zeleny, Formhals,  Baumgarten). Since the 1980s and especially in recent years, electrospinning process has regained more attention probably due to a surging interest in nanotechnology. In fact, name for this process ,,electrospinning'' derived from ,,electrostatic spinning'', is relatively new term, which appeared around 1994.

 

 

How does it work?

 

                                   electrospinning

                                                            Electrospinning Process

Source: Ning Zhu, Xiongbiao Chen ,,Biofabrication of Tissue Scaffolds'', in book entitled ,,Advances in Biomaterials Science and Biomadical Applications''

 

Electrospinning involves the use of a high voltage (commonly from 5 to 30 kV) applied classicaly between a needle of a syringe, filled with moleculary entangled polymer solution. The solution is pumped through a syringe at a constant rate, forming a droplet at the end of the needle. When the surface tension is overcome by electrostatic repulsion of charges within solution, jet is ejected, moving then toward metallic collector which is usually grounded. The solvent evaporates during jet travelling, while the jet is elongated at very high strain rates, ranging from 100 to 1000 s-1. The structure of nanofibers collected finally is far from thermodynamic equilibrium (metastable structures).

The most important forces acting first on a drop and then on a travelling jet are:

- external electrostatic field which acts directly on charges within polymer jet but also results in additional electric polarization of flowing material

- electrostatic repulsion among the charges 

- surface tension, trying to reduce repulsion between charges 
- the forces related to  viscoelasticity of polymer.

Formation of nanofibres is mainly caused by repulsion occuring among charges resulting in high  stretching of the polymer streamStream initially flows in straight. On the way to the collector, the various instabilities occur. The jet is seriously elongated by a bending and whipping processes caused by electrostatic repulsion initiated at small bends in the fiber, until it is finally deposited on the collector. The elongation and thinning of the fiber resulting from this bending/whipping instabilities leads to the formation of uniform fibers. The spiral movement of the stream substantially increases the path between the needle and the collector, resulting in a significant stretching (nanometer diameter). 

Currently, there are two standard electrospinning setups, vertical and horizontal. The process can also be carried out using two several independent syringes/needles with  different solutions. 

                        elektro 99

                   Using a rotating collector (drum) we can affect  the arrangement of fibers

Source: www.cosmobio.co.jp/export_e/products/detail/products_hks_20090105.asp?entry_id=1643

 

Parameters affecting the process of electrospinning

 

The most significant effect for the process of electrospinning have the properties of polymer solution, i.e: 

- solution viscosity depending mostly on  polymer molecular weight as well as polymer concentration,

- solution surface tension, 

- solution electric conductivity,

- dielectric constant of solvent.

Initialization of electrospinning process needs overcoming of surface tension of solution by electric interactions between charges. The surface tension of commonly used solvents does not vary seriously, being for most solvents between 20 and 40 mN/m2, with exception for very high surface tension of water. One of the conditions for electrospinning is that the viscosity should be above some critical value to prevent the breakage of the jet. The higher molecular weight of polymer (viscosity of solution) results in more entanglements, preventing the breakage of the fibers and formation of beads.

The second group of parameters which affect  electrospinning process are processing conditions:

- applied voltage, 

- flow rate of solution, 

- type of a collector, 

- diameter of a needle,

- distance between a needle and a collector. 

 

The third group are environmental parameters: 

- temperature

- humidity, 

- pressure. 

 

 

Materials

 

Among nanofibers from polymers, being very important class of materials for medical applications (scaffolds), both natural like collagen, gelatin, chitosan, fibrine, etc.,   and synthetic like PCL, PLLA, PLA, PGA, PLGA, PU etc. should be mentioned. 

 

 Advantages

 

Main advantages of electrospinning method are:

- simple and low cost equipment,

- possibility of scalling the process,

- possibility to  control fiber morphology,

- practically all kind of polymers with high enough molecular weight can be processed by electrospinning.

 

 

Disadvantages

 

Main disadvantages of electrospinning method are:

- used solvents can be toxic,

- problematic to obtain 3D structures as well as sufficeint size of pores needed for biomedical applications,

- process depends on many variables.

 

 

 Applications  

 

Nanofibres produced by electrospinning can be used for analytical chemistry, tissue enginnering, filtration techniques, electronic or enviromental engineering. In tissue enginnering,  electrospinning of nanofibers  is used for fabrication of scaffolds for tissue regeneration, mimicking natural ECM. Thanks to this material and method, we can create for instance artificial skin, bone and cartilage implants. Nanofibres also can be used for drug delivery system or vascular surgery.   

 

                                                   nanofibres applications-001

                                                 Nanofiber mats used for medical dressings

Source: M.  Mahfuzur Rahman Chowdhury,  ,,Electrospinning Process. Nanofiber and their application'',  http://www.cottonbangladesh.com/January2009/ElectroSpinning.htm

 

 

                                                   nanoelectronic

                                                      Fiber nanogenerator

Source: Sarah Young, ,, New fiber nanogenerators could lead to electric clothing'', Media Relations, February 12, 2010

 

Literature:

1. Bhardwaj N., Kundu S.C., ,, Electrospinning: A Fascinating Fiber Fabrication Technique'', Biotechnology Advances 28 (2010), 325-347

2. Baranowska- Korczyc A., ,,Półprzewodnikowe sensory oparte na nanowłóknach otrzymanych metodą elektroprzędzenia'', Rozprawa doktorska, Instytut Fizyki PAN,  2012 

3. Ramakrishna S., Fujihara K., Teo W.E., Lim T.C., Ma Z., ,, An Introduction to Electrospinning and Nanofibres'', World Scientific 

4. Vasita R., Kattai S.D., ,,Nanofibres and their applications in tissue engineering'', International Journal of Nanomedicine 2006: I (I) 15-30

 

 

 

Tissue engineering is the new, very fast growing interdisciplinary branch of science which combines biology, biotechnology, chemistry, and materials science. The practical aim of this discipline is to create scaffolds for new tissue growth, meaning nothing other than regeneration of damaged tissue. Nowadays, using tissue engineering methods we can replace for instance damaged bone or skin tissue. But still, plenty of questions remain unanswered. Cartillage tissue or nerve tissue are still important challenges for research groups. 


