Fungistatic activity of modified chitosans against Saprolegnia parasitica.

Five chemically modified chitosans were tested for their antifungal activities against Saprolegnia parasitica by the radial growth assay in chitosan-bearing agar, and the fungal growth assay in chitosan-bearing broth. Results indicated that methylpyrrolidinone chitosan, N-carboxymethyl chitosan and N-phosphonomethyl chitosan exerted effective fungistatic action against S. parasitica: in fact the radial growth was nil for 50 h at 20 degrees C, and the fungus was precipitated when the Bacto YM broth contained one of these chitosans. Electron microscopy observations (TEM and SEM) provided evidence of ultrastructural alterations, damaged fungal structures, uptake of modified chitosans, and hyphal distortion and retraction.

N,N-dicarboxymethyl chitosan as delivery agent for bone morphogenetic protein in the repair of articular cartilage.

Bone morphogenetic protein (BMP), associated with N,N-dicarboxymethyl chitosan, is used to induce or facilitate the repair of articular cartilage lesions. This association is intended for the synergistic potentiation of the respective biological effects. Data show that BMP-7 enhances the in vivo proliferation of cells with chondrocytes phenotype in the articular environment, leading to partial healing of the articular surface of the lesions. N,N-dicarboxymethyl chitosan is found to be useful as a molecular carrier or drug delivery agent.

Osteoblast behaviour in the presence of bisphosphonates: ultrastructural and biochemical in vitro studies.

OBJECTIVE: A positive balance in bone remodelling is an important goal of bone metabolism both in the presence of the osteoporotic processes characteristic of ageing and, especially, of prosthetic implants. The aim of the present work was to obtain new information about the initial steps of osteoblastic growth in an in vitro osteoblastic model in the presence of two bisphosphonates. METHODS: Experiments were performed with Alendronate and Neridronate, two molecules used in the therapy of osteoporosis. Since differentiating features into osteoblastic cells are known to parallel the presence in the cytoplasm of alkaline phosphatase and osteocalcin, we also carried out immunohistochemical typing. RESULTS: Good differentiation and osteoblastic activity were generally observed in the cells in contact with these compounds, except for 10(-4) Neridronate, where biochemical data clearly indicated its toxic effect on the cells. CONCLUSION: The detection of osteoblastic markers associated with an ultrastructural picture of correct organellar morphology in our cultures further supports the hypothesis of a metabolically positive action of these molecules on osteoblasts.

Native, industrial and fossil chitins.

Countless living organisms continuously synthesize and degrade chitin enzymatically, for nutritional, morphogenetic and defensive or aggressive purposes. Chemically modified chitins are important in the light of their biochemical significance in medicine and crop protection; their environmentally friendly behaviour permits industrial exploitation of the huge chitinous biomasses generated by fishing activities and biotechnology. Chitin is promptly metabolized in sediments, and fossil chitin is not frequently encountered.

Analytical biochemistry and clinical significance of N-acetyl-beta-D-glucosaminidase and related enzymes.

Human N-acetyl-beta-D-glucosaminidase, N-acetyl-alpha-D-glucosaminidase, endo-beta-N-acetylglucosaminidase, hexosaminidase, beta-N-acetylgalactosaminidase and glucocerebrosidase have not been so widely studied as the beta-N-acetylhexosaminidases in bacteria, fungi and arthropods. Their biochemical role has been elucidated, however, and their urinary and plasma determination is being adopted for the early detection of diseases before clinical manifestation, in particular for hypertension, renal injuries and disorders, depression and lysosomal storage diseases. The spectrophotometric determinations of N-acetyl-beta-D-glucosaminidase, most often done with 3-cresolsulphone phthaleinyl N-acetyl-beta-D-glucosaminide, have been recently simplified and adapted to automatic instruments.

Biochemistry, histology and clinical uses of chitins and chitosans in wound healing.

Biodegradability, biocompatibility and capacity to promote the synthesis of hyaluronan are main characteristics of chitin-derived wound healing materials, whose biological significance in the human body depends largely on the actions that certain hydrolases exert on them. The resulting chitooligomers stimulate various cells, while the released monomers are phosphorylated and incorporated into hyaluronan, keratan sulphate and chondroitin sulphate, components of the intracellular matrix and connective tissue. The healing process favoured by these materials is examined in terms of macrophage activation, cytokine production by macrophages and fibroblasts, antiinflammatory action, angiogenesis stimulation, granulation and scar formation. Current biomedical applications are illustrated by the treatment of leg ulcers, the use of skin substitutes, and the regeneration of bone, nerve and meniscus tissues.

