The chitosan prepared from crab tendons: II. The chitosan/apatite composites and their application to nerve regeneration

The chitosan tubes derived from crab tendons form a hollow tube structure, which is useful for nerve regeneration. However, in order to use the chitosan tubes effectively for nerve regeneration, there remain two problems to be solved. First, the mechanical strength of the tubes is quite high along the longitudinal axis, but is somewhat low for a pressure from side. Second, the chitosan tube walls swell to reduce the inner space of the tubes in vivo. These two problems limit the clinical use of the chitosan tubes. In this study, to solve the problems, apatite was made to react with the chitosan tubes to enhance the mechanical strength of the tube walls. Transmission electron microscopy showed that apatite crystals were formed in the walls of the chitosan tubes. The c-axis of the crystals aligned well in parallel with chitosan molecules. These results indicate that the apatite crystals grow in the tubes starting from the nucleation sites of the chitosan molecules, probably by forming complexes with amino groups of chitosan and calcium ions. Further, the tubes were thermally annealed at 120 degrees C to prevent from swelling, and simultaneously formed into a triangular shape to enhance the stabilization of the tube structure. By these treatments, the hollow tubes could keep their shape even in vivo after implantation. Animal tests using SD rats further showed that the chitosan tubes effectively induced the regeneration of nerve tissue, and were gradually degraded and absorbed in vivo.     

Structure and mechanical properties of crab exoskeletons

The structure and mechanical properties of the exoskeleton (cuticle) of the sheep crab (Loxorhynchus grandis) were investigated. The crab exoskeleton is a natural composite consisting of highly mineralized chitin–protein fibers arranged in a twisted plywood or Bouligand pattern. There is a high density of pore canal tubules in the direction normal to the surface. These tubules have a dual function: to transport ions and nutrition and to stitch the structure together. Tensile tests in the longitudinal and normal to the surface directions were carried out on wet and dry specimens. Samples tested in the longitudinal direction showed a convex shape and no evidence of permanent deformation prior to failure, whereas samples tested in the normal orientation exhibited a concave shape. The results show that the composite is anisotropic in mechanical properties. Microindentation was performed to measure the hardness through the thickness. It was found that the exocuticle (outer layer) is two times harder than the endocuticle (inner layer). Fracture surfaces after testing were observed using scanning electron microscopy; the fracture mechanism is discussed.     

Effect of chemical treatments on tendon cellularity and mechanical properties

Removal of cells may decrease the antigenicity and risk of disease transmission associated with tendon allografts and xenografts. An ideal cell removal method would not compromise graft structure and mechanical properties. This study compared the effects of three extraction chemicals [t-octyl-phenoxypolyethoxyethanol (Triton X-100), tri(n-butyl)phosphate (TnBP), and sodium dodecyl sulfate (SDS)] on tendon cellularity, structure, nativity, and mechanical properties. Rat tail tendons were soaked in extraction solutions for various time periods (12-48 h) and concentrations (0.5-2%), then they were rinsed with distilled water and ethyl alcohol. Histological analysis and tensile tests were performed on control and chemically treated tendons. Changes in collagen nativity were estimated by mechanical testing following incubation in a trypsin solution. Treatment of tendons with 1% Triton X-100 for 24 h disrupted the collagen fiber structure and did not remove cells. Treatment with 1% SDS for 24 h or 1% TnBP for 48 h resulted in an acellular tendon matrix with retention of near normal structure and mechanical properties. Consistent with previous studies demonstrating cell removal from other tissue types using SDS and TnBP, our preliminary results suggest these treatments are potentially useful for removing cells from tendon allografts or xenografts without compromising the graft structure or mechanical properties.     

Nano and micro mechanical properties of uncross-linked and cross-linked chitosan films

The aim of this study is to determine the nano and micro mechanical properties for uncross-linked and cross-linked chitosan films. Specifically, we looked at nanoindentation hardness, microhardness, and elastic modulus. It is important to study the nano and microscale mechanical properties of chitosan since chitosan has been widely used for biomedical applications. Using the solvent-cast method, the chitosan films were prepared at room temperature on the cleaned glass plates. The chitosan solution was prepared by dissolving chitosan in acetic acid 1% (v/v). Tripolyphosphate (TPP) was used to create the cross-links between amine groups in chitosan and phosphate groups in TPP. In this study, atomic force microscopy was used to measure the nanoindentation hardness and surface topography of the uncross-linked and cross-linked chitosan films. Elastic modulus was then calculated from the nanoindentation results. The effective elastic modulus was determined by microhardness with some modifications to previous theories. The microhardness of the chitosan films were measured using Vicker's hardness meter under three different loads. Our results show that the microhardness and elastic modulus for cross-linked chitosan films are higher than the uncross-linked films. However, the cross-linked chitosan films show increased brittleness when compared to uncross-linked films. By increasing the load magnitude, the microhardness increases for both uncross-linked and cross-linked chitosan films.     

