Advanced protein adsorption properties of a novel silicate-based bioceramic: A proteomic analysis.

Silicate bioceramics have been shown to possess excellent cytocompatibility and osteogenic activity, but the exact mechanism is still unclear. Protein adsorption is the first event taking place at the biomaterial-tissue interface, which is vital to the subsequent cellular behavior and further influence the biomaterial-tissue interaction. In this work, the protein adsorption behavior of a novel CPS bioceramic was evaluated using the proteomics technology. The results showed that CPS adsorbed more amount and types of serum proteins than HA. FN1 and IGF1 proteins selected from proteomics results were validated by Western-blot experiment. Pathway analysis also revealed mechanistic insights how these absorbed proteins by CPS help mediate cell adhesion and promotes osteogenic activity. Firstly, the dramatically enhanced adsorption of FN1 could greatly promote cell adhesion and growth. Secondly, IGF1 was uniquely adsorbed on CPS bioceramic and IGF1 could activate Rap1 signaling pathway to promote cell adhesion. Thirdly, the increased adsorption of FN1, IGF1 and COL1A2 proteins on CPS explains its better ability on bone regeneration than HA. Fourthly, the increased adsorption of IGF1, CHAD, COL2A1 and THBS4 proteins on CPS explains its ability on cartilage formation. Lastly, the increased adsorption of immunological related proteins on CPS may also play a positive role in bone regeneration. In addition, CPS had a much better cell adhesion ability than HA, proving that more adsorbed proteins really had a positive effect on cell behavior. The more adsorbed proteins on CPS than HA might indicated a better bone regeneration rate at early stage of implantation.

Multiplexed detection of lung cancer biomarkers based on quantum dots and microbeads.

We have developed a multiplexed fluoroimmunoassay of three lung cancer biomarkers based on multicolor quantum dots (QDs) as detection elements and micro-magnetic beads as immune carriers. QDs have the ability to simplify multiplexed analysis. In our method, the fluorescent signals derived from three cross-talk-free QD conjugated probes with emission maxima at 525, 585 and 625nm could be analyzed to determine the concentrations of the target proteins. With this system, fragments of cytokeratin 19 (CYRFA 21-1), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSE), were simultaneously detected in a single sample with a low detection limit down to the 1.0ng/mL level (364pg/mL for CYRFA 21-1, 38pg/mL for CEA, 370pg/mL for NSE in a single detection). Additional advantages of the presented method include ease of operation, low cost, and a very low sample volume (20µL).

Procyanidins-crosslinked aortic elastin scaffolds with distinctive anti-calcification and biological properties.

Elastin, a main component of decellularized extracellular matrices and elastin-containing materials, has been used for tissue engineering applications due to their excellent biocompatibility. However, elastin is easily calcified, leading to the decrease of life span for elastin-based substitutes. How to inhibit the calcification of elastin-based scaffolds, but maintain their good biocompatibility, still remains significantly challenging. Procyanidins (PC) are a type of natural polyphenols with crosslinking ability. To investigate whether pure elastin could be crosslinked by PC with anti-calcification effect, PC was first used to crosslink aortic elastin. Results show that PC can crosslink elastin and effectively inhibit elastin-initiated calcification. Further experiments reveal the possible mechanisms for the anti-calcification of PC crosslinking including (1) inhibiting inflammation cell attachment, and secretion of inflammatory factors such as MMPs and TNF-α, (2) preventing elastin degradation by elastase, and (3) direct inhibition of mineral nucleation in elastin. Moreover, the PC-crosslinked aortic elastin maintains natural structure with high pore volume (1111 µL/g), large pore size (10-300 µm) and high porosity (75.1%) which facilitates recellularization of scaffolds in vivo, and displays excellent hemocompatibility, anti-thrombus and anti-inflammatory potential. The advantages of PC-crosslinked porous aortic elastin suggested that it can serve as a promising scaffold for tissue engineering.

Crosslinking of saphenous vein ECM by procyanidins for small diameter blood vessel replacement.