Microparticulate drug delivery systems.

Chitosan was proposed as a drug carrier for mucosal administration in ocular, buccal, nasal, gastroenteric and vaginal-uterine therapies based on its bioadhesive properties and biodegradability in vivo under the action of hydrolases. Examples are the delivery of acyclovir via ocular administration, and the delivery of 5-aminosalicylic acid to the colon. Microparticles may need to be cross-linked to retard their degradation in acidic media; yet cross-linking with glutaraldehyde introduces cytotoxic characteristics and depresses bioadhesion. Alternative cross-linking approaches are discussed along with the suitability of chitosan for the oral delivery of vaccines.

Clinical and biochemical evaluation of chitosan for hypercholesterolemia and overweight control.

After providing basic information on enzymes involved in cholesterol homeostasis, and on management of hypertriglyceremia and hypercholesterolemia, with the aid of cholestyramine and fibric acid, this chapter examines the effects of the ingestion of chitosan. Dietary chitosan is effective on serum cholesterol and in atherosclerosis in normal and diabetic mice, and lends itself to the treatment of hypercholesterolemia in humans. It also exhibits antiulcer, antiarthritic, antihypertension and antiuricemic properties. The published human trials, analysed statistically, further indicate that chitosan is effective to control overweight when associated to a diet. This chapter discusses several issues raised against the use of chitosan, namely, depletion of zinc and liposoluble vitamins, as well as advantages such as enhanced absorption of nutrients and competitive inhibition of lipases. It also directs attention to the unexplored areas of fungal and algal chitosans, and the use of chitins instead of chitosans.

Bioactivity of chitosan in dentistry. Preliminary data on chitosan-based cements.

BACKGROUND: The chemical association of chitosan with inorganic salts, such as calcium phosphate, finds a promising application in dentistry as room-temperature self-hardening cement. We present the physical, chemical and crystallographic characterization of newly-developed cements made of 1) calcium-phosphate and a chitosan gel obtained by acetic acid treatment, and 2) calcium phosphate and a chitosan gel obtained by ascorbic acid treatment. Both cements are self-hardening at room temperature. METHODS: The cements were characterized by X-ray diffractography, scanning electron microscopy and fluorine-selective electrode analysis. RESULTS: The chitosan-hydroxyapatite cements had hardness comparable to spongy bone and above that of PMMA. CONCLUSIONS: The cements are promising for application in endodontics and restorative dentistry.

Analysis of practice-role perceptions of physical therapy, occupational therapy, and speech-language therapy students.

The purpose of this study was to determine whether physical therapy (PT), occupational therapy (OT), and speech-language therapy (SLP) students shared common perceptions of the practice roles of the three disciplines. The survey instrument used in this study contained 55 questions that addressed practice-role perceptions. The questions were based on a case study. A total of 172 undergraduate students (PT 71, OT 52, SLP 49) from a southeastern university participated. Chi-square test of association was used to analyze the data. Results showed that PT, OT, and SLP students shared common perceptions of administrative and educational practice roles but differed on their perceptions of assessment and physical/mental treatment roles. Practice-role confusion was particularly acute between OT and PT and between OT and SLP students in these areas.

Preparation and characterization of ampicillin loaded methylpyrrolidinone chitosan and chitosan microspheres.

Ampicillin was embedded in microparticles made of a new derivative of chitosan: methylpyrrolidinone chitosan. They were prepared using different drug-to-polymer weight ratios and by a spray-drying technique. Spray-dried drug-loaded chitosan microspheres were prepared for comparison. The microparticles were characterized by scanning electron microscopy (SEM), particle size analysis, differential scanning calorimetry (DSC) and in vitro drug release. Microbiological assay was performed using different bacterial strains. Spray-dried microspheres of almost spherical shape, smooth surface and narrow size distribution were always obtained. Ampicillin loaded into both polymer matrices showed amorphous behaviour as determined by DSC. Drug-loaded microspheres resulted to control the drug release in a 30-120 min range, depending on chitosan type. Thermal denaturation of the microspheres does not modify drug release rate. The results of the microbiological assay show that the loading of ampicillin into chitosans is able to maintain or improve the anti-bacterial activity of the drug.

