Studies on the gelation of gelatin solutions and on the use of resulting gels for medical scaffolds

This study focuses on the early stages of the gelation of an aqueous type A (Pig skin) gelatin solution. The thermo-reversible mono and triple helix formation was observed by rheology and proton NMR relaxation measurements. At high temperatures (T > 330 K), gelatin molecules form flexible random coils of small hydrodynamic radius, the elastic modulus of the solution is relatively low. On decreasing the temperature, mono helix formation begins, connected with an increase of the storage modulus and the hydrodynamic radius. The absence of a significant concentration dependence of this early variation of the modulus indicates the intramolecular nature of this structural change. The simultaneous decrease of the spin-spin relaxation times of the 1H signals of certain amino acids confirms its effect on the molecular mobility. As this affects especially the signals of arginine and lysine, we conclude that these basic amino acids play a significant role in forming the intramolecular interactions. Triple helix formation occurs at a point at which the viscosity begins to increase rapidly near the gel point (T < 320 K). This process is clearly dominated by intermolecular interactions, as the slope as well as the starting point of the rapid increase significantly depends on the concentration. In this work the coil structure of gelatin in the early stage of gelatin gelation under variation of temperature (283-330 K), pH (3, 6.5, 11) and concentration (1, 3, 5 % w/w) were also evaluated by NMR spectroscopy. In addition, changes in the rheological characteristics such as storage modulus and complex viscosity of the gelatin solutions were measured and the effect of acidity, temperature and concentration are evaluated. The experimental results are analyzed to obtain the polar contribution to the relaxation behavior and the mobility of aminoacids in different temperature and different acidity. It was indicated by spin-spin relaxation time that mobility of amino acids in acidic and basic solution are much more than in neutral solution and some amino acids have different behavior in acidic and basic media. Besides, gelation temperature shifted to lower temperature in both acidic and basic solutions. Rheological behavior also confirms that inter-molecular and intra-molecular interaction will be decreased in acidic and basic media and the gelation time decreased. Therefore, by controlling the temperature, concentration and also pH, folding of the amino acids could be controlled. Gelation process of gelatin after mineralization with nano-particulate hydroxyapatite was monitored and investigation of relaxation behavior of aminoacids in gelatin chains and changes in the coil structure of 3 and 5 % gelatin solution under variation of temperature (298-330 K). The NMR state diagrams, i.e. plots of ln (T2) vs. the reciprocal temperature (1/T), were produced for 3 % and 5 % gelatin solutions mineralized with hydroxyapatite. We found that the different concentrations (3 and 5 %) have no drastic effect on the T2 relaxation time but different amino acids have different patterns, so a different mobility during heat scan can be postulated. It may relate to this fact that they are well designed to have an interaction with phosphate and calcium anion. In our study, we introduced new n-HAp (spherical, mixed shape and rod-like) /gelatin scaffolds coated with n-HAp using Chemical Bath Deposition technique. In this research n-HAp was prepared by wet chemical process and according to the present state of knowledge; the samples prepared at 25˚C show spherical (via continuous method) and mixed shape (via non-continuous method) particles and aggregate readily. The n-HAp particles fabricated at 40, 70˚C and 24 h aging time are needle and rod-like with widths ranging 30-60 nm and lengths from 100-300 nm, respectively. They distribute in gelatin much better than spherical and mixed shape particles, due to their higher surface area and higher reactivity. Therefore, the good mechanical properties of the nano-rod HAp/gelatin scaffold may result from their uniform distribution in the gelatin matrix, from their surface activity and their interface chemical bonding which makes it possible for n-HAp to link with gelatin. Spin-spin relaxation time measurements confirm that n-HAp may link with gelatin by interactions with Ca2+ and phosphate ions. The compressive modulus of the n-HAp/gelatin scaffolds coated with n-HAp was 8.459 and 4.584 MPa for 5 and 3 % gelatin concentration respectively which is comparable to the compressive modulus of a human cancellous bone. Both compressive strength and moduli seem to increase by coating of the scaffolds. N-HAp particles coat the scaffold wall and easily enter the microscopic fractures resulting from inter particle contact and increase the mechanical strength. According to the cell culture experiments, the incorporation of rod-like n-HAp and coating of scaffolds with n-HAp particles enable the prepared scaffolds to possess good biocompatibility, high bioactivity and sufficient mechanical strength in comparison with pure one. Coated scaffolds seem to have a better cell attachment and proliferation. This research suggests that the newly developed rod-like HAp/gelatin coated with n-HAp fulfill most of the requirements for the use as a suitable bone replacement and may be superior for bone tissue engineering

