Biocompatible oligochitosans as cationic modifiers of alginate/Ca microcapsules

In the past, it has been proven that by properly adjusting the molecular mass of the oligochitosan samples, it is possible to optimize the formation of rigid, biocompatible capsules with semipermeable membranes under physiological conditions. In this study, the feasibility of four oligochitosan samples, with varying molar masses (M(n) in range 3-5 kDa), as biocompatible coatings of alginate/Ca capsules was investigated. By selection of appropriate depolymerization and purification methods we obtained oligochitosan samples that appeared to be noncytotoxic for C(2)C(12) myoblasts and did not influence the mammalian cell metabolism especially in relative short time during the process of capsule formation. Furthermore, oligochitosans can be used as a tool to reduce the membrane cut-off of the alginate capsules. However, such reduction, as well as mechanical resistance of formed microcapsules, depend on MM of the cationic polysaccharide and the chemical composition of the alginate (mannuronic/guluronic acid ratio). Here, we address that the use of low molar mass chitosan (< 5000 g/mol) permits the formation of mechanical stable capsules at physiological pH, which represents a strong advantage over other chitosan-based chemistries.     

Collagen and mechanical strength in various organs of rats treated with D-penicillamine or amino-acetonitrile.

After oral treatment with D-penicillamine (D-Pc) or with aminoacetonitrile (AAn) for 10 days, mechanical and chemical parameters were studied simultaneously in various organs of Sprague Dawley rats. Tensile strength of skin strips and of tail tendons, breaking strength of femur bones and tensile strength of granuloma tissue (induced by implanted glass rods) were measured and calculated. In the same tissue the soluble collagen fractions and the insoluble collagen were determined. Total collagen and the ratio insoluble vs. soluble collagen were calculated. Tensile strength of skin, tendon and granuloma tissue were greatly reduced by D-Pc treatment but only minimally influenced by AAN treatment. On the other hand only AAN significantly reduced the breaking strength of bone. All these changes were closely correlated with the content of insoluble collagen in the respective tissues. The correlation coefficients to total collagen were similar but lower. The correlation coefficients between strength and the ratio insoluble vs. soluble collagen were generally still lower. Earlier findings in aged and corticoid treated rats, proving that insoluble collagen content determines mechanical strength of connnective and supporting tissue thus could be confirmed.     

Multi-layered microcapsules for cell encapsulation.

Mechanical stability, complete encapsulation, selective permeability, and suitable extra-cellular microenvironment, are the major considerations in designing microcapsules for cell encapsulation. We have developed four types of multi-layered microcapsules that allow selective optimization of these parameters. Primary hepatocytes were used as model cells to test these different microcapsule configurations. Type-1 microcapsules with an average diameter of 400 microm were formed by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 microm. Cells in these microcapsules exhibited improved cellular functions over those cultured on collagen monolayers. Type-II microcapsules were formed by encapsulating the Type-I microcapsules in another 2-5 microm ter-polymer shell and a approximately 5 microm collagen layer between the two ter-polymer shells to ensure complete cell encapsulation. Type-II microcapsules comprised of a macro-porous exoskeleton with materials such as alumina sol-gel coated on the Type-I microcapsules. Nano-indendation assay indicated an improved mechanical stability over the Type-I microcapsules. Type-IV microcapsules were created by encapsulating Type-III microcapsules in another 2-5 microm ter-polymer shell, with the aim of imparting a negatively charged smooth surface to minimize plasma protein absorption and ensure complete cell encapsulation. The permeability for nutrient exchange, cellular functions in terms of urea production and mechanical stability of the microcapsules were characterized. The advantages and limitations of these microcapsules for tissue engineering are discussed.     

