Retrostructural model to predict biomass formulations for barrier performance.

Barrier performance and retrostructural modeling of the macromolecular components demonstrate new design principles for film formulations based on renewable wood hydrolysates. Hardwood hydrolysates, which contain a fair share of lignin coexisting with poly- and oligosaccharides, offer excellent oxygen-barrier performance. A Hansen solubility parameter (HSP) model has been developed to convert the complex hydrolysate structural compositions into relevant matrix oxygen-permeability data allowing a systematic prediction of how the biomass should be formulated to generate an efficient barrier. HSP modeling suggests that the molecular packing ability plays a key role in the barrier performance. The actual size and distribution of free volume holes in the matrices were quantified in the subnanometer scale with Positron annihilation lifetime spectroscopy (PALS) verifying the affinity-driven assembly of macromolecular segments in a densely packed morphology and regulating the diffusion of small permeants through the matrix. The model is general and can be adapted to determine the macromolecular affinities of any hydrolysate biomass based on chemical composition.

Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces.

In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin.

Conceptual approach to renewable barrier film design based on wood hydrolysate.

Biomass is converted to oxygen barriers through a conceptually unconventional approach involving the preservation of the biomass native interactions and macromolecular components and enhancing the effect by created interactions with a co-component. A combined calculation/assessment model is elaborated to understand, quantify, and predict which compositions that provide an intermolecular affinity high enough to mediate the molecular packing needed to create a functioning barrier. The biomass used is a wood hydrolysate, a polysaccharide-rich but not highly refined mixture where a fair amount of the native intermolecular and intramolecular hemicelluloses-lignin interactions are purposely preserved, resulting in barriers with very low oxygen permeabilities (OP) both at 50 and 80% relative humidity and considerably lower OPs than coatings based on the corresponding highly purified spruce hemicellulose, O-acetyl galactoglucomannan (AcGGM). The component interactions and mutual affinities effectively mediate an immobilization of the chain segments in a dense disordered structure, modeled through the Hansen's solubility parameter concept and quantified on the nanolength scale by positron annihilation lifetime spectrum (PALS).

In vitro and in vivo degradation profile of aliphatic polyesters subjected to electron beam sterilization.

Degradation characteristics in response to electron beam sterilization of designed and biodegradable aliphatic polyester scaffolds are relevant for clinically successful synthetic graft tissue regeneration. Scaffold degradation in vitro and in vivo were documented and correlated to the macroscopic structure and chemical design of the original polymer. The materials tested were of inherently diverse hydrophobicity and crystallinity: poly(L-lactide) (poly(LLA)) and random copolymers from L-lactide and ε-caprolactone or 1,5-dioxepan-2-one, fabricated into porous and non-porous scaffolds. After sterilization, the samples underwent hydrolysis in vitro for up to a year. In vivo, scaffolds were surgically implanted into rat calvarial defects and retrieved for analysis after 28 and 91days. In vitro, poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) samples degraded most rapidly during hydrolysis, due to the pronounced chain-shortening reaction caused by the sterilization. This was indicated by the rapid decrease in both mass and molecular weight of poly(LLA-co-DXO). Poly(L-lactide-co-ε-caprolactone) (poly(LLA-co-CL)) samples were also strongly affected by sterilization, but mass loss was more gradual; molecular weight decreased rapidly during hydrolysis. Least affected by sterilization were the poly(LLA) samples, which subsequently showed low mass loss rate and molecular weight decrease during hydrolysis. Mechanical stability varied greatly: poly(LLA-co-CL) withstood mechanical testing for up to 182 days, while poly(LLA) and poly(LLA-co-DXO) samples quickly became too brittle. Poly(LLA-co-DXO) samples unexpectedly degraded more rapidly in vitro than in vivo. After sterilization by electron beam irradiation, the three biodegradable polymers present widely diverse degradation profiles, both in vitro and in vivo. Each exhibits the potential to be tailored to meet diverse clinical tissue engineering requirements.

Minimization of residual tin in the controlled Sn(II)octoate-catalyzed polymerization of epsilon-caprolactone.

