Mineralization in a Critical Size Bone-Gap in Sheep Tibia Improved by a Chitosan-Calcium Phosphate-Based Composite as Compared to Predicate Device.

Deacetylated chitin derivatives have been widely studied for tissue engineering purposes. This study aimed to compare the efficacy of an injectable product containing a 50% deacetylated chitin derivative (BoneReg-Inject™) and an existing product (chronOS Inject®) serving as a predicate device. A sheep model with a critical size drill hole in the tibial plateau was used. Holes of 8 mm diameter and 30 mm length were drilled bilaterally into the proximal area of the tibia and BoneReg-Inject™ or chronOS Inject® were injected into the right leg holes. Comparison of resorption and bone formation in vivo was made by X-ray micro-CT and histological evaluation after a live phase of 12 weeks. Long-term effects of BoneReg-Inject™ were studied using a 13-month live period. Significant differences were observed in (1) amount of new bone within implant (p < 0.001), higher in BoneReg-InjectTM, (2) signs of cartilage tissue (p = 0.003), more pronounced in BoneReg-InjectTM, and (3) signs of fibrous tissue (p < 0.001), less pronounced in BoneReg-InjectTM. Mineral content at 13 months postoperative was significantly higher than at 12 weeks (p < 0.001 and p < 0.05, for implant core and rim, respectively). The data demonstrate the potential of deacetylated chitin derivatives to stimulate bone formation.

Bone remodeling effect of a chitosan and calcium phosphate-based composite.

Chitosan is a biocompatible polymer that has been widely studied for tissue engineering purposes. The aim of this research was to assess bone regenerative properties of an injectable chitosan and calcium phosphate-based composite and identify optimal degree of deacetylation (%DDA) of the chitosan polymer. Drill holes were generated on the left side of a mandible in Sprague-Dawley rats, and the hole was either left empty or filled with the implant. The animals were sacrificed at several time points after surgery (7-22 days) and bone was investigated using micro-CT and histology. No significant new bone formation was observed in the implants themselves at any time points. However, substantial new bone formation was observed in the rat mandible further away from the drill hole. Morphological changes indicating bone formation were found in specimens explanted on Day 7 in animals that received implant. Similar bone formation pattern was seen in control animals with an empty drill hole at later time points but not to the same extent. A second experiment was performed to examine if the %DDA of the chitosan polymer influenced the bone remodeling response. The results suggest that chitosan polymers with %DDA between 50 and 70% enhance the natural bone remodeling mechanism.

Partially acetylated chitooligosaccharides bind to YKL-40 and stimulate growth of human osteoarthritic chondrocytes.

Recent evidences indicating that cellular kinase signaling cascades are triggered by oligomers of N-acetylglucosamine (ChOS) and that condrocytes of human osteoarthritic cartilage secrete the inflammation associated chitolectin YKL-40, prompted us to study the binding affinity of partially acetylated ChOS to YKL-40 and their effect on primary chondrocytes in culture. Extensive chitinase digestion and filtration of partially deacetylated chitin yielded a mixture of ChOS (Oligomin™) and further ultrafiltration produced T-ChOS™, with substantially smaller fraction of the smallest sugars. YKL-40 binding affinity was determined for the different sized homologues, revealing micromolar affinities of the larger homologues to YKL-40. The response of osteoarthritic chondrocytes to Oligomin™ and T-ChOS™ was determined, revealing 2- to 3-fold increases in cell number. About 500 µg/ml was needed for Oligomin™ and around five times lower concentration for T-ChOS™, higher concentrations abolished this effect for both products. Addition of chitotriose inhibited cellular responses mediated by larger oligosaccharides. These results, and the fact that the partially acetylated T-ChOS™ homologues should resist hydrolysis, point towards a new therapeutic concept for treating inflammatory joint diseases.

Endotoxins affect bioactivity of chitosan derivatives in cultures of bone marrow-derived human mesenchymal stem cells.

Biomaterials research has been expanding over the last decade, in part to provide improved medical devices for the treatment of orthopedic tissue injuries. In the quest to provide the best performance combined with low cost for medical implants, an increasing number of non-chemists have entered the field of biomaterials research without the profound knowledge of chemistry needed to understand the complex interaction mechanisms and characteristics of natural substances. Likewise, non-biologists often lack understanding when it comes to the presence of the contaminating biota frequently found in natural substances. This lack of knowledge by researchers in the field, combined with sensitive in vitro cell-based assays, can lead to inaccurate evaluation of biomaterials. Hence, there should be both an active effort to assemble multi-disciplinary teams and a genuine concern for the possible effects of contamination on in vitro assays. Here, we show that the presence of bacterial endotoxins in chitosan derivatives can result in false-positive results, profoundly altering product performance in in vitro assays. False-positive results through uncritical use of natural substances in vitro can be avoided by proper endotoxin testing and careful evaluation of cytokine secretion patterns.

Selection of a practical assay for the determination of the entire range of acetyl content in chitin and chitosan: UV spectrophotometry with phosphoric acid as solvent.