The mechanical strength of collagen gels containing glycosaminoglycans and populated with fibroblasts

Collagen gels provide a biocompatible matrix for replacing soft tissues, but it is essential to determine whether the mechanical properties of the matrix can be retained after cell ingrowth into the collagen scaffold. We have determined the mechanical strength of four collagen gel compositions (plain collagen; collagen-chrondroitin-6-sulphate glycosaminoglycan (GAG); collagen crosslinked with carbodiimide and putrescine, and collagen-GAG with the crosslinkers) in the presence of either 3T3 mouse fibroblasts or human skin fibroblasts, to determine whether cellular activity influences the mechanical properties of the matrix, and whether the crosslinking processes alter the effects of the cells. The presence of GAG and the crosslinkers increased the strength and stiffness of the unseeded gels, but there was no evidence for synergy between these treatments. In all cases, the gels became significantly weaker after 6 days in the presence of either human or mouse fibroblasts, as judged by the decrease in the values of the maximum load and stress before failure, and the stiffness decreased as shown by the lower values of the incremental modulus. With most parameters, the effect of the cells was independent of gel composition, and the presence of crosslinkers or GAG did not impart resistance to the cell-induced decrease in strength.     

Biocompatible polyphosphazenes by radiation-induced graft copolymerization and heparinization.

Investigations were carried out on the radiation-induced graft copolymerization by direct irradiation of dimethylaminoethyl methacrylate on to poly(bis(trifluoroethoxy)phosphazene) and on to poly (bis(phenoxy)phosphazene). Kinetics of grafting were followed with the polyphosphazenes immersed in monomer - methanol mixtures of various composition. The grafted film samples were quaternized with methyl iodide and, to the produced ammonium group, heparin was ionically bonded with high yield. On the grafted and heparinized-grafted film samples an evaluation of hydrophilicity, mechanical properties, biocompatibility and anticoagulating properties was carried out.     

Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. III. Biocompatibility Of smaller versus standard microcapsules.

Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.     

Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength

Hydroxyapatite–chitosan/gelatin (HA:Chi:Gel) nanocomposite scaffold has potential to serve as a template matrix to regenerate extra cellular matrix of human bone. Scaffolds with varying composition of hydroxyapatite, chitosan, and gelatin were prepared using lyophilization technique where glutaraldehyde (GTA) acted as a cross-linking agent for biopolymers. First, phase pure hydroxyapatite–chitosan nanocrystals were in situ synthesized by coprecipitation method using a solution of 2% acetic acid dissolved chitosan and aqueous solution of calcium nitrate tetrahydrate [Ca(NO₃)₂,4H₂O] and diammonium hydrogen phosphate [(NH₄)₂H PO₄]. Keeping solid loading constant at 30 wt% and changing the composition of the original slurry of gelatin, HA–chitosan allowed control of the pore size, its distribution, and mechanical properties of the scaffolds. Microstructural investigation by scanning electron microscopy revealed the formation of a well interconnected porous scaffold with a pore size in the range of 35–150 μm. The HA granules were uniformly dispersed in the gelatin–chitosan network. An optimal composition in terms of pore size and mechanical properties was obtained from the scaffold with an HA:Chi:Gel ratio of 21:49:30. The composite scaffold having 70% porosity with pore size distribution of 35–150 μm exhibited a compressive strength of 3.3–3.5 MPa, which is within the range of that exhibited by cancellous bone. The bioactivity of the scaffold was evaluated after conducting mesenchymal stem cell (MSC) – materials interaction and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay using MSCs. The scaffold found to be conducive to MSC’s adhesion as evident from lamellipodia, filopodia extensions from cell cytoskeleton, proliferation, and differentiation up to 14 days of cell culture.     

Synthesis and characterization of a novel fast-set proline-derivative-containing glass ionomer cement with enhanced mechanical properties

