A microplate compression method for elastic modulus measurement of soft and viscoelastic collagen microspheres.

Hydrogel-based microspheres are commonly used for drug and cell delivery in regenerative medicine. Characterization of their physical and mechanical properties is important in monitoring their quality during fabrication and in predicting their performance upon injection. However, existing methods have limitations in measuring these micron-sized, soft and viscoelastic spherical structures. In this study, a protocol is developed to measure the elastic modulus of non-linear viscoelastic spheres by microplate compression, and is applied to collagen microspheres fabricated with or without cells. During the measurement, a microsphere is placed on a rigid surface and is compressed by a calibrated flexible microplate gripped to a rigid end. A step increase in the displacement rate of the rigid end of the flexible microplate is introduced and the reduced elastic modulus of the microsphere is calculated from the deformation response of the microsphere, using an equation derived in this study. The reduced elastic modulus of collagen microspheres with and without mesenchymal stem cells measured by this method was 9.1 kPa and 132 Pa, respectively.

Development of a mathematical model for the water distribution in freeze-dried solids.

PURPOSE: Development of a mathematical model to provide information about the amount of water associated with a protein and an excipient in a lyophilized product. METHODS: The moisture content of the product and the mass fraction of each component were used to derive a model for the calculation of the mass of water associating with each component. The model was applied to lyophilized formulations of rhDNase containing various amounts of mannitol or lactose. The total water content was investigated by thermogravimetry, crystalline properties by X-ray powder diffraction and water uptake behaviour using a moisture microbalance system. RESULTS: Calculations based on the model suggest that in a lyophilized rhDNase-mannitol formulation where the sugar is crystalline, most of the water is taken up by the protein. However, in the lyophilized rhDNase-lactose formulation where the sugar is amorphous, water is taken up by both the sugar and protein to a comparative extent. At high relative humidities when the amorphous sugar undergoes crystallization, the model can accommodate such a change by allowing for the formation of an additional crystalline phase. CONCLUSIONS: The rhDNase-sugar formulations show excellent conformity to the model which provides quantitative information about the distribution of water in the lyophilized binary protein-excipient products.

Effects of additives on heat denaturation of rhDNase in solutions.

PURPOSE: To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions. METHODS: Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T(m)) and enthalpy (H(m)) of rhDNase. The effects of denaturants (guanidine HCl and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6-7. RESULTS: The T(m) and H(m) of rhDNase in pure water were measured as 67.4 degrees C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca(2+) stabilizes rhDNase against thermal denaturation and elevates T(m) and H(m) while Mg(2+), Mn(2+) and Zn(2+) destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T(m) and H(m). All destabilization of rhDNase can be prevented by adding Ca(2+) to the solutions. CONCLUSIONS: CaCl(2) and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HCl destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca(2+) the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.