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Photopolymers for Tissue Engineering Purposes

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    Tissue Engineering offers the potential to grow the cartilage in a precise shape and requires minimal operative time. In most of the preliminary studies, a prefabricated mold is used to grow the chondrocytes and obtain a tissue-engineered cartilage. However using the mold techniques is time consuming, does not provide an aesthetic framework for growing the cartilage and there is an uneven growth of cartilage tissue over the framework. 3D CAD manufacturing provides an alternative technique whereby one can accurately fabricate an ear shaped scaffold similar to the normal ear. Approach in scaffold design must be able to create porous structures to attain desired mechanical properties and to produce these structures within arbitrary and complex three-dimensional (3D) anatomical shapes. Material chemistry along with fabrication technique determines the properties that a scaffold can achieve and how cells interact with the scaffold .There are many techniques which are used in additive manufacturing like Stereolithography, Fused deposit modeling, selective laser sintering. Stereolithography exhibits the capability to control the spatial organization of multicellular material compositions with precise porous structures and defined shape according to patient obtained from any medical imaging modality data. In this study, we accomplished stereolithographic fabrication of hybrid scaffolds using visible light excitation by using a commercially available low cost 3D printer. The scaffolds fabricated as such will be suitable as a photo curable material that could offer an ideal environment suitable for cell growth and provide the mechanical support for the regenerative process. The table shows current studies that have made use of photo curable biomaterial that can be used for tissue engineering process.

  Figure 6.1 Current studies involving photopolymers for tissue engineering purposes

      As explained in the above table there are many studies, which use biodegradable polymers that can be fabricated using the stereolithography technique. However, in most of these studies there are no such combinations of natural and synthetic polymers. Also in many studies, the material has been cross-linked in the lab by using a light source or by a modified/custom made 3D printer. For this study, we decided to use a low cost and a commercially available 3D printer (Formlabs Form 1+) and natural and synthetic available polymers without making any modifications.

       PEG is one of the most commonly used synthetic photo polymers for tissue engineering applications. For photo polymerization process the end group of PEG are modified into methacrylates, di acrylates, fumarates,vinyl esters etc  and used for the polymerization. The reactivity of vinyl monomers towards free-radical chain polymerization follows this sequence: acrylate  >  vinyl ester ∼ vinyl carbonate  >  methacrylate  >  fumarate. Due to the high reactivity rate we have decided to make use of acrylated PEG. Acrylated PEG enables photo polymerization with variable mechanical properties, but by itself, PEG cannot provide an ideal environment for cell growth  despite having possessing properties like nontoxicity, low protein adhesion, and nonimmunogenicity. Also PEG does not possess the ability to degrade by itself.

      When it comes to biocompatibility issues, natural polymers are generally thought to be advantageous over synthetic hydrogels since natural gels may offer biological property to surrounding cells. Most naturally-derived polymers are either components of natural ECM or provide similar properties that can mimic the ECM properties. One such natural, biocompatible,and biodegradable polymer used to generate hybrid hydrogels is chitosan, an N-deacetylated derivative of the polysaccharide chitin. Although there is a  study that

shows the photopolymerization of oligomeric chitosan with PEGDA polymeric chitosan has not been successfully polymerized with PEGDA. Chitosan is structurally similar to glycosaminoglycans (GAGs) found in cartilage and is degradable by enzymes in humans. The objective of the study was to get a hybrid copolymer of Chitosan and PEGD which can be 3D printed by stereolithography.

        To create the resin we dissolved the chitosan in acetic acid.  The acetate anions

deprotonate the primary amino groups of chitosan. So it became necessary to dialyze the chitosan solution in a strong basic group solution like a sodium acetate. Dialysis of chitosan solutions in sodium acetate partially neutralizes the protonated primary amino groups. Such partial de-protonation of chitosan enabled mixing of photo initiators for polymerization of PEGDA without quenching the radicals formed by protonated amino groups. Because of the high degree of crosslinking of short chain PEGDA, caused by a higher concentration diacrylate groups compared to long chain PEGDA a commercially available PEGDA 575 was used. In absence of  Chitosan the minimum concentration required to create the  printable resin was 30% (w/v) . However as shown in table the amount of  PEGDA using Chitosan was reduced from 30%  to  6-9 % . Once the printable formulation was obtained it was necessary to test the mechanical and cellular properties of these scaffolds.

Schematic of cross linked hydrogel with mesh size and crosslinking distance

When a hydrogel is kept in the solvent the solvent molecules try to enter inside by the capillary action. As more molecules enter the hydrogel the mesh size  increases and more of the solvent is absorbed. However, the swelling is not a continuous process and when the capillary forces balances the elastic forces of the network the equilibrium is reached.

                                              Q1/3 * (γ2)1/2

where Q =swelling ratio and    γ = distance between two crosslinking points.

As evident from the figure and the equation there is a direct relation between the swelling ratio and mesh size. As the amount of the PEGDA concentration increases, the degree of the crosslinking has increased. Highly cross-linked hydrogels will have a tighter structure, and will swell less compared to the same hydrogels with lower crosslinking ratios. Crosslinking hinders the mobility of the polymer chain and hence lowers the swelling ratio.

   As evident from Fig the mechanical modulus of the hydrogel was inversely related to the swelling ratio. As the ratio of PEGDA increased from 5 to 15, the elastic modulus increased by approximately seven times in both LMWC and HMWC Chitosan. As the swelling ratio decreases the increased resistance of the hydrogel contributes to the increase in Youngs modulus. Diluted PEGDA, without chitosan, at 30% (w/v) had the highest stiffness with a compression modulus of 1125 ± 68.05 kPa (Mean ± SD). It was observed that the gel was capable of recovering to its original length following even with a 50% strain deformation.It is evident that increasing the ratio of the initiator will increase the crosslinking density which will reduce the mesh size and in turn increase the modulus of the hydrogel.As evident from the swelling ratio the hybrid hydrogel had a higher swelling ratio than pure PEGDA which led to higher pore size which was proved with the SEM Imaging side

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