Three-dimensional hierarchical composite scaffolds consisting of polycaprolactone, β-tricalcium phosphate, and collagen nanofibers: fabrication, physical properties, and in vitro cell activity for bone tissue regeneration

β-Tricalcium phosphate (β-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to β-TCP's inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/β-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the β-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter = 160 nm) were well layered between the composite struts. By accommodating the β-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/β-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/β-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and β-TCP particles in the scaffold provide good synergistic effects for cell activity.     

Ectopic Osteoid and Bone Formation by Three Calcium-Phosphate Ceramics in Rats,Rabbits and Dogs

Calcium phosphate ceramics with specific physicochemical properties have been shown to induce de novo bone formation upon ectopic implantation in a number of animal models. In this study we explored the influence of physicochemical properties as well as the animal species on material-induced ectopic bone formation. Three bioceramics were used for the study: phase-pure hydroxyapatite (HA) sintered at 1200°C and two biphasic calcium phosphate (BCP) ceramics, consisting of 60 wt.% HA and 40 wt.% TCP (β-Tricalcium phosphate), sintered at either 1100°C or 1200°C. 108 samples of each ceramic were intramuscularly implanted in dogs, rabbits, and rats for 6, 12, and 24 weeks respectively. Histological and histomorphometrical analyses illustrated that ectopic bone and/or osteoid tissue formation was most pronounced in BCP sintered at 1100°C and most limited in HA, independent of the animal model. Concerning the effect of animal species, ectopic bone formation reproducibly occurred in dogs, while in rabbits and rats, new tissue formation was mainly limited to osteoid. The results of this study confirmed that the incidence and the extent of material-induced bone formation are related to both the physicochemical properties of calcium phosphate ceramics and the animal model.     

Functionalization of biomimetic calcium phosphate bone cements with alendronate

Bisphosphonates (BPs) are widely employed for the treatment of a variety of bone disorders. We have previously successfully added small amounts of BPs into calcium phosphate bone cements in order to enhance their bio-functionality. In this work we were able to increase greatly the amount of BP introduced in the cement, thanks to suitable modifications of composition. In particular, we utilized biomimetic α-tricalcium phosphate (α-TCP) cements at different gelatin contents (10, 15 and 20 wt.%) to introduce Disodium Alendronate up to a concentration of 25 mM. Due to the small liquid/powder ratio (0.22 ml/g) the lengthening of the setting times due to alendronate is quite modest. The rate of transformation of α-TCP into calcium deficient hydroxyapatite slightly decreases as a function of alendronate content, whereas it increases with increasing gelatin concentration. Moreover, relatively high alendronate concentrations provoke significant reduction of the compressive strength of the cements. The results of in vitro tests indicate that alendronate-containing cements significantly affect osteoclast proliferation and differentiation, whereas they promote osteoblast differentiation, to an extent which depends on cement composition.     

In vitro cytotoxicity of a novel injectable and biodegradable alveolar bone substitute

The unsaturated polyphosphoester (UPPE) polymer is being investigated as an injectable and biodegradable system for alveolar bone repair in the treatment of periodontal diseases. The incorporation of β-tricalcium phosphate (β-TCP) particles into the UPPE polymer was previously shown to significantly increase the material’s mechanical properties. Moreover, in vitro experiments demonstrated that the UPPE/β-TCP composite was capable of zero-order release of tetracycline for over 2 weeks. In this study, we investigated the in vitro cytotoxicity of each individual component, the resulting cross-linked network and the degradation products of the UPPE/β-TCP composite using an AlamarBlue viability assay. We confirmed that each individual component except β-TCP and the in vitro degradation products of the composite displayed a dose-dependent cytotoxic response. Once cross-linked, however, the composite did not demonstrate an adverse response. Our results suggest that the UPPE/β-TCP composite holds great promise for use as an injectable and biodegradable alveolar bone substitute.     

