Dynamics of in vitro polymer degradation of polycaprolactone-based scaffolds : accelerated versus simulated physiological conditions

The increasing use of biodegradable devices in tissue engineering and regenerative medicine means it is essential to study and understand their degradation behaviour. Accelerated degradation systems aim to achieve similar degradation profiles within a shorter period of time, compared with standard conditions. However, these conditions only partially mimic the actual situation, and subsequent analyses and derived mechanisms must be treated with caution and should always be supported by actual long-term degradation data obtained under physiological conditions. Our studies revealed that polycaprolactone (PCL) and PCL-composite scaffolds degrade very differently under these different degradation conditions, whilst still undergoing hydrolysis. Molecular weight and mass loss results differ due to the different degradation pathways followed (surface degradation pathway for accelerated conditions and bulk degradation pathway for simulated physiological conditions). Crystallinity studies revealed similar patterns of recrystallization dynamics, and mechanical data indicated that the scaffolds retained their functional stability, in both instances, over the course of degradation. Ultimately, polymer degradation was shown to be chiefly governed by molecular weight, crystallinity susceptibility to hydrolysis and device architecture considerations whilst maintaining its thermodynamic equilibrium.

Vibrational spectroscopic studies of wool

Fourier transfonn (FT) Raman, Raman microspectroscopy and Fourier transform infrared (FTIR) spectroscopy have been used for the structural analysis and characterisation of untreated and chemically treated wool fibres. For FT -Raman spectroscopy novel methods of sample presentation have been developed and optimised for the analysis of wool. No significant fluorescence was observed and the spectra could be obtained routinely. The stability of wool keratin to the laser source was investigated and the visual and spectroscopic signs of sample damage were established. Wool keratin was found to be extremely robust with no signs of sample degradation observed for laser powers of up to 600 m W and for exposure times of up to seven and half hours. Due to improvements in band resolution and signal-to-noise ratio, several previously unobserved spectral features have become apparent. The assignment of the Raman active vibrational modes of wool have been reviewed and updated to include these features. The infrared spectroscopic techniques of attenuated total reflectance (ATR) and photoacoustic (P A) have been used to examine shrinkproofed and mothproofed wool samples. Shrinkproofing is an oxidative chemical treatment used to selectively modifY the surface of a wool fibre. Mothproofing is a chemical treatment applied to wool for the prevention of insect attack. The ability of PAS and A TR to vary the penetration depth by varying certain instrumental parameters was used to obtain spectra of the near surface regions of these chemically treated samples. These spectra were compared with those taken with a greater penetration depth, which therefore represent more of the bulk wool sample. The PA and ATR spectra demonstrated that oxidation was restricted to the near-surface layer of wool. Extensive curve fitting of ATR spectra of untreated wool indicated that cuticle was composed of a mixed protein conformation, but was predominately that of an a.-helix. The cortex was proposed to be a mixture of both a.helical and ~-pleated sheet protein conformations. These findings were supported by PAS depth profiling results. Raman microspectroscopy was used in an extensive investigation of the molecular structure of the wool fibre. This included determining the orientation of certain functional groups within the wool fibre and the symmetry of particular vibrations. The orientation ofbonds within the wool fibre was investigated by orientating the wool fibre axis parallel and then perpendicular to the plane of polarisation of the electric vector of the incident radiation. It was experimentally determined that the majority of C=O and N-H bonds of the peptide bond of wool lie parallel to the fibre axis. Additionally, a number of the important vibrations associated with the a-helix were also found to lie parallel to the fibre axis. Further investigation into the molecular structure of wool involved determining what effect stretching the wool fibre had on bond orientation. Raman spectra of stretched and unstretched wool fibres indicated that extension altered the orientation ofthe aromatic rings, the CH2 and CH3 groups of the amino acids. Curve fitting results revealed that extension resulted in significant destruction of the a-helix structure a substantial increase in the P-pleated sheet structure. Finally, depolarisation ratios were calculated for Raman spectra. The vibrations associated with the aromatic rings of amino acids had very low ratios which indicated that the vibrations were highly symmetrical.

Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

The effects of bioactive akermanite on physiochemical, drug-delivery and biological properties of poly (lactide-co-glycolide) beads

Poly(lactide-co-glycolide) (PLGA) beads have been widely studied as a potential drug/protein carrier. The main shortcomings of PLGA beads are that they lack bioactivity and controllable drug-delivery ability, and their acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Akermanite (AK) (Ca(2) MgSi(2) O(7) ) is a novel bioactive ceramic which has shown excellent bioactivity and degradation in vivo. This study aimed to incorporate AK to PLGA beads to improve the physiochemical, drug-delivery, and biological properties of PLGA beads. The microstructure of beads was characterized by SEM. The effect of AK incorporating into PLGA beads on the mechanical strength, apatite-formation ability, the loading and release of BSA, and the proliferation, and differentiation of bone marrow stromal cells (BMSCs) was investigated. The results showed that the incorporation of AK into PLGA beads altered the anisotropic microporous structure into homogenous one and improved their compressive strength and apatite-formation ability in simulated body fluids (SBF). AK neutralized the acidic products from PLGA beads, leading to stable pH value of 7.4 in biological environment. AK led to a sustainable and controllable release of bovine serum albumin (BSA) in PLGA beads. The incorporation of AK into PLGA beads enhanced the proliferation and alkaline phosphatase activity of BMSCs. This study implies that the incorporation of AK into PLGA beads is a promising method to enhance their physiochemical and biological property. AK/PLGA composite beads are a potential bioactive drug-delivery system for bone tissue repair.