Bioactivity modulation of bioactive materials in view of their application in osteoporotic patients.

The application of bioactive ceramic coatings to prostheses confers strength to a material (ceramic or biological glass) that exerts beneficial effects on bone-tissue growth but that itself lacks the toughness and stability required of an implant device. The rate of bioactivity is related to the chemical reactivity of the material and causes interface dissolution, precipitation and ion-exchange reactions. Ceramics may differ in sintering temperature and thus exhibit differences in their in vitro dissolution features and in vivo performance. To test these effects, in vitro and in vivo studies were carried out on two biocompatible biological glasses and a ceramic of proven bioactivity in view of their potential utilization as covering materials. In addition, a modified chitosan was adsorbed on the surface of a series of hydroxyapatite (HA) samples. Human fibroblasts and/or osteoblasts were used for the in vitro tests, and normal (INT) and osteoporotic (OVX) rats, normal rabbits and sheep for the in vivo studies. Similar chemical changes were observed in both glasses, suggesting that these materials underwent modifications directly dependent on their biological environment. The in vivo tests point to the possibility of improving the bioactivity of ceramic substrates with chitosan. However, the different behaviour of the materials in vitro and in vivo suggests that these tests should be conducted in parallel.

Effect of low-molecular-weight chitosans on the adhesive properties of oral streptococci.

It was previously shown that a low-molecular-weight chitosan and its derivatives N-carboxymethyl chitosan and imidazolyl chitosan inhibit Streptococcus mutans adsorption to hydroxyapatite. The ability of the same molecules to interfere with adhesive properties of other oral streptococci (Streptococcus sanguis, Streptococcus gordonii, Streptococcus constellatus, Streptococcus anginosus, Streptococcus intermedius, Streptococcus oralis, Streptococcus salivarius, Streptococcus vestibularis) was tested. When saliva-coated or -uncoated hydroxyapatite beads were treated with N-carboxymethyl chitosan, a reduction varying from 60% to 98% depending on strains was observed. Low-molecular-weight chitosans and imidazolyl chitosan did not have any effect. Growth in N-carboxymethyl chitosan-supplemented medium (final concentrations ranging from 20 to 500 micrograms.ml-1) caused a dose related reduction in the adsorption of all strains to hydroxyapatite and in their affinity towards xylene. No effect was observed with low-molecular-weight chitosans and imidazolyl chitosan. In contrast to what observed with S. mutans, the three polysaccharides did not affect detachment from hydroxyapatite beads and adherence to cheek epithelial cells of the other streptococci. These results suggest that low-molecular-weight chitosans and/or imidazolyl chitosan, selectively affecting S. mutans adsorption to hydroxyapatite, may be very interesting as potential anti-dental caries agents.

Human enzymatic activities related to the therapeutic administration of chitin derivatives.

Three cases are presented where modified chitins have been extensively administered to volunteers, as dressings for wounded soft and bone tissues, as anticholesterolemic dietary foods, and in the controlled delivery of anti-inflammatory drugs. The interactions of the modified chitins with human enzymes is critically examined. In the context of drug carrier resorption and wound healing, chitooligomers and monomers, generated by lysozyme, N-acetylglucosaminidase and human chitinase, activate macrophages and stimulate fibroblasts, respectively; the effects are production of smooth, vascularized and physiologically normal tissues. In the dietary food area, lipase, amylase, 3-hydroxy-3-methylglutaryl CoA reductase, glucokinase and the enzymes of prostaglandin synthesis are involved in the oral administration of chitosan: lipid adsorption is depressed mainly because of the physical form of the chitosan-lipid aggregates, which are unsuitable as substrates. When chitosan is used as a drug carrier, chitosan-drug complexes are present. The uniqueness of chitosan among polysaccharides is underlined in terms of susceptibility to enzymatic depolymerization, cationicity, supply of cell-activating oligomers, and supply of N-acetylglucosamine for rebuilding of other biopolymers. Advances in molecular recognition and biocompatibility are also presented.