 

                                   Calciumposphate scaffold 

                                                                    Porous cell scaffold

                                                                                    Source: http://www.cellsupports.com/

 

Transplantaton is still one of the most commonly used method to replace damaged tissue. However, this method entails a lot of serious problems. The most important is the need for a greate number of procedures and possibility of graft rejection. All problems connected with this way of treatment forced to search for new solutions.

The idea of replacing tissue by materials like polymers appeared many centuries ago, but even 50 years ago biomaterials as we think of them did not exist.  However, in recent years, together with the development of new technologies and improvement of biomaterials, new idea of tissue regeneration had appear. Nowadays, in tissue engineering plenty of various materials and technologies are used. Reffering to current trends in tissue engineering, the most promising materials are polymers, both synthetic and biopolymers like PLA, PLGA, PGA, PCL, PU, chitosan, collagen, or gelatin. Thanks to their wide spectrum of properties (chemical, physical and mechanical), biocompability and biodegradability, they found use in such applications like scaffolds for bone and cartilage, skin tissue, heart tissue etc. The use of degradable polymers to induce tissue regeneration is particularly promising in case of s.c. critical size, i.e. non-healing defects. Once implanted, polymer should act as a temporary substitute of the native extracellular matrix, enabling the ingrowth of a new tissue by attracting cells from surrounding and providing suitable conditions for proliferation. The polymer scaffold is expected to degradate gradually as the formation of neotissue with native, self-produced ECM occurs. Sometimes polymer scaffolds are combined with  ceramics (hydroxyapatite, calcium triphoshate) for special applications.

 

The main techniques for scaffold fabication are as follows:

- solvent casting,

- particulate leaching techniqes,

- gas foaming,

- phase separation,

- electrospinnig,

- porogen leaching,

- fiber mesh,

- fiber bonding,  

- self assembly,

- rapid prototyping,

- melt moding,

- membrane lamination,

- freeze drying.

 

Not every method of scaffolds production can be used in a given case. The method of scaffold formation leading to a particular morphology, internal structure and hence final properties of a product, should be correlated with specific application. For instance, the most important methods of scaffold preparation used in the regeneration of nerve tissue include electrospinning and phase separation technique. Common methods for preparing scaffolds for new blood vessel formation is the filament winding method and electrospinning. 

                                             hodowla   

                                                                        Cell culture           

                                         Source: http://animal.sggw.pl/2013/04/19/letni-kurs-hodowli-komorek/         

                                             scaffold bone

                                               Bone-forming osteoblasts on the scaffold

                                                                    Source: http://www.ammrf.org.au/innerspace/img17.html

 

Finally, scaffold has to be populated by cell which will create new tissue. Initially cells have to be isolated, then amplified at in vitro conditions and introduced into scaffold structure. In the next stage scaffold with cells has to be placed in a bioreactor. There in suitable enviroment of growth and nutrient factors cells start grow up and proliferate. Finally all new tissue can be placed into place of tissue defect.    

 

Although, we are potentially able to regenerate many different tissues, some applications are still extremely difficult. Currently, the most problematic is the reconstruction of complex organs such as the heart or kidneys. The main problem is to reconstruct a complex network of blood vessels and innervation of these tissues. Which are essential for keeping her alive. Nevertheless, numerous studies are conducted which show promise. Looking for new solutions for organs regeneration such as the liver, researchers use a so-called ,,natural skaffold''. Using dead tissue  or tissue containing dead cells. Using a special procedure, all cells and cell debris are removed from the liver. Then, healthy stem cells are seeded to the empty tissue and vasculature is restored to promote growth. Lungs have also been successfully replaced using this technique (animal trials). 

 

Restoration the specific conditions in which cells build the particular tissue is essential for the success of therapy. In addition to an appropriate chemical environment of living (nutrient, enzymes, hormones)  also physical factors (loads, pressure, temperature) are very important for cells differentation. For example, articular cartilage cells differentiate into the correct way in the presence of loads which are relevant in the joints.

 

There is still a lot of fundamental research in the scaffolds design. Also plenty of clinical trials.that must be followed by extensive. This branch of science is changing rapidly, so the opportunities for the use of tissue engineering will surely change year-by-year.

 

  Literature:

1.  Buddy D. Ratner, Allan S. Hoffman, Frederick J. Schoen, Jack E. Lemons, ,,Biomaterials Science: An Introduction to Materials in Medicine '', Academic Press

2. B. Subia, J. Kundu and S. C. Kundu, ,,Biomaterial scaffold fabrication techniques for potential tissue engineering applications'', Interchopen,  

3. http://www.bioscaffold.com/Natural-Bioscaffold.html 

4. http://www.regenerativemedicine.net/Tissue.html

  

 

 

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