Co-electrospun nanofiber fabrics of poly(L-lactide-co-epsilon-caprolactone) with type I collagen or heparin

Functionally designed elastomeric nanofiber fabrics made of the equimolar copolyester, poly(L-lactide-co-epsilon-caprolactone) (PLCL), with type I collagen or the tri-n-butylamine salt of heparin (heparin-TBA) were co-electrospun using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as a solvent. The co-electrospun fabrics (mixing ratio: 0, 5, 10, 30, 50, 70, and 100 wt % of collagen to PLCL) consisted of nanoscale fibers with a mean diameter ranging from approximately 120 to 520 nm. An increase in collagen content in the solution resulted in a decrease in the mean diameter of fibers. Transmission electron microscopy (TEM) showed that collagen in a co-electrospun fiber was phase-separated to form a dispersed phase, which was localized in the interior and peripheral region in the continuous matrix phase of fibers. The tensile strength was decreased with increasing collagen content. Human umbilical vein endothelial cells (HUVECs) were highly elongated and well spread on the fibrous surfaces of fabrics made of PLCL with 5 wt % or 10 wt % collagen. Heparin-TBA (mixing ratio: 1, 5, and 10 wt % to PLCL), soluble in HFIP, was co-electrospun with PLCL to form a fabric. TEM observation showed that heparin-TBA formed as a dispersed phase in a PLCL nanofiber. The releasing rate, released amount, and surface content of heparin-TBA were increased with increasing heparin-TBA content in co-electrospun fabrics. The potential biomedical application of co-electrospun PLCL with type I collagen or heparin-TBA was discussed.     

Beta-tricalcium phosphate exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway

Beta-tricalcium phosphate (β-TCP) has been clinically used as a bone graft substitute for decades because of its excellent osteoconductivity. However, the exact mechanism(s) by which β-TCP exerts osteoconductivity are not fully documented. This study was aimed to investigate the molecular mechanism(s) by which β-TCP modulates the biological response of primary human osteoblasts (HOBs). It was showed that HOBs seeded into the β-TCP scaffolds expressed significantly higher levels of osteogenic genes, compared to those cultured on tissue culture plastic; meanwhile these cells showed 7-fold increase in α2 integrin subunit gene expression and the activation of the mitogen-activated protein kinase (MAPK)/extracellular related kinase (ERK) signaling pathway. In addition, the osteogenic conduction by β-TCP scaffolds was attenuated directly by inhibiting MAPK/ERK or indirectly by blocking the α2β1 integrin signaling pathway. We concluded that β-TCP scaffold exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway, suggesting a feasible approach to consider when designing or fabricating the scaffolds for bone tissue engineering.     

<Emphasis Type="Italic">In vitro</Emphasis> behaviour of osteoblast cells seeded into a COL/β-TCP composite scaffold

The purpose of the present study was to investigate the effect of a collagen/β-tricalcium phosphate (COL/β-TCP) composite on osteoblast growth and proliferation. The COL/β-TCP composite was prepared by mixing COL type I with β-TCP, in 1:1 (w/w) ratio and conditioned as sponge by freeze-drying. The osteoblast culture was obtained from rat calvaria bones by enzymatic digestion and cells were seeded in the COL/β-TCP composite. The cell morphology and viability, alkaline phosphatase and osteocalcin, as markers of osteoblast proliferation were evaluated at 3, 7 and 25 days of culture. Histological sections revealed that cell colonization progressively increased inside the COL/β-TCP scaffold, and osteoblasts had a random distribution throughout the scaffold. Cells cultured into the COL/β-TCP scaffold presented osteoblast phenotype, intense staining of alkaline phosphatase and increased production of osteocalcin. Transmission electron micrographs revealed intimate contacts between osteoblasts and the scaffold. MTT test indicated that the viability of the cells cultivated in the presence of COL/β-TCP scaffold was similar to that of the control. All these results show that our COL/β-TCP composite act as a good substrate for rat osteoblast proliferation and migration and could be a promising substitute for bone repair.     

Synthesis and in vitro behavior of β-TCP zirconia/polymeric biocomposites for bio-applications

Novel biocomposites were fabricated by impregnating β-tricalcium phosphate (β-TCP)/zirconia particles into the polymers matrix. The composite materials were characterized using thermo-gravimetric analysis (TGA), X-ray diffraction (XRD), Fourier Transform Infrared (FT-IR) analyzes and Scanning Electron Microscopy (SEM). The results confirmed the conversion of hydroxyapatite (HA) to β-TCP at a sintering temperature of 1150 °C with or without zirconia powder. The in vitro behavior was assessed via measurement of calcium and phosphorus ions in SBF (simulated body fluid). FT-IR and SEM of the composites were performed pre and post immersion in SBF. The results prove that the bone like apatite layer formation was enhanced on the β-TCP-Z20/polymeric composite surface more than that on the β-TCP-Z10/polymeric composite. Therefore, the data confirmed that zirconia plays an important role in the enhancement of the apatite formation. The conclusions proved that the β-TCP-Z20/polymeric biocomposites, containing 20% of zirconia, are promising for bone remodeling applications.     