Xenogenic decellularized vessels, mainly composed of extracellular matrices (ECMs), are thought to be one of the alternative resources of small-diameter blood vessels due to abundant source, tubular configuration, vascular microstructure, and good cytocompatibility. However, the main shortcomings of ECM vessels are their low chemical stability, easy calcification, immunogenicity, and high risk of thrombogenicity. Previous studies have shown that, glutaraldehyde (GA), as a crosslinking agent, led to significant calcification and cytotoxicity for the prepared ECM substitutes. To overcome the drawbacks of pure and GA-crosslinked vascular alternatives of small-diameter blood vessels, procyanidins (PC), a naturally derived polyphenol with anti-inflammatory and platelet aggregation inhibiting bioactivities, was applied to crosslink the decellularized bovine saphenous vein ECM (svECM). After crosslinking, the obtained svECM substitutes exhibited natural tubular configuration with significantly improved mechanical properties, proper resistance to proteolysis, high chemical stability, and excellent anticalcification property. The PC-crosslinked svECM substitutes were cytocompatible for cells adhesion and proliferation, and blood compatible for erythrocytes with far less hemolysis than that of safety standard. Furthermore, the PC-crosslinked svECM substitutes showed distinct antithrombosis and anti-immunogenicity potential. With these advantages, it is suggested that the PC-crosslinked svECM may be used as a practical substitutes of small diameter blood vessels.

Stimulatory effects of the ionic products from Ca-Mg-Si bioceramics on both osteogenesis and angiogenesis in vitro.

Ideal biomaterials for bone tissue engineering should have the capability to guide the osteogenic differentiation of mesenchymal stem cells and, at the same time, to stimulate angiogenesis of endothelia cells. In this study it was found that three Ca-Mg-Si-containing bioceramics (bredigite Ca7MgSi4O16, akermanite Ca2MgSi2O7 and diopside CaMgSi2O6) had osteogenic and angiogenic potential. The effects of three silicate ceramics on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and the angiogenesis of human aortic endothelial cells (HAECs) were explored in comparison with ß-tricalcium phosphate (ß-TCP) bioceramics. The proliferation, alkaline phosphatase (ALPase) activity and bone-related gene expression (COL1, ALPase, OP, BSP and OC) of hBMSCs were significantly enhanced upon stimulation with ionic extracts of these silicate bioceramics. In addition, the results showed that extracts from the three silicate bioceramics also stimulated HAEC proliferation and in vitro angiogenesis with improved NO synthesis and angiogenic gene expression (KDR, FGFR1, ACVRL1 and NOS3). Among the three silicate ceramics bredigite showed the highest osteogenic and angiogenic potential and with the highest extract Si (possibly Si(OH)3O(-)) concentration, while diopside had the lowest osteogenic and angiogenic potential with the lowest extract Si concentration. Furthermore, it was found that the concentration of Si ions in extracts of the three silicate bioceramics was obviously higher than that of ß-TCP ceramics, indicating an important role of Si ions in stimulating cell proliferation, osteogenic differentiation and angiogenesis. The results suggest that the silicate-based akermanite and bredigite ceramics might be good scaffold biomaterials for bone tissue engineering applications due to their distinctive dual functions of osteogenesis/angiogenesis stimulation.

The effect of plaster (CaSO4 ·1/2H2O) on the compressive strength, self-setting property, and in vitro bioactivity of silicate-based bone cement.

Bone cements have been widely used for orthopedic applications. Previous studies have shown that calcium silicon-based bone cements (CSC) were injectable, bioactive, biodegradable, and mechanically strong in the long term, while their short-term compressive strength was low and setting time was too long. On the other hand, plaster (CaSO(4)·1/2H(2)O, POP) sets quickly upon contact with water and has excellent short-term compressive strength. The aim of this study is to prepare CSC/POP composite cements and investigate the effect of POP on the compressive strength, setting time, injectability, degradation, and in vitro bioactivity of the composite cements. The results have shown that POP content plays an important role to modulate the physicochemical property of CSC. The addition of POP into CSC significantly decreased the initial and final setting time and enhanced the short-term compressive strength and degradation rate. The obtained composite cement with 30% POP has been found to possess optimal setting time and short-term compressive strength. In addition, the prepared composite cements still maintain apatite-mineralization ability in simulated body fluids and their ionic extracts have no significant cytotoxicity to L929 cells. The results suggested that the addition of POP into CSC is a viable method to improve their setting properties and short-term compressive strength. The obtained composite cements with the optimized composition of 70% CSC and 30% POP could be potentially used for bone repair application.