By using less catalyst in the ring-opening polymerization of epsilon-caprolactone, a residual tin content of 5 ppm was reached without the need for additional purification. The initial amount of tin (II) 2-ethylhexanoate [Sn(Oct)(2)] was varied using catalyst:monomer ratios of 1:1000, 1:10,000, and 1:20,000. The impact on the final conversion, reaction control, average molecular weight, and polydispersity was studied. The amount of Sn(Oct)(2) could be significantly reduced without influencing the reaction results. The residual amount of tin was reduced from 176 ppm with a catalyst:monomer ratio of 1:1000 in the polymer, to 5 ppm with the ratio 1:10,000. It was thus concluded that a catalyst:monomer ratio of 1:10,000 or lower is required to achieve a polymer with tin content suitable for biomedical applications. The materials were also tested in a proliferation study with mesenchymal stem cells from mouse. Porous scaffolds were fabricated from the polymers, using a salt leaching technique, and the cell growth on the porous scaffolds as well as on homogeneous films was determined by light absorbance measurements. In this study, the cell proliferation results showed that cells could grow on all polymers with an efficiency equal to or better than that on normal tissue culture plastic.

Finalizing the properties of porous scaffolds of aliphatic polyesters through radiation sterilization.

Porous scaffolds made of various L,L-lactide (LLA), 1,5-dioxepane-2-one (DXO) and epsilon-caprolactone (CL) copolymers were sterilized by EB- and gamma-irradiation. Differences in the comonomers, composition and the microstructure of the starting materials were used to influence the degradation mechanism and susceptibility towards irradiation and by this means to achieve sterilized scaffolds with predicted end-properties. The chemical changes and the formation of low-molecular-weight products were determined by SEC, 1H nuclear magnetic resonance (NMR), 13C NMR and gas chromatography-mass spectrometry (GC-MS). The degradation mechanism changed from random chain scission to cross-linking depending on the choice of monomers, the copolymer composition and the monomer sequences. Copolymerization of LLA with small amounts of CL or DXO increased the stability compared to that of the LLA homopolymer. Changing DXO to CL in a LLA copolymer also increased the stability. The type of radiation and the microstructure of the copolymer chains determined which of the monomer sequences were more prone to degrade. The most abundant low-molecular-weight product identified after sterilization was DXO monomer. Traces of LLA and CL monomers were also identified. Modification of the copolyester microstructure changed the degradation mechanism and the susceptibility towards irradiation. This allows the use of radiation sterilization to finalize the scaffold properties.

Surface functionalization of degradable polymers by covalent grafting.

With a new non-destructive and solvent-free photografting technique, N-vinylpyrrolidone was covalently grafted onto the surfaces of degradable polymers; poly(l-lactide), poly(epsilon-caprolactone), poly(lactide-co-glycolide), and poly(trimethylene carbonate). The modified surfaces were characterized by XPS, ATR-FTIR, SEM, and cell growth tests. The wettability was markedly improved, as static contact angles changed from about 80 degrees for the pristine substrates to around 30 degrees after 30min of grafting. Well-defined surface topographies, such as micro-patterns, are preserved in the process since the graft layers are thin. The biological response, measured as cytotoxicity, showed that the modified films provide good substrates, comparable with optimized cell culture plastics, for the adhesion and proliferation of normal human keratinocytes and skin fibroblasts.

Solvent free vapor phase photografting of maleic anhydride onto poly(ethylene terephthalate) and surface coupling of fluorinated probes, PEG, and an RGD-peptide.

Poly(ethylene terephthalate) (PET) was photografted in a solvent free vapor of maleic anhydride and benzophenone. After hydrolysis of the initially grafted succinic anhydride groups, the carboxylic PET surfaces were modified by coupling reactions in organic and aqueous solutions. 2,2,2-Trifluoroethylamine and diamino PEGs of molecular weight 3400 and 2000 were reacted with acid chloride groups obtained by treating the PET-COOH surface with PCl(5). Furthermore, fluoro substituted thiols and a cystein terminated RGD containing peptide were bound to PET-COOH surfaces via a disulfide link by a three step coupling sequence. Coupling yields and surface concentrations of the fluoro substituted ligands were calculated from ESCA data. The RGD-peptide surfaces were evaluated by cultivation with rat smooth muscle cells.