Inhibition of Streptococcus mutans adsorption to hydroxyapatite by low-molecular-weight chitosans.

The role of Streptococcus mutans in the initiation of dental caries has been recognized and attributed, at least in part, to its ability to colonize the tooth surface. Therefore, factors which prevent S. mutans attachment to hydroxyapatite (HA) are of considerable interest for the prophylaxis of this infectious disease. Chitosan, a chitin derivative by N-deacetylation, is an interesting candidate in this respect, since it stimulates the ordered regeneration of oral soft tissues, prevents the deleterious action of organic acid, and exhibits bactericidal action against several pathogens. In the present work, the efficacy of a low-molecular-weight chitosan (LMWC) and its derivatives N-carboxymethyl chitosan (NCMC) and imidazolyl chitosan (IMIC) in preventing S. mutans attachment to HA beads was assessed. The effects of chitosan on both sucrose-dependent and -independent adherence were evaluated. In both cases, when saliva-coated or uncoated HA beads were treated with any of the chitosans, a reduction in S. mutans adsorption ranging from 47 to 66% was observed. When HA beads were coated with saliva after the treatment with chitosan, neither carbohydrate caused a statistically significant reduction in S. mutans adsorption, suggesting that saliva deposition restores HA binding properties. Bacteria grown in the presence of chitosan subminimal inhibitory concentrations (sub-MICs) ranging from 12 to 500 micrograms mL-1 adsorbed poorly to HA and exhibited a lower affinity toward xylene than untreated controls. In the presence of chitosan sub-MICs up to 60 micrograms mL-1, an increase in the percentage of detached bacteria from two- to nine-fold was observed. The desorptive effect of chitosan was weaker when S. mutans had adhered to saliva-coated HA in the presence of sucrose. These results demonstrate that the presence of minor amounts of modified chitosans prevents S. mutans adsorption to HA and suggest that colonization of the tooth surface might be impaired by the use of toothpastes, mouthrinses, or chewing gums containing any of the tested polysaccharides.

Stimulatory effect on bone formation exerted by a modified chitosan.

A novel modified chitosan carrying covalently linked imidazole groups (average molecular weight 700,000, degree of substitution 0.28, degree of acetylation 0.08) was used to stimulate bone formation in an animal model. Lesions (7 mm diameter) were surgically made in the femoral condyle of sheep and treated with the modified chitosan. Within 40 d after surgery, the neoformed tissue occluded the surgical hole and assumed a trabecular structure in the peripheral area of the lesion, while looking like a mineralization nodule in the central part in association with a fibrous component. In the control, no sign of osteoinduction or reparative process was observed and bone marrow was rich in adipocytes.

Solubility and structure of N-carboxymethylchitosan.

N-Carboxymethylchitosan from crab and shrimp chitosans was obtained in water-soluble form by proper selection of the reactant ratio, i.e. using equimolar quantities of glyoxylic acid and amino groups. HPLC determinations of glyoxylic and glycolic acids, in conjunction with NMR analysis, permitted identification of the structure of the product, which is partly N-mono-carboxymethylated (0.3), N,N-dicarboxymethylated (0.3) and N-acetylated depending on the level of deacetylation of the starting chitosan (0.08-0.15). The preparation can be made successfully even in the presence of large concentrations of glycolic acid. The use of enzymes exerting hydrolysing activity on the high-molecular-weight fractions helps to avoid gel formation during storage and precipitate formation on addition of anti-microbial agents.

Chitin-based poly(urea-urethane)s.

Chitins of various origins in DMA-LiCl solution have been reacted with excess 1,6-diisocyanatohexane (three or twelve equivalents per repeating unit) for 4-20 h. The resulting solutions were exposed to water vapor for 2 days and flexible and opaque materials were produced, which upon drying yielded powders whose main characteristics were insolubility in aqueous and organic solvents, remarkable crystallinity, typical infrared spectrum, high N/C ratio (0.287), and a high degree of substitution (0.29). Under the SEM structural features reminiscent of chitin were absent but no thermoplastic behavior was found by differential scanning calorimetry. Chitosan was similarly treated under heterogeneous conditions in anhydrous pyridine, and yielded reaction products with a lower degree of substitution (0.17). With partially hydrolysed chitosan, highly crystalline products were obtained.