Degradation of hollow mesoporous silica nanoparticles in human umbilical vein endothelial cells.

Hollow mesoporous silica nanoparticles (HMSNs) are considered a potential drug delivery system owing to their recognized advantages in drug loading and releasing. However, whether HMSN could be degraded inside the cells remains unknown. In this study, based on the observations by transmission electron microscopy, fluorescence staining, enzymatic proteolysis, and inductively coupled plasma atomic emission spectroscopy, HMSNs were proved to be degradable in human umbilical vein endothelial cells. The degradation first took place in cytoplasm and lysosomes, and secondly in lysosomes only. The Si content in culture medium increased as the time increases, suggesting that the degradation product inside the cells could be excreted into the culture medium. The degrading rate is fast in the first 2 days and slow after 2 days. The present results provided a clue to further research on the metabolic way and cytotoxicity of silica nanoparticles.

Silicate bioceramics induce angiogenesis during bone regeneration.

The capacity to induce rapid vascular ingrowth during new bone formation is an important feature of biomaterials that are to be used for bone regeneration. Akermanite, a Ca-, Mg- and Si-containing bioceramic, has been demonstrated to be osteoinductive and to promote bone repair. This study further demonstrates the ability of akermanite to promote angiogenesis and investigates the mechanism of this behavior. The akermanite ion extract predominantly caused Si-ion-stimulated proliferation of human aortic endothelial cells. The Si ion in the extract was the most important component for the effect and the most effective concentration was found to be 0.6-2 µg ml(-1). In this range of Si ion concentration, the stimulating effect of the ceramic ion extract was demonstrated by the morphology of cells at the primary, interim and late stages during in vitro angiogenesis using ECMatrix™. The akermanite ion extract up-regulated the expression of genes encoding the receptors of proangiogenic cytokines and also increased the expression level of genes encoding the proangiogenic downstream cytokines, such as nitric oxide synthase and nitric oxide synthesis. Akermanite implanted in rabbit femoral condyle model promoted neovascularization after 8 and 16 weeks of implantation, which further confirmed its stimulation effect on angiogenesis in vivo. These results indicate that akermanite ceramic, an appropriate Si ion concentration source, could induce angiogenesis through increasing gene expression of proangiogenic cytokine receptors and up-regulated downstream signaling. To our knowledge, akermanite ceramic is the first Si-containing ceramic demonstrated to be capable of inducing angiogenesis during bone regeneration.

In vitro degradation behavior and bioactivity of magnesium-Bioglass(®) composites for orthopedic applications.

To improve the bioactivity and degradation behavior of biodegradable magnesium, biodegradable metal matrix composites with the ZK30 magnesium alloy as the matrix and bioactive glass (BG, 45S5) as the reinforcement were prepared. The microstructures of the ZK30-BG composites showed homogeneous dispersion of BG particles throughout the matrix. XRD and EDX analyses confirmed the retention of the morphological characteristics and composition of BG particles in the composites. Immersion tests in the minimum essential medium with Earle's balanced salts at 37°C showed that the composites with 5 and 10% BG had lower rates of degradation and hydrogen evolution than the matrix alloy. In addition, the tests confirmed that the composites possessed an enhanced ability to induce calcium and phosphate ion deposition on sample surfaces during degradation, suggesting accelerated surface mineralization that would lead to improved bioactivity when compared with the matrix alloy. In vitro cytotoxicity tests showed that the ionic products of the composites formed during degradation possessed a superior ability to support the survival, proliferation, and osteoblastic differentiation of bone marrow stromal cells to those of the ZK30 alloy. The ZK30-BG composites with enhanced bioactivity and reduced degradation rate could be promising biodegradable materials for orthopedic implants.

Hierarchically structured nanocrystalline hydroxyapatite assembled hollow fibers as a promising protein delivery system.