Polyesters based on diacid monomers.

Polymers with ester linkages in their main chain comprise a family of polymers with immense diversity and versatility. This review deals with the preparation of such polymers from dicarboxylic acid monomers, and the result in terms of properties and applicability. Polyesters alone, and their copolymers with amides, anhydrides, urethanes, imides, ethers or other functional groups, offer countless opportunities to tune the properties of the resulting material within a broad range. Of particular interest is the inherent biodegradability of the ester linkage. Biodegradability is sought after in a wide range of applications, above all in the preparation of environmentally friendly polymers and biomedical materials for temporary surgical use and in drug delivery.

Sterilization, storage stability and in vivo biocompatibility of poly(trimethylene carbonate)/poly(adipic anhydride) blends.

Biodegradable blends of poly(trimethylene carbonate) (PTMC) and poly(adipic anhydride) (PAA) have been proven to be strong candidates for controlled drug delivery polymers in vitro. We now report on the stability, sterilizability and in vivo local tissue response of these matrices. Blend matrices were sterilized by beta-radiation or ethylene oxide gas treatment, stored at different times and temperatures, and analyzed for changes in physicochemical properties. Moisture uptake at different relative humidities and storage times was determined. Sterilization procedures induced hydrolysis of the matrices. Ethylene oxide gas sterilization had a significantly more marked effect upon the matrix properties than radiation treatment. The onset of degradation was reflected in a decrease of crystallinity and molecular weight along with a change of blend composition. A similar onset of matrix degradation was observed upon storage in air. The physicochemical properties of the blends were well preserved upon storage under argon atmosphere. Biocompatibility of PTMC/PAA implants was assessed in the anterior chamber of rabbits eyes for 1 month. At selected post-operative time points, aqueous humor was analyzed for white blood cells and the corneal thickness was measured. The results suggest good biocompatability of PTMC-rich matrices, whereas fast eroding PAA-rich matrices caused inflammatory responses, due to a burst release of degradation products.

Great advantages in using a natural rubber instead of a synthetic SBR in a pro-oxidant system for degradable LDPE.

Different pro-oxidant systems are used in degradable low-density polyethylene (LDPE). The main question is the degradation products and not the degradation time from the used materials. The pro-oxidant formulation used consisted of manganese stearate and natural rubber (NR) or manganese stearate and a synthetic, styrene-butadiene copolymer rubber (SBR). The samples were heated in air at 100 degrees C in sealed glass vials. The molecular weight changes were measured by size exclusion chromatography (SEC). The volatile and nonvolatile degradation products have been identified by gas chromatography-mass spectrometry (GC/MS). A wide variety of degradation products were identified, including ketones, carboxylic acids, keto acids, dicarboxylic acids, and furanones as a homologous series. Benzaldehyde, acetophenone, benzoic acid, benzyl benzoate, and two benzene derivative compounds were identified only in the LDPE-SBR system. These aromatic compounds originate from the styrene part of SBR. The advantages using pro-oxidant containing NR are more effective degradation of LDPE without any aromatic degradation products.

Encapsulation of rotavirus into poly(lactide-co-glycolide) microspheres.

Two small-scale double emulsion techniques for incorporation of formaldehyde-inactivated rotavirus particles (FRRV) into poly(lactide-co-glycolide) (PLG) microspheres were developed and optimised. The effects of high-speed homogenisation versus vortex mixing on the double emulsion stability, microsphere size, entrapment efficiency and in vitro release of FRRV in the second emulsification step were studied. A stable double emulsion was verified only when using vortex mixing in this step. Slow removal of the organic phase allowed measurement of the size of the emulsion droplets and subsequent prediction of the size of the resulting microspheres. Microspheres in the size range of 1-10 microm were prepared using both techniques. The homogenisation technique was sensitive to changes in the operating time, the emulsification energy and the volume of the outer aqueous phase, while the vortex technique was more robust. Rotavirus was released in vitro in a triphasic manner with both techniques. The more robust vortex technique was selected for preparation of PLG microspheres containing rotavirus for in vivo studies. After immunisation of mice with a single intramuscular injection, the PLG-FRRV microspheres elicited an IgG antibody response in serum detected by ELISA equally high as that elicited with FRRV alone. These results indicate that the antigenicity of FFRV was retained after incorporation into PLG microspheres using the vortex technique.