壳聚糖／魔芋葡甘聚糖共混膜细胞毒性和机械性能研究

目的利用天然高分子材料壳聚糖（CS）和魔芋葡甘聚糖（KGM）制备两者含量不同比例的生物膜,测定其细胞毒性和机械性能。方法流延法制备CS与KGM含量不同比例的混合膜。并将其与人脐带间充质干细胞联合培养,观察其细胞毒性：测定其断裂伸长率、拉伸强度及吸水率。结果CS/KGM含量比例分别为3：0、2：1、1：1和1：2制备的8种共混膜,具有良好的细胞毒性：膜平均厚度0．110mm,拉伸强度为35．84～48．17MPa,断裂伸长率为4．84％-6．25％,吸水率均随着CS含量增加而增加。结论CS／KGM含量比为3：0、2：1、1：1和1：2制备的共混膜,都具有良好的细胞毒性,初步具备了引导组织再生膜的基本性能。     

Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles

Although total joint replacement has become commonplace in recent years, bacterial infection remains a significant complication following this procedure. One approach to reduce the incidence of joint replacement infection is to add antimicrobial agents to the bone cement used to fix the implant. In this in vitro study, we investigated the use of chitosan nanoparticles (CS NP) and quaternary ammonium chitosan derivative nanoparticles (QCS NP) as bactericidal agents in poly(methyl methacrylate) (PMMA) bone cement with and without gentamicin. The antibacterial activity was tested against Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). A 10(3)-fold reduction in the number of viable bacterial cells upon contact with the surface was achievable using QCS NP at a nanoparticle/bone cement weight ratio of 15%. The inhibition of S. aureus and S. epidermidis growth on the surface of the CS NP and QCS NP-loaded bone cements was clearly shown using the LIVE/DEAD Baclight bacterial viability kits and fluorescence microscopy. The CS NP and QCS NP also provided a significant additional bactericidal effect to gentamicin-loaded bone cement. The antibacterial effectiveness remained high even after the modified bone cements had been immersed for 3 weeks in an aqueous medium. No cytotoxic effect of the CS NP- and QCS NP-loaded cements was shown in a mouse fibroblast MTT cytotoxicity assay. Mechanical tests indicated that the addition of the CS and QCS in nanoparticulate form allowed the retention of a significant degree of the bone cement's strength. These results indicate a new promising strategy for combating joint implant infection.     

Model of protease I from the crab Uca pugilator

Collagenolytic protease I from the fiddler crab Uca pugilator is one of the serine proteases of the trypsin family. A graphic molecular model was built on the basis of the sequences and crystalline structures of four homologous proteins which were superimposed in order to identify structurally conserved regions. The sequence of protease I was matched to sequences of the reference proteins, without allowing any deletions or insertions in these regions. For structurally variable regions, the most similar sequences of the four reference proteins were selected. Intramolecular steric clumping due to replacement of reference side-chains by protease I side-chains were corrected by adjusting side-chain conformations. The model was optimized by energy minimization. The conformation of the primary specificity pocket for protease I predicted by the model indicated a preference for P1 hydrophobic or positively charged substrates. This prediction is consistent with biochemical findings. Because soya bean trypsin inhibitor (STI) has been shown to inhibit protease I, a tentative model of the complex was constructed and possible protease I-STI interactions were analyzed.     

Changes in the mechanical properties or rat tail tendon during postnatal ontogenesis

Summary The ultimate tensile strength, elasticity modulus and ultimate elongation of tail tendon in rats aged 1–18 months were measured with an Instron tensile apparatus. An increase in all these parameters was observed during the period of maturation, with a later levelling off of the tensile strength and the ultimate elongation. The value of the elasticity modulus attained a maximum during sexual maturation, and then decreased and stabilized.     

