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.

In vitro bioactivity of different degree of deacetylation chitosan, a potential coating material for titanium implants.

Clinical treatment of orthopaedic tissue injuries often involves the use of titanium and titanium alloys with considerable research focusing on the surface modification of these materials. Chitosan, the partly deacetylated form of chitin, is one of the materials under investigation as surface coating for orthopaedic implants in order to improve osteo-integration and cellular attachment. In this study, we determined the effects of the degree of deacetylation (DD) of chitosan membranes on attachment, proliferation and osteogenic differentiation of MC3T3-E1 mouse preosteoblasts. Chitosan membranes were coated with fibronectin to promote biocompatibility and cellular attachment. Membranes were characterized in terms of wettability and surface topography using water contact angle measurements and atomic force microscopy. The results in this study indicate that the surface roughness and fibronectin adsorption increase with increased DD. A higher DD also facilitates attachment and proliferation of cells, but no induction of spontaneous osteogenic differentiation was observed. Lower DD chitosan membranes were successfully prepared to sustain attachment and were modified by crosslinking with glutaraldehyde to promote long-term studies. The chitosan membranes used in this study are suitable as a potential coating for titanium implants.

Chitosan and Chitin Hexamers affect expansion and differentiation of mesenchymal stem cells differently.

Chitooligosaccharides are of interest as potential drugs due to their bioactivity and water solubility. We compared the effect of acetylated and deacetylated chitooligomers (Hexamers) on short-term expansion (7 days) and osteogenic differentiation of bone-marrow derived, human mesenchymal stem cells in terms of gene expression, cytokine secretion and quality of osteogenic differentiation. We show that chitooligomers affect hMSC gene expression and cytokine secretion, but not mineralization. The effect of chitooligomers was shown to be dependent on the acetylation degree, with significantly stronger effects when cells are stimulated with chitin-derived Hexamers (N-Acetyl Chitohexaose) than with Chitosan Hexamers (Chitohexaose).

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus.

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiADeltasp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiADeltasp were 70 degrees C and 4.5-5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70 degrees C, with half-lives of 3 h at 90 degrees C and 45 min at 95 degrees C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl2, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.

Sequence analysis of chitooligosaccharides by matrix-assisted laser desorption ionization postsource decay mass spectrometry.

Chitin/chitosan oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D-glucopyranose (GlcN) were prepared by chemical degradation of chitin or chitosan and separated by gel permeation chromatography. Oligosaccharides obtained after enzymatic hydrolysis of chitosan [F(A) 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The composition of a hexamer D3A3 was ca. 65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X not equal Y) are mutually either D or A. This structure motif was also observed in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)(3)-D-A-A.