In this study, a methacryloyl derivative of l-proline was synthesized, characterized and incorporated into a conventional glass ionomer cement (GIC) with a polyacid composition. Subsequently, the effects of incorporation of synthesized N-methacryloyl-proline and terpolymer on the GIC's mechanical and working properties were studied. 1-Methacryloylpyrrolidone-2-carboxylic acid was synthesized and used in a polymerization reaction with acrylic acid and itaconic acid in order to form terpolymer which was used in Fuji II commercial GIC formulations. Chemical structural characterization of the resulting products was performed using (1)H nuclear magnetic resonance and Fourier transform infrared spectroscopy. The viscosity and molecular weight of the terpolymer were also measured. The mechanical strength properties of the modified GICs were evaluated after 24h or 1 week of immersion in distilled water at 37 degrees C. Analysis of variance was used to study the statistical significance of the mechanical strengths and working properties, and to compare them with a control group. Results showed that N-methacryloyl-proline modified GICs exhibited significantly higher compressive strength (CS; 195-210MPa), higher diametral tensile strength (DTS; 19-26MPa) and higher biaxial flexural strength (38-46MPa) in comparison to Fuji II GIC (161-166MPa in CS, 12-14MPa in DTS and 13-18MPa in biaxial flexural strength). The working properties (setting and working time) of the modified samples showed that the modified cement was a fast-set cement. It was concluded that a novel amino acid-containing GIC has been developed in this study with 27%, 94% and 170% increases in values for compressive, diametral tensile and biaxial flexural strength, respectively, in comparison to commercial Fuji II GIC.     

Biocompatible poly(N-vinyllactam)-based materials with environmentally-responsive permeability

Biocompatible polymer-based materials whose properties respond to environmental stimuli are of great value in biomedical and biotechnological applications (e.g., controlled release of drugs and sensitive reactants, bioseparations, spontaneous adjusting of flow of liquids including flow control in microfluidic devices). This study describes preparation and properties of biocompatible poly(N-vinyllactam)-based environment responsive composite membranes. The non-charged network N-vinyl lactam polymers form hydrogels that can exhibit swelling and de-swelling behaviour and, thus, regulate porosity of the membrane. The first part of the study has involved investigation of bulk polymerization reactivity of N-vinylcaprolactam (VCL)-based systems. VCL has been combined with up to 20 mol% of N-vinylpyrrolidone (N-vinylbutyrolactam). These synthetic processes have been investigated as thermal polymerizations (with and without a free-radical initiator) and photopolymerizations. Subsequently, the most reactive systems have been used to prepare composite materials by polymerization-modifying borosilicate microfibre membranes. The modified membranes have been characterized by the net mass gain, infrared spectra, and change in permeability in response to changes in ionic strength of the aqueous media. The permeability experiments have been carried out at constant fluxes and the resultant changes in trans-membrane pressure observed. Those membranes that are ionic-strength responsive could potentially be used for bio-separations and other types of biomedical and biotechnological applications.     

Biocompatible poly(N-vinyllactam)-based materials with environmentally-responsive permeability

Biocompatible polymer-based materials whose properties respond to environmental stimuli are of great value in biomedical and biotechnological applications (e.g., controlled release of drugs and sensitive reactants, bioseparations, spontaneous adjusting of flow of liquids including flow control in microfluidic devices). This study describes preparation and properties of biocompatible poly(N-vinyllactam)-based environment responsive composite membranes. The non-charged network N-vinyl lactam polymers form hydrogels that can exhibit swelling and de-swelling behaviour and, thus, regulate porosity of the membrane. The first part of the study has involved investigation of bulk polymerization reactivity of N-vinylcaprolactam (VCL)-based systems. VCL has been combined with up to 20 mol% of N-vinylpyrrolidone (N-vinylbutyrolactam). These synthetic processes have been investigated as thermal polymerizations (with and without a free-radical initiator) and photopolymerizations. Subsequently, the most reactive systems have been used to prepare composite materials by polymerization-modifying borosilicate microfibre membranes. The modified membranes have been characterized by the net mass gain, infrared spectra, and change in permeability in response to changes in ionic strength of the aqueous media. The permeability experiments have been carried out at constant fluxes and the resultant changes in trans-membrane pressure observed. Those membranes that are ionic-strength responsive could potentially be used for bio-separations and other types of biomedical and biotechnological applications.     