Improving the mechanical properties of chitosan-based heart valve scaffolds using chitosan fibers

Chitosan is being widely studied for tissue engineering applications due to its biocompatibility and biodegradability. However, its use in load-bearing applications is limited due to low mechanical properties. In this study, we investigated the effectiveness of a chitosan fiber reinforcement approach to enhancing the mechanical properties of chitosan scaffolds. Chitosan fibers were fabricated using a solution extrusion and neutralization method and incorporated into porous chitosan scaffolds. The effects of fiber/scaffold mass ratio, fiber mechanical properties and fiber length on scaffold mechanical properties were studied. The results showed that incorporating fibers improved scaffold strength and stiffness in proportion to the fiber/scaffold mass ratio. A fiber-reinforced, heart valve scaffold achieved leaflet tensile strength values of 220±17 kPa, comparable to the radial values of human pulmonary valve leaflets. Additionally, the effects of 2 mm fibers were found to be up to threefold greater than 10 mm fibers at identical mass ratios. Heparin crosslinking of fibers produced a reduction in fiber strength, and thus failed to produce additional improvements to fiber-reinforced scaffold properties. Despite this reduction in fiber strength, heparin-modified fibers still improved the mechanical properties of reinforced scaffolds, but to a lesser extent than unmodified fibers. The results demonstrate that chitosan fiber reinforcement can be used to achieve porous chitosan scaffold strength approaching that of tissue, and that fiber length and mechanical properties are important parameters in defining the degree of mechanical improvement.     

The effect of cell seeding density on the cellular and mechanical properties of a mechanostimulated tissue-engineered tendon

The initial seeding density is a critical variable in functional tissue engineering. A sufficient number of cells uniformly distributed throughout the scaffold is a key requirement to achieve homogeneous extracellular matrix deposition in vitro. However, high initial seeding densities might have negative repercussions on nutrient availability, cellular metabolism, and cell viability. In the current study, our aim was to understand the implications of using high seeding densities (3, 5, and 10 million cells/mL) in a human umbilical vein (HUV) tendon model subjected to 1?h of cyclic stretching per day at 2% strain and a frequency of 0.0167?Hz in a mechanostimulating bioreactor, on nutrient availability, cell viability and metabolism, and construct properties. Mechanostimulated constructs seeded with 3 million cells/mL had significantly higher cell number than the static controls and resulted in a 20-fold increase in proliferation rates and a 3-fold increase in tensile strength values after 1 week of culture in the bioreactor. However, higher seeding densities resulted in cell death, degraded extracellular matrix, and poorer mechanical properties. Nutrient and growth factor mass transport limitations are implicated in the inability of the decellularized HUV to support high cell numbers. The effective diffusion coefficient for glucose was measured to be 0.21±0.04?cm(2)/day. In the absence of convective flow, proteins and growth factors with a molecular radius larger than 4.9?nm could not diffuse through the HUV. Cells seeded in the HUV consumed 10.5±0.5?ng/cell/day of glucose. Glucose diffusion coefficient and glucose consumption rates in the HUV indicated the presence of glucose mass transport limitations when cell seeding densities exceed 3 million cells/mL.     

Experimental measurement and modeling analysis on mechanical properties of tensor tympani tendon

In this paper, we report mechanical properties of the tensor tympani tendon of human ear measured from uniaxial tensile, stress relaxation and failure tests. The hyperelastic Ogden model and digital image correlation method were employed to analyze experimental data. The constitutive equation of the tendon was derived through data iteration processes, and Young's modulus was presented as a function of stress. The viscoelastic property of the tendon was described by stress relaxation function and hysteresis. Furthermore, three-dimensional finite element analysis was carried out on five tendon models to investigate relationship between the structure and properties. The dimensions of the tendon were also measured by image processing techniques and presented with statistic significance. The structure and properties of the tensor tympani tendon reported in this study add new data into the study of ear tissue biomechanics.     

Leaching and mechanical properties characterization of dental composites

The objective of this study was to determine the leaching of Ba, Si, and Sr from four dental composites: Restolux (RX), Micronew (M), Renew (RW), and Choice (C) and to correlate the effects of such leaching with flexure strength and modulus of elasticity. The specimens were 3 x 3 x 25-mm bars, polished with 120- and 240-grit SiC paper, and were aged for 4, 6, and 8 months in artificial saliva, distilled water, and a 50/50 mixture of ethanol and distilled water. Inductively coupled plasma with mass spectroscopy (ICP/MS) was used to determine the ion concentrations after aging. The greatest release of Ba and Sr occurred after aging in 50/50 volume fraction mixture of ethanol/water and for Si in artificial saliva. The 50/50 solution caused a breakdown of the resin and glass matrix, resulting in an increase of Ba and Sr, whereas aging in artificial saliva resulted in an ion charge balance, which allowed an elevated and continuous release of Si. The flexure strength and the elastic modulus showed a gradual decrease over time, with the greatest decreases occurring following aging in the 50/50 water/ethanol solution, but little correlation with the leaching of the filler ions.     