Bioactive heparin surfaces from derivatization of polyacrylamide-grafted LLDPE.

Primary amine groups were introduced into polyacrylamide-LLDPE films, using the Hofmann degradation synthesis. The Hofmann degradation was studied at room temperature using sodium hypochlorite and sodium hydroxide at different concentrations. Diazotized heparin was covalently bound to the grafted LLDPE film via the primary amine groups. Surfaces were analysed with ESCA, ATR-IR, chloride titration and Toluidine Blue. Evaluation of the biological activity of the heparinized surfaces was made by measuring the capacity for binding antithrombin (AT) and inhibition of the activated coagulation factor XII (FXIIa). The heparinized surfaces were able to bind up to 3 pmol cm-2 of AT in solution with ionic strengths of I = 0.15 and I = 0.40. No activation of the adsorbed FXII was detected.

Copolymers of 1,5-dioxepan-2-one and L- or D,L-dilactide: in vivo degradation behaviour.

Copolymers of 1,5-dioxepan-2-one (DXO) and L- or D,L-dilactide have been synthesized and characterized. The molar ratio of the two monomers was around 20/80 of DXO and L- or D,L-lactide respectively. In vivo studies on rats revealed significant differences in tissue response between the semicrystalline DXO/L-LA copolymer and the amorphous DXO/D,L-LA copolymer. The materials were characterized pre- and post-implantation by size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and light-microscopy. Degradation seems to follow an autocatalytic hydrolysis mechanism. The amorphous copolymer was degraded at a faster rate and gave a less pronounced foreign body reaction as compared to the semicrystalline one.

Detection by high-performance liquid chromatography of polyamines formed by clostridial putrefaction of caseins.

Casein incorporated in building materials is degraded by species of alkali-tolerant Clostridia. A whole range of compounds have previously been detected in degraded building materials containing casein as an additive by gas chromatography (GC), including volatile and non-volatile organic acids, alcohols, ketones, aldehydes and monoamines. Using high-performance liquid chromatography (HPLC) it was possible, however, also to detect polyamines formed in degraded caseins. Histamine, agmatine, serotonine, tyramine, tryptamine, putrescine and cadaverine were detected in solutions containing casein in which the alkali-tolerant Clostriadia had been grown. Uninoculated, sterile incubated caseins contained no detectable amounts of polyamines. This gives clear evidence of the role of the biotic environment in the degradation of caseins. A combination of GC and HPLC therefore, provides a convenient set of techniques for studying the degradation products of casein.

Synthetic polymers as drugs.

Biologically active compounds have been incorporated into synthetic polymers. Bithionol, a well-known and potent antibacterial agent, has been used as bisphenol monomers for polyesters, polyphosphates, and phosphonates; the bischloroformate of bithionol has been converted into copolycarbonates and polyurethanes. The copolycarbonates of bithionol, optimally designed polymer structures with poly(ethylene oxide) glycols as the other comonomer, have been hydrolyzed under physiological conditions at a degradation rate of about 1% per day. Primaquine, an antimalarial, and amantadine, an antiviral agent, when reacted with isocyanates gave polymeric biurets as do other primary aliphatic amines. Primaquine also underwent nucleophilic substitution reactions on polyepichlorohydrin. 3-Vinyl-, 4-vinyl-, and 5-vinylsalicylic acids have been synthesized, polymerized, and copolymerized to polymeric antibacterials. Copolymers of vinylsalicylic acid can be selective in their activity, depending on the comonomer. Selectivity can also be achieved by derivation of the poly(vinylsalicylic acid).