Preparation of surgical meshes using self-regulating technology based on reaction-diffusion processes.

While reaction-diffusion processes are utilized in multiple scientific fields, these phenomena have seen limited practical application in the polymer industry. Although self-regulating processes driven by parallel reaction and diffusion can lead to patterned structures, most polymeric products with repeating subunits are still prepared by methods that require complex and expensive instrumentation. A notable, high-added-value example is surgical mesh, which is often manufactured by weaving or knitting. In our present work, we demonstrate how the polymer and the biomedical industry can benefit from the pattern-forming capabilities of reaction-diffusion. We would like to propose a self-regulating method that facilitates the creation of surgical meshes from biocompatible polymers. Since the control of the process assumes a thorough understanding of the underlying phenomena, the theoretical background, as well as a mathematical model that can accurately describe the empirical data, is also introduced and explained. Our method offers the benefits of conventional techniques while introducing additional advantages not attainable with them. Most importantly, the method proposed in this paper enables the rapid creation of meshes with an average pore size that can be adjusted easily and tailored to fit the intended area of application.

Quantitative determination of release kinetics from fibrous poly(3-hydroxybutyrate) scaffolds.

A fibrous scaffold was prepared from poly(3-hudroxybutirate) (PHB) by wet spinning from chloroform solution. The fibers were precipitated into excess ethanol and they had a regular, cylindrical shape. The diameter of the fibers was regulated by the rate of spinning. A model drug, quercetin, was dissolved in the fiber in various amounts. The rate of drug release could be controlled by the adjustment of fiber diameter. A novel approach is proposed in the paper for the quantitative analysis of release kinetics. The model is based on Fick's laws, and does not use any simplification or introduce empirical constants. The prediction of the new model agreed well with experimental results and proved that the approach offers an exact description of experimental data. Although the method allows the estimation of the diffusion coefficient as well, the value obtained is biased because of the large surface to volume ratio of the scaffold. The comparison of the proposed model to empirical or semi-empirical approaches published earlier showed that the latter are less accurate and predict false results at various stages of the release study. The proposed method of fiber preparation and the model makes possible the prediction and control of the release rate of a drug from fibrous scaffolds.

Controlled degradation of poly-ε-caprolactone for resorbable scaffolds.

A lipase from Burkholderia cepacia was successfully adsorbed on the surface of halloysite nanotubes and the coated tubes were incorporated into poly-ε-caprolactone (PCL). The efficiency of the halloysite in the adsorption of the enzyme was characterized by the total protein content determined with the Bradford method. The activity of the adsorbed enzyme was estimated by the kinetic resolution of racemic 1-phenylethanol. The immobilized enzyme was mixed with the polymer and compression molded films were prepared at 70 °C. Activity measurements proved that the enzyme remains active even after adsorption; in fact, larger activities were measured for the immobilized enzyme than for the neat enzyme preparation. The supported enzyme degraded PCL efficiently, the rate of degradation depended on the amount of enzyme adsorbed. The kinetics of degradation was described quantitatively with an appropriate model accounting for two of the three steps of the process, i.e. degradation and the denaturation of the enzyme. The determination of time constants allows the adjustment of degradation rate. This is the first time that the enzyme, which catalyzes degradation, is incorporated into the polymer, and not into the degradation medium, thus allowing the preparation of resorbable scaffolds with controlled lifetime.

The role of enzyme adsorption in the enzymatic degradation of an aliphatic polyester.

The enzyme catalyzed degradation of poly(3-hydroxybutyrate) (PHB) is a two-step process consisting of the adsorption of the enzyme on the surface of a PHB substrate and the cleavage of ester bonds. A deactivated enzyme was prepared by point mutagenesis to separate the two steps from each other. Measurements carried out with active and inactive enzymes on PHB particles proved that mutagenesis was successful and the modified enzyme did not catalyze degradation. Based on the Michaelis-Menten approach a kinetic model was proposed which could describe the processes quantitatively, the agreement between prediction and the measured data was excellent. The separation of the two processes allowed the determination of the adsorption kinetics of the enzyme; the rate constants of the adsorption and desorption process were determined for the first time. Comparison of these constants to reaction rates showed that adsorption is not instantaneous and can be the rate-determining step. The area occupied by an enzyme molecule was also determined (13.1 nm2) and it was found to be smaller than the value published in the literature (17 ± 8 nm2). The separation of the two steps makes possible the prediction and control of the degradation process.

Enzymatic degradation of poly-[(R)-3-hydroxybutyrate]: Mechanism, kinetics, consequences.

Poly-[(R)-3-hydroxybutyrate] (PHB) films prepared by compression molding and solvent casting, respectively, were degraded with the intracellular depolymerase enzyme natively synthetized by the strain Bacillus megaterium. Quantitative analysis proved that practically only (R)-3-hydroxybutyric acid (3-HBA) forms in the enzyme catalyzed reaction, the amount of other metabolites or side products is negligible. The purity of the product was verified by several methods (UV-VIS spectroscopy, liquid chromatography, mass spectroscopy). Degradation was followed as a function of time to determine the rate of enzymatic degradation. Based on the Michaelis-Menten equation a completely new kinetic model has been derived which takes into consideration the heterogeneous nature of the enzymatic reaction. Degradation proceeds in two steps, the adsorption of the enzyme onto the surface of the PHB film and the subsequent degradation reaction. The rate of both steps depend on the preparation method of the samples, degradation proceed almost twice as fast in compression molded films than in solvent cast samples. The model can describe and predict the formation of the reaction product as a function of time. The approach can be used even for the commercial production of 3-HBA, the chemical synthesis of which is complicated and expensive.

The novel technique of vapor pressure analysis to monitor the enzymatic degradation of PHB by HPLC chromatography.

A novel method was introduced for the quantitative determination of substances in aqueous solutions by using the evaporative light scattering (ELS) detector of a high performance liquid chromatograph (HPLC). The principle of the measurement is the different equilibrium vapor pressure of the solvent and the analyte resulting in decreasing evaporation rate, larger droplets and stronger signal with increasing concentration. The new technique based on vapor pressure analysis was validated with traditional UV-Vis detection carried out with a diode array detector (DAD). The new technique was used for monitoring the concentration of solutions obtained during the enzymatic degradation of poly(3-hydroxybutyrate) yielding the 3-hydroxybutyrate monomer as the product. The accuracy of the measurement allowed the determination of degradation kinetics as well. The results obtained with the two techniques showed excellent agreement at small concentrations. Deviations at larger concentrations were explained with the non-linear correlation between analyte concentration and detector signal and the linear regression used for calibration. Mathematical analysis of the method made possible the determination of the evaporation enthalpy of the analyte as well. The new approach is especially suitable for the quantitative analysis of compounds, which do not absorb in the detection range of the DAD detector or if their characteristic absorbance is close to the lower end of its wavelength range.