The rapidly expanding use of chitomaterials in biomedical applications demands accurate and precise analytical methods to determine physico-chemical characteristics, especially the acetyl content of the sample. The analytical methods available for the determination of the acetyl content of the biomaterials are quite different in efficiency, accuracy and precision. Out of 22 analytical methods reviewed, XRD, DSC, FTIR (KBr pellet), solid state (13)C CP/MAS NMR, and acid hydrolysis-HPLC and the spectrophotometry assay using phosphoric acid as solvent (PUV) were selected in this study. The validity and applicability of these methods were investigated with a wide range of chitin and chitosan samples varying acetyl content, preparation methods, and sources. The XRD, DSC, and FTIR (KBr pellet) methods showed poor accuracy with the samples of diverse preparations and sources. The PUV method was modified and accuracy of the method was examined against absolute methods: solid state (13)C CP/MAS NMR and acid hydrolysis-HPLC methods. The correlations between these three methods were >0.9. Therefore, the PUV method was selected as the most generally acceptable method based on its accuracy, reliability, simplicity, and instrument availability.

Evaluation of an improved acid hydrolysis-HPLC assay for the acetyl content in chitin and chitosan.

Functional properties of the amino polysaccharides, chitin and chitosan, vary significantly with their acetyl content. The acid hydrolysis-HPLC method offers good accuracy and precision to assay the acetyl content regardless of the solubility of the sample. In this research, the hydrolysis parameters were changed, and the analytical method was counterchecked with other methods. Complete hydrolysis was achieved by mixing chitosan with 1.4 mM oxalic acid and 12 M sulfuric acid followed by treatment at 110 degrees C for 40 min. A sealed glass ampule was used instead of a vacuum hydrolysis tube. The acetic acid released during acid hydrolysis was measured quantitatively by HPLC. A high correlation (r(2) = 0.98) was obtained between the modified HPLC assay and the solid-state (13)C CP/MAS NMR method for the samples of various crustacean sources with a wide range of acetyl contents. The modified HPLC method was also highly correlated (r(2) = 0.99) with the first derivative UV method for soluble chitosan.

Functional characteristics of shrimp chitosan and its membranes as affected by the degree of deacetylation.

The functional properties of three shrimp chitosan preparations with different degrees of deacetylation (75%, 87% and 96% DD) but with a constant molecular weight (about 810 kDa) were investigated. Chitosan with 75% DD had a 1.5 times higher water absorption, probably due to its 20% lower level of crystallinity. Membranes cast from this chitosan also exhibited 1.5 times more water absorption and 2 times higher permeability. However, chitosan with 87% and 96% DD had 1.5-2 times higher absorption of fat and the orange II dye. This is attributed to the higher content of positively charged amine groups in the polymer. Cast into membrane, chitosan of higher degree of deacetylation showed a higher tensile strength and a higher elongation at break, probably due to the higher level of crystallinity.

Size-controlled hydroxyapatite nanoparticles as self-organized organic-inorganic composite materials.

This paper presents some results concerning the size-controlled hydroxyapatite nanoparticles obtained in aqueous media in a biopolymer matrix from soluble precursors salts. Taking the inspiration from nature, where composite materials made of a polymer matrix and inorganic fillers are often found, e.g. bone, shell of crustaceans, shell of eggs, etc., the feasibility on making composite materials containing chitosan and nanosized hydroxyapatite was investigated. A stepwise co-precipitation approach was used to obtain different types of composites by means of different ratio between components. The synthesis of hydroxyapatite was carried out in the chitosan matrix from calcium chloride and sodium dihydrogenphosphate in alkaline solutions at moderate pH of 10-11 for 24 h. Our research is focused on studying and understanding the structure of this class of composites, aiming at the development of novel materials, controlled at the nanolevel scale. The X-ray diffraction technique was employed in order to study the kinetic of hydroxyapatite formation in the chitosan matrix as well as to determine the HAp crystallite sizes in the composite samples. The hydroxyapatite synthesized using this route was found to be nano-sized (15-50 nm). Moreover, applying an original approach to analyze the (002) XRD diffraction peak profile of hydroxyapatite by using a sum of two Gauss functions, the bimodal distribution of nanosized hydroxyapatite within the chitosan matrix was revealed. Two types of size distribution domains such as cluster-like (between 200 and 400 nm), which are the habitat of ''small'' hydroxyapatite nanocrystallites and scattered-like, which are the habitat of ''large'' hydroxyapatite nanocrystallites was probed by TEM and CSLM. The structural features of composites suggest that self-assembly processes might be involved. The composites contain nanosized hydroxyapatite with structural features close to those of biological apatites that make them attractive for bone tissue engineering applications.

Characterization of decrystallized chitosan and its application in biosorption of textile dyes.

Decrystallized chitosan was produced from shrimp shells with a low degree of crystallinity (10%) and a high anionic dye binding capacity. Raw, mixed dye wastewater from a textile factory was efficiently decolorized using decrystallized chitosan that was more efficient than using normal chitosan and activated carbon. Decolorization reached 90% within 10 min and could be carried out from pH 4.5 to 8.1. Decrystallized chitosan can be regenerated by 2 M H2SO4 and was reusable more than 10 times. It is, therefore, an attractive candidate for the removal of dyes from textile wastewater.

Quantification and characterization of insoluble chitinous materials in viscous chitosan solutions.

Insoluble chitinous materials in highly viscous chitosan solutions can be quantified using the viscosity-lowering action of transglucosidase (EC 2.4.1.24). In chitosan, commonly produced by high temperature deacetylation (90 degrees C), between 70-90% of insoluble chitinous materials were recovered by this enzymatic method whereas only 25% recovery was obtained by the nitrous acid method. The insoluble material recovered after enzyme treatment had a higher degree of deacetylation and a lower degree of crystallization than that after nitrous acid treatment. The results are explained by difference in penetration by enzyme and nitrous acid into the insoluble particle.