Engineering of perfluorooctylbromide polypyrrole nano-/microcapsules for simultaneous contrast enhanced ultrasound imaging and photothermal treatment of cancer

A versatile oil-in-water emulsion method has been explored for constructing water-dispersible polypyrrole (PPy) nano-/microcapsules with a soluble PPy complex as multifunctional photothermal agents for tumor ablation. In this work, both PPy nanocapsules (280.4 ± 79.0 nm) and microcapsules (1.31 ± 0.45 μm) with liquid perfluorooctylbromide (PFOB) core could be obtained by simply tuning the process energy for emulsion formation from ultrasonication to homogenization. Owing to the encapsulated liquid PFOB and strong near-infrared (NIR) absorption of PPy shell, the resulted PPy capsules showed great promise in ultrasound imaging guided photothermal ablation of tumor cells without inducing any significant side effect. Thus, it is anticipated that fine-tuning of the other encapsulated drugs or functional materials in PPy capsules would foster avenues for the development of multifunctional platforms for cancer treatments.     

Short-term training for explosive strength causes neural and mechanical adaptations

This study investigated the neural and peripheral adaptations to short-term training for explosive force production. Ten men trained the knee extensors with unilateral explosive isometric contractions (1 s 'fast and hard') for 4 weeks. Before and after training, force was recorded at 50-ms intervals from force onset (F(50), F(100) and F(150)) during both voluntary and involuntary (supramaximal evoked octet; eight pulses at 300 Hz) explosive isometric contractions. Neural drive during the explosive voluntary contractions was measured with the ratio of voluntary/octet force, and average EMG normalized to the peak-to-peak M-wave of the three superficial quadriceps. Maximal voluntary force (MVF) was also measured, and ultrasonic images of the vastus lateralis were recorded during ramped contractions to assess muscle-tendon unit stiffness between 50 and 90% MVF. There was an increase in voluntary F(50) (+54%), F(100) (+15%) and F(150) (+14%) and in octet F(50) (+7%) and F(100) (+10%). Voluntary F(100) and F(150), and octet F(50) and F(100) increased proportionally with MVF (+11%). However, the increase in voluntary F(50) was +37% even after normalization to MVF, and coincided with a 42% increase in both voluntary/octet force and agonist-normalized EMG over the first 50 ms. Muscle-tendon unit stiffness between 50 and 90% MVF also increased. In conclusion, enhanced agonist neural drive and MVF accounted for improved explosive voluntary force production in the early and late phases of the contraction, respectively. The increases in explosive octet force and muscle-tendon unit stiffness provide novel evidence of peripheral adaptations within merely 4 weeks of training for explosive force production.     

Factors related to contraction and mechanical strength of collagen gels seeded with canine endotenon cells

Fibroblasts can construct a hydrated collagen lattice to a tissue-like structure that is greatly influenced by initial culture conditions. The purpose of this study was to investigate the effects of cell concentration and collagen concentration on the contraction kinetics and mechanical properties of resultant endotenon-derived fibroblast-seeded collagen lattice. The experiment was designed to evaluate the effect of cell concentration (0, 0.25, 0.5, and 1.0 x10(6) cells/mL) and collagen concentration (0.5, 1.0, 1.5, and 2.0 mg/mL). Collagen lattice contraction was recorded for 42 days, after which time the lattices were mechanically tested. The collagen lattices seeded with higher initial cell concentration had a shorter contraction lag phase (p < 0.01), and exhibited a higher ultimate stress (p < 0.01) and instantaneous and equilibrium modulus (p < 0.01) than those seeded with a lower initial cell concentration. The collagen lattices cultured with a lower initial collagen concentration also had a shorter contraction lag phase, and exhibited greater instantaneous and equilibrium modulus (p < 0.01) than those cultured with higher initial collagen concentration. The collagen lattices of initial 0.5 mg/mL collagen concentration had the highest value of ultimate stress (p < 0.03).     

Biocompatible implants for the sustained zero-order release of narcotic antagonists.

Implantable, sustained release drug delivery devices offer benefits not obtained through oral ingestion or injection. These include delivery at a constant therapeutic rate, thus avoiding adverse intermittent and massive dose effects, as well as reliance upon patients taking their prescribed dosages. The drawbacks to their widespread acceptance have been their inability to maintain a zero-order release rate over an extended period of time and poor biocompatibility. Devices capable of satisfying these requirements have been developed and tested extensively for in vitro release of the narcotic antagonist cyclazocine. By using implant models prepared from Hydron, a hydrophilic polymer known to exhibit excellent tissue compatibility, we have found that the release rate could be precisely regulated by proper geometry, copolymer composition, concentration of ionogenic groups and cross-link density. Devices in such varied forms as capusles, barrier-film coated tablets and bulk polymerized rods have been tested in vitro for periods approaching 1 year.     