Effect of Solution Surface Tension on Morphology of PLGA and Gelatin Electrospun Fibers

Based on specifc applications of electrospinning technology in tissue engineering there is a need for more crucial advances to be made in electrospinning of biocompatible polymer systems. In this study we investigated the effect of surface tension of different solvent systems on the morphology of electrospun nanofbers of gelatin and PLGA. At first the best solvent system was established to optimize nanofber formation of the polymers. The overall approach of this research was to prepare several solutions of identical concentrations with different solvent systems for electospinning into nanofbes. Finally, the changes in electrospun fbe

Nanomechanical Properties of Photo-Cured Cyanoacrylate Bioadhesives Crosslinked by TMPTMA or POSS Nanostructures

The  surface  modulus  and  hardness  of  photo-crosslinkable  cyanoacrylate bioadhesives  containing  TMPTMA  and  POSS  as  crosslinking  agents  were studied  using  nanoindentation  technique.  Adhesives  containing  2-octyl cyanoacrylate  (2-OCA)  and  different  percentages  of  POSS  nanostructures  and TMPTMA as crosslinking agents were prepared. 1-Phenyl-1,2-propanedione  (PPD) was incorporated as photo-initiator into the adhesive in 4 wt%. Mechanical properties of  the  surface  of  the  adhesives  were  measured  using  nanoindentation  technique applying a Berkovich tip. The results (three replicates) were analyzed and compared using one-way ANOVA  and Tukey  test  at  a  signifcance  level of 0.05. The  results showed  that by  incorporation of crosslinking agents, surface modulus and hardness increased and force loss during holding decreased. The incorporation of crosslinking agents  into  the  adhesives  enhanced  their  elastic  properties.  The  surface  modulus and  hardness  increased  by  increasing  crosslinking  concentration.  The  results  also indicated that the elastic work increased, and plastic work dropped due to increasing crosslinking density. The samples which had been stored in water for 24 h before the tests, showed reduced elastic properties due to the plasticization effect of water

Study of Mesenchymal Stem Cell Proliferation and Bone Differentiation on Composite Scaffolds of PLLA and Nano Hydroxyapatite with Different Morphologies

Objective: Nowadays, bone constructs elaborated according to tissue engineering principlesare being regarded as an ideal choice for the reconstruction of segmental bonedefects. In this study, proliferation and bone differentiation of marrow-derived mesenchymalstem cells (MSCs) were compared in different composite scaffolds containing varyingmorphologies of nano hydroxyapatite (nHAP).Materials and Methods: Needle nHAP/PLLA (poly (L-lactide acid)), spherical nHAP/PLLA and rod nHAP/PLLA scaffolds were prepared and 3D cultures of passaged-3 ratMSCs were established using the scaffolds. The loading of the cells onto the scaffoldinternal spaces was confirmed with microscopy and their proliferation was determined byMTT assay. To compare the osteogenic differentiation of the cells on the scaffold surfaces,osteogenic 3D cultures were established and kept for 21 days. At the end of this periodculture mineralization and relative bone-related gene expression were quantified usingthe alizarin red quantification assay and semi quantitative RT-PCR analysis respectively.ANOVA was used to compare the data.Results: According to the MTT assays, cells adhered to all the studied scaffold surfacestended to proliferate. In this respect the microenvironment provided by the needle nHAP/PLLA appeared much better than that of either the spherical or rod nHAP/PLLA scaffolds(P<0.05). Similarly, mineralization was observed to be heavier for the needle nHAP/PLLAscaffold compared to the two other composite scaffolds. In addition, the relative expressionof coll I, osteocalcin, runx2 and ALP genes all appeared to be significantly higher in the cellscultivated on needle nHAP/PLLA scaffolds versus their spherical and rod counterparts.Conclusion: Overall, needle nHAP/PLLA scaffolds appear to provide the most appropriatematrix for producing bone construct using MSCs