Nanocrystalline hydroxyapatite assembled hollow fibers (NHAHF) in the membrane form were fabricated by combining the electrospinning technique and the hydrothermal method. This novel hierarchical tubular structure of hydroxyapatite exhibited excellent protein loading capacity and long-term sustained release property.

Effect of tricalcium silicate on the proliferation and odontogenic differentiation of human dental pulp cells.

INTRODUCTION: This study was to investigate the effects of tricalcium silicate (Ca(3)SiO(5)) on proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) in vitro. METHODS: The hDPCs were seeded in culture medium with or without Ca(3)SiO(5) extract and calcium hydroxide (Ca(OH)(2)) extract. Proliferation of the hDPCs was measured by methyl-thiazol-tetrazolium (MTT) assay. Odontogenic differentiation of hDPCs was evaluated by real-time polymerase chain reaction by using odontogenic marker genes such as dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP 1), osteocalcin (OC), alkaline phosphatase (ALP), and collagen type I (Col I), which were verified by ALP activity assessment, mineralization assay, and immunocytochemistry staining for dentin sialoprotein (DSP). RESULTS: The MTT assay showed that hDPCs cultured with Ca(3)SiO(5) extract proliferated more significantly as compared with Ca(OH)(2) extract. Analysis of odontogenic marker genes indicated that Ca(3)SiO(5) enhanced the expression of those genes. Moreover, the extract of Ca(3)SiO(5) stimulated mineralization and increased ALP and DSP production conspicuously. CONCLUSIONS: These results reveal that Ca(3)SiO(5) can induce the proliferation and odontogenic differentiation of hDPCs in vitro and might be a potential candidate for preparation of a new type of Ca(3)SiO(5-)based cement as a pulp-capping agent.

The stimulation of osteogenic differentiation of human adipose-derived stem cells by ionic products from akermanite dissolution via activation of the ERK pathway.

Our previous study indicates that akermanite, a type of Ca-, Mg-, Si-containing bioceramic, can promote the osteogenic differentiation of hASCs. To elucidate the underlying mechanism, we investigated the effect of the extract from akermanite, on proliferation and osteogenic differentiation of hASCs. The original extract was obtained at 200 mg akermanite/ml LG-DMEM and further diluted with LG-DMEM. The final extracts were denoted as 1/2, 1/4, 1/8, 1/16, and 1/32 extracts based on the concentrations of the original extract. The LDH assay and live/dead stain were used to reveal the cytotoxicity of the different extracts on hASCs, while the DNA assay was carried out to quantitatively evaluate the proliferation of cells after being cultured with the extracts for 1, 3 and 7 days. Flow cytometry for cell cycle analysis was carried out on cells cultured in two media (GM and 1/2 extract) in order to further analyze the effect of the extract on cell proliferation behaviors. Osteogenic differentiation of hASCs cultured in the extracts was detected by ALP expression and calcium deposition, and further confirmed by real-time PCR analysis. It was shown that Ca, Mg and Si ions in the extract could suppress the LDH release and proliferation of hASCs, whereas promote their osteogenic differentiation. Such effects were concentration-dependent with the 1/4 extract (Ca 2.36 mM, Mg 1.11 mM, Si 1.03 mM) being the optimum in promoting the osteogenic differentiation of hASCs. An immediate increase in ERK was observed in cells cultured in the 1/4 extract and such osteogenic differentiation of hASCs promoted by released ions could be blocked by MEK1-specific inhibitor, PD98059. Briefly, Ca, Mg and Si ions extracted from akermanite in the concentrations of 2.36, 1.11, 1.03 mM, respectively, could facilitate the osteogenic differentiation of hASCs via an ERK pathway, and suppress the proliferation of hASCs without significant cytotoxicity.

Procyanidins inhibit tumor angiogenesis by crosslinking extracellular matrix.