Effects of neutral sodium hydrogen phosphate on setting reaction and mechanical strength of hydroxyapatite putty.

The setting reaction and mechanical strength in terms of diametral tensile strength (DTS) of hydroxyapatite (HAP) putty made of tetracalcium phosphate, dicalcium phosphate anhydrous, and neutral sodium hydrogen phosphate (Na1.8H1.2PO4) solution containing 8 wt % sodium alginate were evaluated as a function of the Na1.8H1.2PO4 concentration. In one condition, HAP putty was placed in an incubator kept at 37 degrees C and 100% relative humidity. In the other condition, immediately after mixing HAP putty was immersed in serum kept at 37 degrees C. Longer setting times and lower DTS values were observed when HAP putty was immersed in serum regardless of the Na1.8H1.2PO4 concentration. The setting times of the HAP putty in both conditions became shorter with an increase in the Na1. 8H1.2PO4 concentration, reaching approximately 7-13 min when the Na1. 8H1.2PO4 concentration was 0.6 mol/L or higher. The DTS value of HAP putty was relatively constant (10 MPa) regardless of the Na1.8H1. 2PO4 concentration (0.2-1.0 mol/L) when HAP putty was kept in an incubator. In contrast, when HAP putty was immersed in serum, the DTS value was dependent on the Na1.8H1.2PO4 concentration. It increased with the Na1.8H1.2PO4 concentration and reached approximately 5 MPa when the Na1.8H1.2PO4 concentration was 0.6 mol/L, after which it showed a relatively constant DTS value. We therefore would recommend a HAP putty that uses 0.6 mol/L Na1.8H1. 2PO4 since at that concentration the putty's setting time (approximately 10 min) is proper for clinical use and it shows good DTS value (approximately 5 MPa) even when it is immersed in serum immediately after mixing.     

Immunoprotection of pancreatic islet grafts within artificial microcapsules.

To circumvent pancreatic islet graft-directed immune destruction we enveloped porcine islets within highly biocompatible and selectively permeable algin/polyaminoacid microcapsules. These special microspheres were deposited between the inner (permeable) and the outer (impermeable) layers of an artificial, coaxial vascular prosthesis, directly anastomized to blood vessels. Five dogs with spontaneous, insulin-dependent diabetes received microencapsulated porcine islets in arterio-vein iliac prosthesis by-passes. One showed complete and the remainder partial, sustained reversal of hyperglycemia. Microencapsulation may be a potential solution to immunological problems related to islet transplantation.     

Osmotic pressure test: a simple, quantitative method to assess the mechanical stability of alginate microcapsules

Implantation of microencapsulated, nonautologous cells and tissues is an effective method to deliver therapeutic proteins in vivo. Its success depends on the maintenance of the immunoisolating barrier provided by the microcapsule. Thus, one goal in the development of this technology is to create mechanically stable microcapsules. We have developed an osmotic pressure test to quantify the strength of microcapsules by exposing alginate microcapsules to a graded series of hypotonic solutions and quantifying the percentage of broken microcapsules. The test was validated by confirming the relative strengths of different types of alginate capsules, previously known from implantation in dogs to have differing mechanical stability in vivo. Thus, solid alginate microcapsules crosslinked with Ba(2+) were shown to be stronger than those crosslinked with Ca(2+), which in turn were shown to be stronger than the corresponding hollow alginate microcapsules. The incorporation of cells was demonstrated to reduce the mechanical stability of the microcapsules significantly. Hence, this test provides a simple and quantitative method for rapidly determining the strength of a large number of microcapsules. Thus, it is suitable for monitoring the mechanical stability of various types of microcapsules, predicting the performance of microcapsules in vivo, and for quality control of microcapsules during scale-up productions.