Long-term effects of hydrogel properties on human chondrocyte behavior

Hydrogels provide a 3-dimensional network for embedded cells and offer promise for cartilage tissue engineering applications. Nature-derived hydrogels, including alginate, have been shown to enhance the chondrocyte phenotype but are variable and not entirely controllable. Synthetic hydrogels, including polyethylene glycol (PEG)-based matrices, have the advantage of repeatability and modularity; mechanical stiffness, cell adhesion, and degradability can be altered independently. In this study, we compared the long-term in vitro effects of different hydrogels (alginate and Factor XIIIa-cross-linked MMP-sensitive PEG at two stiffness levels) on the behavior of expanded human chondrocytes and the development of construct properties. Monolayer-expanded human chondrocytes remained viable throughout culture, but morphology varied greatly in different hydrogels. Chondrocytes were characteristically round in alginate but mostly spread in PEG gels at both concentrations. Chondrogenic gene (COL2A1, aggrecan) expression increased in all hydrogels, but alginate constructs had much higher expression levels of these genes (up to 90-fold for COL2A1), as well as proteoglycan 4, a functional marker of the superficial zone. Also, chondrocytes expressed COL1A1 and COL10A1, indicative of de-differentiation and hypertrophy. After 12 weeks, constructs with lower polymer content were stiffer than similar constructs with higher polymer content, with the highest compressive modulus measured in 2.5% PEG gels. Different materials and polymer concentrations have markedly different potency to affect chondrocyte behavior. While synthetic hydrogels offer many advantages over natural materials such as alginate, they must be further optimized to elicit desired chondrocyte responses for use as cartilage models and for development of functional tissue-engineered articular cartilage.

The effect of various pH, ionic strength and temperature on papain hydrolysis of salivary film

Stimulated human whole saliva (WS) was used to study the dynamics of papain hydrolysis at defined pH, ionic strength and temperature with the view of reducing an acquired pellicle. A quartz crystal microbalance with dissipation (QCM-D) was used to monitor the changes in frequency due to enzyme hydrolysis of WS films and the hydrolytic parameters were calculated using an empirical model. The morphological and conformational changes of the salivary films before and after enzymatic hydrolysis were characterized by atomic force microscopy (AFM) imaging and grazing angle infrared spectroscopy (GA-FTIR) spectra, respectively. The characteristics of papain hydrolysis of WS films were pH-, ionic strength- and temperature-dependent. The WS films were partially removed by the action of enzyme, resulting thinner and smoother surfaces. The IR data suggested that hydrolysis-induced deformation did not occur onto the remnants salivary films. The processes of papain hydrolysis of WS films can be controlled by properly regulating pH, ionic strength and temperature.

Immobilised anatase on clay mineral particles as a photocatalyst for herbicides degradation

Materials consisting of anatase linked to Laponite particles were synthesized by the reaction of TiOSO4 with Laponite, and were used for the degradation of pesticides. All these materials were characterized by XRD, FTIR, Raman, TEM, specific surface area and porosity determinations. Based on the amount of photoactive phase per unit mass of the clay mineral, not based on the total weight of the catalysts, these porous catalysts were displaying a high degradation rate than commercial P25. The TiO2 immobilized clay mineral catalysts can sediment in few minutes and could be readily separated out from a slurry system after the photocatalytic reaction. Settling properties of these catalysts are enormously high in aqueous media in contrast to P25.

Environmental degradation of lignin/poly(hydroxybutyrate) blends

Blends of lignin and poly(hydroxybutyrate) (PHB) were obtained by melt extrusion. They were buried in a garden soil for up to 12 months, and the extent and mechanism of degradation were investigated by gravimetric analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR) over the entire range of compositions. The PHB films were disintegrated and lost 45 wt% of mass within 12 months. This value dropped to 12 wt% of mass when only 10 wt% of lignin was present, suggesting that lignin both inhibited and slowed down the rate of PHB degradation. TGA and DSC indicated structural changes, within the lignin/PHB matrix, with burial time, while FTIR results confirmed the fragmentation of the PHB polymer. XPS revealed an accumulation of biofilms on the surface of buried samples, providing evidence of a biodegradation mechanism. Significant surface roughness was observed with PHB films due to microbial attack caused by both loosely and strongly associated micro-organisms. The presence of lignin in the blends may have inhibited the colonisation of the micro-organisms and caused the blends to be more resistant to microbial attack. Analysis suggested that lignin formed strong hydrogen bonds with PHB in the buried samples and it is likely that the rate of breakdown of PHB is reduced, preventing rapid degradation of the blends.