Chemical evaluation of partially acidulated phosphate rocks and their impact on dry matter yield and phosphorus uptake of maize

Previous studies investigated the direct application of phosphate rock and its partially acidulated to enhance its solubility compared to soluble fertilizers. However, the interaction between the effect of particles diameter and partial acidulation of phosphate rock on phosphorus (P) availability and its effect on dry matter yield and P uptake is still elusive. This study was conducted to assess the effect of partially acidulated Egyptian phosphate rocks with different particle size diameters on P availability and its effect on dry matter yield and P uptake of maize (Zea mays L.). A pot experiment was conducted on maize plants grown on light clay soil for 42 days. Acidulation was done by mixing phosphate rock with single superphosphate or triple superphosphate at a total rate of 200 mg P kg^?1 with five acidulation mix ratios (100:0, 75:25, 50:50, 25:75, and 0:100). Different particle size diameters of phosphate rocks (500, 212, 75, and <45 µm included nano-particles ranged from 69.3 to 25.7 nm) were used. We found that dry matter yield and P uptake increased significantly due to the use of partially acidulated phosphate rocks especially when triple superphosphate was used for acidulation and the mixing ratio of 50:50 was the best. We also found that maize yield and P uptake increased significantly with decreasing particle size. It is recommended to use finely grounded partially acidulated phosphate rocks with particles diameter less than 45 µm at acidulation ratio 50% and no need to increase acidulation ratio above that as a slow-release phosphate fertilizer.     

Optimization of a biomimetic bone cement: role of DCPD

We previously proposed a biomimetic α-tricalcium phosphate (α-TCP) bone cement where gelatin controls the transformation of α-TCP into calcium deficient hydroxyapatite (CDHA), leading to improved mechanical properties. In this study we investigated the setting and hardening processes of biomimetic cements containing increasing amounts of CaHPO₄·2H₂O (DCPD) (0, 2.5, 5, 10, 15 wt.%), with the aim to optimize composition. Both initial and final setting times increased significantly when DCPD content accounts for 10 wt.%, whereas cements containing 15 wt.% DCPD did not set at all. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), thermogravimetry (TG) and scanning electron microscopy (SEM) investigations were performed on samples maintained in physiological solution for different times. DCPD dissolution starts soon after cement preparation, but the rate of transformation decreases on increasing DCPD initial content in the samples. The rate of α-TCP to CDHA conversion during hardening decreases on increasing DCPD initial content. Moreover, the presence of DCPD prevents gelatin release during hardening. The combined effects of gelatin and DCPD on the rate of CDHA formation and porosity lead to significantly improved mechanical properties, with the best composition displaying a compressive strength of 35 MPa and a Young modulus of 1600 MPa.     

Molecular modeling of the axial and circumferential elastic moduli of tubulin

Microtubules play a number of important mechanical roles in almost all cell types in nearly all major phylogenetic trees. We have used a molecular mechanics approach to perform tensile tests on individual tubulin monomers and determined values for the axial and circumferential moduli for all currently known complete sequences. The axial elastic moduli, in vacuo, were found to be 1.25 GPa and 1.34 GPa for α- and β-bovine tubulin monomers. In the circumferential direction, these moduli were 378 MPa for α- and 460 MPa for β-structures. Using bovine tubulin as a template, 269 homologous tubulin structures were also subjected to simulated tensile loads yielding an average axial elastic modulus of 1.10 ± 0.14 GPa for α-tubulin structures and 1.39 ± 0.68 GPa for β-tubulin. Circumferentially the α- and β-moduli were 936 ± 216 MPa and 658 ± 134 MPa, respectively. Our primary finding is that that the axial elastic modulus of tubulin diminishes as the length of the monomer increases. However, in the circumferential direction, no correlation exists. These predicted anisotropies and scale dependencies may assist in interpreting the macroscale behavior of microtubules during mitosis or cell growth. Additionally, an intergenomic approach to investigating the mechanical properties of proteins may provide a way to elucidate the evolutionary mechanical constraints imposed by nature upon individual subcellular components.     