Morphology and mechanical properties of chitosan fibers obtained by gel-spinning: influence of the dry-jet-stretching step and ageing

The morphology and mechanical properties of chitosan fibers obtained by gel-spinning are reported. The objectives were both to understand how the microstructure of the fibers could be modified and how the mechanical properties were improved by means of a dry-jet-stretching step. A highly deacetylated chitosan (degree of acetylation=2.7%) from squid-pens, with a high weight-average molecular weight (M(w)=540,000g/mol) was dissolved in an aqueous acetic acid solution, spun using gaseous ammonia as the coagulant, and then directly dried under hot air. A "post-drying" of 1week was necessary to stabilize the fibers in ambient atmosphere. A dry-jet-stretching ratio applied during the monofilament coagulation (maximal value of 4.3) allowed us to increase the density and favor the orientation of chains along the fiber axis. This allowed us to improve the mechanical properties of the fibers (Young's modulus of 82g/denier and tenacity of 2g/denier). The ageing in ambient atmosphere played an important role in the crystalline microstructure in relation to: the kinetics of ammonium acetate hydrolysis, the formation of a weak fraction of the anhydrous allomorph of chitosan, and an increase of the crystallinity index, whereas the Young's modulus was increased and the tenacity was slightly lowered. In addition, gel-jet-stretched or dry-jet-stretched fibers could be stored at least 3months in ambient atmosphere without any significant degradation.     

明胶联合壳聚糖纤维对磷酸钙骨水泥力学性能的影响

背景：已有多种纤维被用于提高磷酸钙骨水泥的强度及抗断裂性能。 目的：了解明胶联合壳聚糖纤维对磷酸钙骨水泥力学性能的影响,寻找较为合适的配比。 方法：采用2×4析因设计,将质量比为0(蒸馏水),5%的明胶,体积比为0,10%,30%和50%的壳聚糖纤维分别混入磷酸钙骨水泥,检测复合物的抗弯曲强度,扫描电子显微镜观察各组试样断口形态并进行电子能谱分析。 结果与结论：各明胶组间抗弯强度差异有非常显著性意义(P < 0.001);各体积比纤维间抗弯强度差异有非常显著性意义(P < 0.001),其中5%明胶和30%壳聚糖纤维构成的复合物抗弯曲强度最大,达 12.31 MPa。以蒸馏水为液相的磷酸钙骨水泥固化后,表面可见不规则颗粒,平均微孔直径小于5 μm,添加明胶后颗粒似乎黏在一起,微孔直径与前者相似,但是数目少于前者。磷酸钙骨水泥-5%明胶-30%纤维复合物的断口扫描可见拔出纤维的表面黏附有大量颗粒,磷酸钙骨水泥-蒸馏水-30%纤维复合物拔出纤维表面的颗粒明显减少。表明明胶与壳聚糖纤维可提高磷酸钙骨水泥的抗弯曲强度,5%明胶和30%壳聚糖纤维为这种增强模式较为合适的比例。     

Role of storage on changes in the mechanical properties of tendon and self-assembled collagen fibers

Fibrous collagen networks are the major elements that provide mechanical integrity to tissues; they are composed of fiber forming collagens in combination with proteoglycans (PGs). Using uniaxial tensile tests we have studied the viscoelastic mechanical properties of rat tail tendon (RTT) fibers and self-assembled collagen fibers that were stored at 22 degrees C and 1 atm of pressure. Our results indicate that storage of RTT and self-assembled type I collagen fibers results in increased elastic and viscous components of the stress-strain behavior consistent with the hypothesis that storage causes the introduction of crosslinks. Analysis of the elastic and viscous mechanical data suggests that the elastic constant of the collagen molecule in RTT is about 7.7 GPa. Measurement of the viscous component of the stress-strain curves for RTTs and self-assembled collagen fibers suggests that PGs may increase the viscous component and effectively increase the collagen fibril length.