Electrospinning and crosslinking of low-molecular-weight poly(trimethylene carbonate-co-L-lactide) as an elastomeric scaffold for vascular engineering

The growth of suitable tissue to replace natural blood vessels requires a degradable scaffold material that is processable into porous structures with appropriate mechanical and cell growth properties. This study investigates the fabrication of degradable, crosslinkable prepolymers of l-lactide-co-trimethylene carbonate into porous scaffolds by electrospinning. After crosslinking by γ-radiation, dimensionally stable scaffolds were obtained with up to 56% trimethylene carbonate incorporation. The fibrous mats showed Young’s moduli closely matching human arteries (0.4–0.8 MPa). Repeated cyclic extension yielded negligible change in mechanical properties, demonstrating the potential for use under dynamic physiological conditions. The scaffolds remained elastic and resilient at 30% strain after 84 days of degradation in phosphate buffer, while the modulus and ultimate stress and strain progressively decreased. The electrospun mats are mechanically superior to solid films of the same materials. In vitro, human mesenchymal stem cells adhered to and readily proliferated on the three-dimensional fiber network, demonstrating that these polymers may find use in growing artificial blood vessels in vivo.

Cross-linked poly(trimethylene carbonate-co-L-lactide) as a biodegradable, elastomeric scaffold for vascular engineering applications

A series of copolymers of trimethylene carbonate (TMC) and l-lactide (LLA) were synthesized and evaluated as scaffolds for the production of artificial blood vessels. The polymers were end-functionalized with acrylate, cast into films, and cross-linked using UV light. The mechanical, degradation, and biocompatibility properties were evaluated. High TMC polymers showed mechanical properties comparable to human arteries (Young’s moduli of 1.2–1.8 MPa and high elasticity with repeated cycling at 10% strain). Over 84 days degradation in PBS, the modulus and material strength decreased gradually. The polymers were nontoxic and showed good cell adhesion and proliferation over 7 days using human mesenchymal stem cells. When implanted into the rat peritoneal cavity, the polymers elicited formation of tissue capsules composed of myofibroblasts, resembling immature vascular smooth muscle cells. Thus, these polymers showed properties which were tunable and favorable for vascular tissue engineering, specifically, the growth of artificial blood vessels in vivo.

methoxy-poly(ethylene glycol) modified poly(L-lactide) enhanced cell affinity of human bone marrow stromal cells by the upregulation of 1-cadherin and delta-2-catenin

Poly(l-lactide) (PLLA), a versatile biodegradable polymer, is one of the most commonly-used materials for tissue engineering applications. To improve cell affinity for PLLA, poly(ethylene glycol) (PEG) was used to develop diblock copolymers. Human bone marrow stromal cells (hBMSCs) were cultured on MPEG-b-PLLA copolymer films to determine the effects of modification on the attachment and proliferation of hBMSC. The mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analyzed using RT-qPCR to understand the underlying mechanisms. It was found that MPEG-b-PLLA copolymer films significantly improved cell adhesion, extension, and proliferation.This was found to be related to the significant upregulation of two adhesion genes, CDH1 and CTNND2, which encode 1-cadherin and delta-2-catenin, respectively, two key components for the cadherin-catenin complex. In summary, MPEG-b-PLLA copolymer surfaces improved initial cell adhesion by stimulation of adhesion molecule gene expression.

Hydrolysis of triasulfuron, metsulfuron-methyl and chlorsulfuron in alkaline soil and aqueous solutions

The hydrolysis of triasulfuron, metsulfuron-methyl and chlorsulfuron in aqueous buffer solutions and in soil suspensions at pH values ranging from 5.2 to 11.2 was investigated. Hydrolysis of all three compounds in both aqueous buffer and soil suspensions was highly pH-sensitive. The rate of hydrolysis was much faster in the acidic pH range (5.2-6.2) than under neutral and moderately alkaline conditions (8.2-9.4), but it increased rapidly as the pH exceeded 10.2. All three compounds degraded faster at pH 5.2 than at pH 11.2. Hydrolysis rates of all three compounds could be described well with pseudo-first-order kinetics. There were no significant differences (P =0.05) in the rate constants (k, day-1) of the three compounds in soil suspensions from those in buffer solutions within the pH ranges studied. A functional relationship based on the propensity of nonionic and anionic species of the herbicides to hydrolyse was used to describe the dependence of the 'rate constant' on pH. The hydrolysis involving attack by neutral water was at least 100-fold faster when the sulfonylurea herbicides were undissociated (acidic conditions) than when they were present as the anion at near neutral pH. In aqueous buffer solution at pH > 11, a prominent degradation pathway involved O-demethylation of metsulfuron-methyl to yield a highly polar degradate, and hydrolytic opening of the triazine ring. It is concluded that these herbicides are not likely to degrade substantially through hydrolysis in most agricultural (C) 2000 Society of Chemical Industry.

Degradation of monogalactosyl diglyceride and diagalactosyl diglyceride by sheep pancreatic enzymes

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca2+ had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of a- and b-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of a-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by b-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.