Molecular Modelling of a Multiphosphorylated Sequence Motif Bound to Hydroxyapatite Surfaces

Proteins and peptides containing the multiphosphorylated motif -Ser(P)-Ser(P)- Ser(P)--Glu-Glu- stabilise amorphous calcium phosphate (ACP) in body fluids and bind with high affinity to crystalline calcium phosphate phases such as hydroxyapatite (HA) regulating crystal growth. Binding of this motif to hydroxyapatite surfaces was investigated in this study using molecular modelling techniques. Using a three-step computational procedure, we have determined the relative binding energies of the motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu to different crystalline surfaces of HA. This analysis revealed preferences of the motif for (100) and (010) surfaces of the crystal and preferences for particular orientations on a given surface. These preferences are principally governed by electrostatic interactions between the crystal lattice and the peptide with the most stable conformers adopting structures where alternate residues exhibit backbone angles characteristic of a -strand and values of an -helix or a distorted -helix, allowing maximal interaction between the acidic side groups and surface calciums. The results of this study are consistent with experimentally-derived data on the interaction of multiphosphorylated proteins/peptides with HA and have implications for the role of these proteins/peptides in calcium phosphate stabilisation and biomineralisation processes.Electronic Supplementary Material available.     

Mechanical, microstructural and solubility properties of pectin/poly(vinyl alcohol) blends

Citrus pectin was blended and cast into films with poly(vinyl alcohol) (PVOH). PVOH and pectin were miscible in all proportions. Dynamic mechanical analysis revealed that pectin controls exhibited no thermal transitions, whereas PVOH controls exhibited a glass transition temperature (T_g) over a broad temperature range commencing at about 0 °C and ending about 50 °C. A mixture of 49% pectin, 21% PVOH and 30% glycerol exhibited lower storage moduli and more flexibility than comparable mixtures of either pectin/PVOH or pectin/glycerol. Scanning electron microscopy and phase contrast optical microscopy indicated that the mixture was biphasic and a compatible composite either of PVOH in pectin or pectin in PVOH depending on which macromolecule was in excess. Elongation to break measurements revealed that pectin/PVOH films underwent a brittle to ductile transition with increasing PVOH composition. The addition of glycerol to pectin/PVOH films increased ductility significantly when films were relatively brittle. Initial moduli (IM) as a function of composition gave complex curves which exhibited either one or two local maxima depending on such factors as degree of hydrolysis and molar mass of the PVOH in addition to the moisture content of the film. Solubility studies in water revealed that, at 30 and 50 °C, only films with 30% PVOH or less were soluble. At 70 °C, all compositions were soluble but films containing pectin dissolved more rapidly than those without. The solution kinetics of pectin/PVOH films with 30% or less PVOH were approximated with zero-order kinetics and activation energies were about 3–5 kcal mol⁻¹. In general, addition of PVOH to pectin films resulted in films with more PVOH-like properties and addition of pectin to PVOH films resulted in films with more pectin-like properties.     

Construction of viscoelastic biocompatible films via the layer-by-layer assembly of hyaluronan and phosphorylcholine-modified chitosan

Films of hyaluronan (HA) and a phosphorylcholine-modified chitosan (PC-CH) were constructed by the polyelectrolyte multilayer (PEM) deposition technique and their buildup in 0.15 M NaCl was followed by atomic force microscopy, surface plasmon resonance spectroscopy (SPR), and dissipative quartz crystal microbalance (QCM). The HA/PC-CH films were stable over a wide pH range (3.0-12.0), exhibiting a stronger resistance against alkaline conditions as compared to HA/CH films. The loss and storage moduli, G' and G", of the films throughout the growth of eight bilayer assemblies were derived from an impedance analysis of the QCM data recorded in situ. Both G' and G" values were one order of magnitude lower than the moduli of HA/CH films. The fluid gel-like characteristics of HA/PC-CH multilayers were attributed to their high water content (50 wt %), which was estimated by comparing the surface coverage values derived from SPR and QCM measurements. Given the versatility of the PEM methodology, HA/PC-CH films are attractive tools for developing biocompatible surface coatings of controlled mechanical properties.     

Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors

The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.     

The structure and mechanical properties of the proteins of lamprey cartilage

The cyanogen bromide‐resistant proteins of lamprey cartilage are biochemically related to the mammalian elastic protein, elastin. This study investigates their mechanical properties and enquires whether, like elastin, long‐range elasticity arises in them from a combination of entropic and hydrophobic mechanisms. Branchial and pericardial proteins resembled elastin mechanically, with elastic moduli of 0.13–0.35 MPa, breaking strains of 50%, and low hysteresis. Annular and piston proteins had higher elastic moduli (0.27–0.75 MPa) and larger hysteresis. Exchanging solvent water for trifluoroethanol increased the elastic moduli, whereas increasing temperature lowered the elastic moduli. Raman microspectrometry showed small differences in side‐chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were like those of elastin, preponderantly unordered, which probably confer the conformational flexibility necessary for entropy elasticity. Piston and annular proteins showed the strongest interactions with water, suggesting, together with the mechanical testing data, a greater role of hydrophobic interactions in their mechanics. Two‐photon imaging of intrinsic fluorescence and dye injection experiments showed that annular and piston proteins formed closed‐cell honeycomb structures, whereas the branchial and pericardial proteins formed open‐cell structures, which may account for the differences in mechanical properties. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 187–202, 2015.     

