nan Zainuddin

Advancing Towards a Tissue-engineered Tympanic Membrane: Silk Fibroin as a Substratum for Growing Human Eardrum Keratinocytes

Human tympanic membrane cells (hTMCs), harvested from tympanic membrane (TM) explants, were grown in culture and then seeded on membranes prepared from silkworm (Bombyx mori) silk fibroin (BMSF) and on tissue-culture plastic membranes (PET). Fibroin was isolated from silk cast into membranes with a thickness of 10—15 μm. The hTMCs were cultured on both materials for 15 days in a serum-containing culture medium. The cells grown on both substrata were subjected to nuclear staining (DAPI) and counted. Further, the cultures were immunostained for a number of protein markers related to the epithelial/keratinocyte phenotype and cell adhesion complexes. The BMSF membranes supported levels of hTMC growth higher than that observed on the PET membranes. The immunofluorochemical analysis indicated unequivocally that BMSF is a more suitable substratum than PET with respect to the growth patterns, proliferation, and cell—cell contact and adhesion. BMSF appear as a promising substratum in the tissue-engineered constructs for the replacement of TM in case of nonhealing perforations.

The behavior of aged regenerated Bombyx mori silk fibroin solutions studied by 1H NMR and rheology

As part of a project to utilize the regenerated silk fibroin (RSF) membranes as a supporting matrix for the attachment and growth of corneal stem/progenitor cells in the development of tissue engineered constructs for the surgical restoration of the ocular surface, the behavior of the aged RSF solutions has been investigated. The solutions were produced from domesticated silkworm (Bombyx mori) cocoons according to a protocol involving successive dissolution steps, filtration and dialysis. The solutions were kept at 4 degrees C in a refrigerator for a certain period of time until near the gelation time. The changes in molecular conformation were studied by solution-state (1)H NMR, while the flow of the solutions was characterized by rheological method. Upon ageing turbidity developed in solutions and the viscosity continuously decreased prior to a drastic increased near the gelation time. The (1)H resonances of aged solutions showed a consistent downfield shift as compared to the (1)H resonances of the fresh solution. Shear thinning with anomalous short recovery within a certain range of low shear rates occurred in both fresh and aged solutions. While the solutions behave as pseudo-plastic materials, the chain conformation in aged solutions adopted all secondary configurations with beta-strand being predominant.

Silk as Substratum for Cell Attachment and Proliferation

Silk fibroin (SF), isolated from silkworm (Bombyx mori) cocoons, is a natural biodegradable polymer. Over the past decade, there was some interest in using SF as a biomedical material. As part of a project to develop tissue-engineered constructs for the surgical restoration of the ocular surface (cornea, conjunctiva), we have investigated the capacity of SF to function as a substratum for the attachment and growth of corneal stem/progenitor cells harvested from the corneoscleral limbus of donor human corneal tissue. SF membranes were produced from cocoons following a protocol involving successive dissolution steps, filtration, dialysis, evaporation, and methanol treatment. Human limbal epithelial cells were harvested from donor tissue and seeded onto SF membranes. After 5 days, the culture was fixed and stained with specific agents to visualize the cells. The study indicated profuse cellular attachment and growth. SF membranes appear to be suitable as a substratum for the repair of damaged ocular surface.

Spontaneous calcification of acrylic hydrogels in abiotic calcifying media and the relevance of ionic solute effects

The calcification of acrylic hydrogels in abiotic calcifying media was analyzed. The calcification in hydrogels was independent of the physiological microenvironment and biological factors. In all the experiments high purity, sterile and nonpyrogenic water was used for injections with osmolality zero. The results show that all ionic species involved in the calcification of PHEMA hydogels in a metastable solution of CaCl and NaPO induced salting-out effects.

PHEMA Hydrogels Modified through the Grafting of Phosphate Groups by ATRP Support the Attachment and Growth of Human Corneal Epithelial Cells

Converting the surface of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel into a cell-adhesive surface has been successfully achieved through a method based on atom transfer radical polymerization (ATRP) grafting. Following activation of the surface hydroxyl groups of PHEMA by bromination, surface-initiated ATRP of mono(2-methacryloyloxyethyl) phosphate (MMEP) was conducted in a methanol—water system with Cu(I)Br as catalyst at room temperature. The conversion of PHEMA hydroxyl groups to brominated isobutyryl groups and the occurrence of grafting of PMMEP were confirmed by infrared and X-ray photoelectron spectroscopies. Cell attachment experiments were conducted by culturing human corneal limbal epithelial cells on the PMMEP-grafted PHEMA, and on unmodified PHEMA and tissue culture plastic for comparison. The results showed that the grafted PMMEP was homogeneously distributed, and the phosphate groups appeared to significantly promote the attachment, spreading and growth of cells, at a level comparable to the tissue culture plastic.

The States, Diffusion, and Concentration Distribution of Water in Radiation‐Formed PVA/PVP Hydrogels

Abstract Hydrogels with various compositions of polyvinyl alcohol (PVA) and poly(1‐vinyl‐2‐ pyrrolidinone) (PVP) were prepared by irradiating mixtures of PVA and PVP in aqueous solutions with gamma‐rays from 60Co sources at room temperature. The states of water in the hydrogels were characterized using DSC and NMR T2 relaxation measurements and the kinetics of water diffusion in the hydrogels were studied by sorption experiments and NMR imaging. The DSC endothermic peaks in the temperature range −10 to +10°C implied that there are at least two kinds of freezable water present in the matrix. The difference between the total water content and the freezable water content was referred to as bound water, which is not freezable. The weight fraction of water at which only nonfreezable water is present in a hydrogel with FVP=0.19 has been estimated to be gH2O/gPolymer=0.375. From water sorption experiments, it was demonstrated that the early stage of the diffusion of water into the hydrogels was Fickian. A curve‐fit of the early‐stage experimental data to the Fickian model allowed determination of the water diffusion coefficient, which was found to lie between 1.5×10−11 m2 s−1 and 4.5×10−11 m2 s−1, depending on the polymer composition, the cross‐link density, and the temperature. It was also found that the energy barrier for diffusion of water molecules into PVA/PVP hydrogels was ≈24 kJ mol−1. Additionally, the diffusion coefficients determined from NMR imaging of the volumetric swelling of the gels agreed well with the results obtained by the mass sorption method.