OBJECTIVE: Procyanidins (PC) are widely available natural polyphenols. The present study is designed to investigate if PC can inhibit angiogenesis in lung adenocarcinoma xenografts through crosslinking vascular extracellular matrix (ECM) and preventing proteolysis by matrix metalloproteinases (MMPs). METHODS: Using the in vitro MMP-2 proteolysis and in vivo subcutaneous implantation models, we investigated if PC crosslinking inhibits MMP-mediated proteolysis. Using a cultured cell detachment assay, an in vitro angiogenesis assay, and a cell proliferation assay, we investigated if PC inhibits MMP-2-mediated endothelial cell detachment, angiogenesis, and cell proliferation, respectively. Using tumor xenografts, we evaluated if PC can inhibit growth of lung adenocarcinoma. RESULTS: PC crosslink vascular ECM proteins, protecting them against proteolysis by MMPs in vitro and in vivo, protecting cultured human umbilical vein endothelial cells from detachment by MMP-2, and inhibiting in vitro angiogenesis. However, PC (0.75-100 µg/ml) did not inhibit vascular and tumor cells proliferation. PC injections (30 mg PC/kg bodyweight) in situ had anticancer effects on xenografts of lung adenocarcinoma, most likely by inhibiting angiogenesis during ECM proteolysis by MMPs. CONCLUSION: The results suggest that PC may be important MMP inhibitors that can be used as therapeutic anticancer agents.

Flower-like hierarchically nanostructured hydroxyapatite hollow spheres: facile preparation and application in anticancer drug cellular delivery.

Flower-like nanostructured hydroxyapatite hollow spheres (NHHS) assembled with nanosheets with a hierarchical morphology are fabricated by a rapid microwave-assisted hydrothermal route. The presence and concentration of block copolymer poly(lactide)-block-poly(ethylene glycol) (PLA-PEG) are important parameters for the formation of the hollow structure. The possible formation mechanism of NHHS is proposed. The NHHS are explored as anticancer drug carriers for cellular delivery of mitoxantrone (MIT). The MIT-loaded NHHS exhibit sustained-drug-release behavior in vitro and the intracellular drug-distribution tests indicate that the MIT loaded in NHHS carriers can enter the cells efficiently. The experiments also show that the NHHS have a good biocompatibility, and therefore, they are promising anticancer drug carriers in cancer chemotherapy.

The effect of Zr content on the microstructure, mechanical properties and cell attachment of Ti-35Nb-xZr alloys.

Beta-type low elastic modulus alloys of the Ti-Nb-Zr system have recently attracted much attention for both orthopedic and dental applications. In the present study, meta-stable beta alloys of Ti-35Nb-xZr with different Zr contents were developed. The effect of Zr content on the microstructure, mechanical properties and cell attachment was investigated. It was found that the addition of Zr improved the tensile strength and elongation of Ti-35Nb-xZr alloys, and simultaneously reduced the elastic modulus. Moreover, the Zr element helped to stabilize the beta phase. Cell culture work indicated that the addition of Zr enhanced the attachment and spreading of bone marrow stem cells. Cell attachment and spreading on the surface of titanium alloys were dominated not only by the wettability but also by the inherent biocompatibility of alloying elements. The peak-aged alloy with 5 wt% Zr had a highest tensile strength of 874 MPa, while its elastic modulus was only 65 GPa, presenting a much higher strength/modulus ratio than Ti-6Al-4V. The Ti-35Nb-5Zr alloy exhibited a great potential for orthopedic and dental applications.

Hydroxyapatite nanorods/poly(vinyl pyrolidone) composite nanofibers, arrays and three-dimensional fabrics: electrospun preparation and transformation to hydroxyapatite nanostructures.

Electrospinning has been recognized as an efficient technique for fabricating polymer nanofibrous biomaterials. However, the study of electrospun inorganic biomaterials with well-designed three-dimensional (3-D) structures is still limited and little reported. In this study hydroxyapatite (HAp) nanorods with an average diameter of approximately 7 nm and length of approximately 27 nm were synthesized through a simple precipitation method and used for the fabrication of inorganic/organic [poly(vinyl pyrolidone) (PVP)] composite nanofibers by electrospinning in ethanol solution. 3-D fabrics and aligned nanofiber arrays of the HAp nanorods/PVP composite were obtained as precursors. Thereafter, 3-D single phase HAp fabrics, tubular structures and aligned nanofiber arrays were obtained after thermal treatment of the corresponding composite precursors. Cytotoxicity experiments indicated that the HAp fabric scaffold had good biocompatibility. In vitro experiments showed that mesenchymal stem cells could attach to the HAp fabric scaffold after culture for 24h.