磷酸三钙涂层镁合金材料的细胞相容性研究

目的:制备磷酸三钙(β-TCP)涂层镁合金材料,评价材料表面的特性及体外的细胞生物适应性。方法 电化学法制备β-TCP涂层镁合金材料(β-TCP-Mg-AI-Zn),观测金属材料表面微观结构特性和能谱分析,小鼠颅骨源成骨细胞与材料直接接触培养,荧光染色观察材料表面细胞生长状况,检测成骨细胞增殖和碱性磷酸酶(ALP)活性。结果 β-TCP涂层Mg-AI-Zn材料表面呈多孔状,材料表面含有镁、钙和磷等元素;成骨细胞与材料直接接触培养24 h及48 h后,材料表面有大量的成骨细胞粘附、伸展、汇合;与Mg-AI-Zn材料比较,β-TCP-Mg-AI-Zn材料明显地促进细胞增殖、显著地增加成骨细胞中ALP活性 (P＜0.05)。结论 β-TCP涂层改善了Mg-AI-Zn镁合金材料表面特性及体外的细胞相容性,有望成为新一代可降解医用金属材料。     

Magainin 2 Revisited: A Test of the Quantitative Model for the All-or-None Permeabilization of Phospholipid Vesicles

The all-or-none kinetic model that we recently proposed for the antimicrobial peptide cecropin A is tested here for magainin 2. In mixtures of phosphatidylcholine (PC)/phosphatidylglycerol (PG) 50:50 and 70:30, release of contents from lipid vesicles occurs in an all-or-none fashion and the differences between PC/PG 50:50 and 70:30 can be ascribed mainly to differences in binding, which was determined independently and is ∼20 times greater to PC/PG 50:50 than to 70:30. Only one variable parameter, β, corresponding to the ratio of the rates of pore opening to pore closing, is used to fit dye release kinetics from these two mixtures, for several peptide/lipid ratios ranging from 1:25 to 1:200. However, unlike for cecropin A where it stays almost constant, β increases five times as the PG content of the vesicles increases from 30 to 50%. Thus, magainin 2 is more sensitive to anionic lipid content than cecropin A. But overall, magainin follows the same all-or-none kinetic model as cecropin A in these lipid mixtures, with slightly different parameter values. When the PG content is reduced to 20 mol %, dye release becomes very low; the mechanism appears to change, and is consistent with a graded kinetic model. We suggest that the peptide may be inducing formation of PG domains. In either mechanism, no peptide oligomerization occurs and magainin catalyzes dye release in proportion to its concentration on the membrane in a peptide state that we call a pore. We envision this structure as a chaotic or stochastic type of pore, involving both lipids and peptides, not a well-defined, peptide-lined channel.     

Characteristics of a 1:30 mixing ratio stopped flow apparatus

The annular mixer of a small stopped flow apparatus for following changes in transmission is presented together with test results on mixing NaOH with ferricytochromec in the volume ratio 130.This work was supported in part by the Chicago and Illinois Heart Associations (C70-66) and by the National Science Foundation (GB-28023)     

Synthesis and Characterization of PVA-HA-Silk Composite Hydrogel by Orthogonal Experiment

PVA-HA-Silk composite hydrogel was synthesized with polyvinyl alcohol (PVA),nano-hydroxyapatite (HA) and natural silk by using the method of repeated freezing and thawing.A series of tests were performed to study water content,stress relaxation behavior,elastic modulus,and creep characteristics of PVA-HA-Silk composite hydrogel.Orthogonal experimental design method was used to analyze the influence degree of PVA,HA and silk (three kinds of raw materials) on mechanical properties and water content of the PVA-HA-Silk composite hydrogel to select the best material ratio according to their overall performance.The results demonstrate that the mass percentage of PVA has the greatest impact on the water content,followed by HA and silk.Compression stress-strain variation of PVA-HA-Silk composite hydrogel presents a nonlinear relationship,which proves that it is a typical viscoelastic material.Comparing the mechanical properties of 16 formulas,the formula of PVA-HA-silk composite hydrogel with mass percentage of PVA 15％,HA 2.0％ and silk 1.0％ is the best.