Synthesis, in vitro hydroxyapatite forming ability, and cytocompatibility of strontium silicate powders.

Phase-pure strontium silicate (SrSiO(3)) powders were synthesized by chemical precipitation method. In vitro bioactivity of the powders were evaluated by examining the hydroxyapatite forming ability on their surface soaking simulated body fluid for various periods. The results showed that SrSiO(3) could induce hydroxy carbonate apatite formation after soaking for 7 days. Both L929 and rabbit bone marrow stromal cells (rMSCs) were used to test the in vitro cytocompatibility of SrSiO(3). L929 culture experiment showed that the ionic product of SrSiO(3) did not exhibit cytotoxicity except at high ion concentrations (Si 3.75 mM and Sr 0.12 mM). Moreover, at certain ion concentrations (Si 1.87-0.12 mM and Sr 0.12-3.75 x 10(-3) mM), the ionic product of SrSiO(3) stimulated the proliferation of rMSCs. All the results indicated that SrSiO(3) might be used as a new bioactive material for bone regeneration applications.

Preparation and characterization of a novel willemite bioceramic.

Willemite (Zn(2)SiO(4)) ceramics were prepared by sintering the willemite green compacts. The effects of sintering temperature on the linear shrinkage, porosity and mechanical strength of the ceramics were examined. With the sintering temperature increased, the linear shrinkage of the ceramics increased and the porosity decreased. When sintered at 1,300 degrees C, willemite ceramics showed mechanical properties of the same order of magnitude as values for human cortical bone, as measured by bending strength (91.2 +/- 4.2 MPa) and Young's modulus (37.5 +/- 1.5 GPa). In addition, the adhesion and proliferation of rabbit bone marrow stromal cells (BMSCs) on willemite ceramics was investigated. The results showed that the ceramics supported cell adhesion and stimulated the proliferation. All these findings suggest that willemite ceramics possess suitable mechanical properties and favorable biocompatibility and might be a promising biomaterial for bone implant applications.

Crosslinking effect of Nordihydroguaiaretic acid (NDGA) on decellularized heart valve scaffold for tissue engineering.

Decellularized heart valve scaffolds possess many desirable properties in valvular tissue engineering. However, their current applications were limited by short durability, easily structural dysfunction and immunological competence. Although crosslinking with chemical reagents, such as glutaraldehyde (GA), will enhance the mechanical properties, the low long-term stability and cytotoxicity of the scaffolds remains potential problem. Nordihydroguaiaretic acid (NDGA) is a bioactive natural product which is able to crosslink collagen and was proven to be effective in preparation of scaffold for tendon tissue engineering. In this paper, NDGA crosslinked decellularized heart valve scaffolds demonstrated higher tensile strength, enzymatic hydrolysis resistance and store stability than the non-crosslinked ones. Its mechanical properties and cytocompability were superior to that of GA-crosslinked heart valve matrix. Below the concentration of 10 microg/ml, NDGA has no visible cytotoxic effect on both endothelial cells (EC) and valvular interstitial cells (VIC) and its cytotoxicity is much less than that of GA. The LC50 (50% lethal concentration) of NDGA on ECs and VICs are 32.6 microg/ml and 47.5 microg/ml, respectively, while those of GA are almost 30 times higher than NDGA (P < 0.05). ECs can attach to and maintain normal morphology on the surface of NDGA-crosslinked valvular scaffolds but not GA-crosslinked ones. This study demonstrated that NDGA-crosslinking of decellularized valvular matrix is a promising approach for preparation of heart valve tissue engineering scaffolds.

Quercetin-crosslinked porcine heart valve matrix: mechanical properties, stability, anticalcification and cytocompatibility.

Bioprosthetic heart valves, prepared by glutaraldehyde (GA) crosslinking, have some limitations due to poor durability, calcification and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of a natural product, quercetin, on decellularized porcine heart valve extracellular matrix (ECM). After crosslinking, the mechanical properties, stability, anticalcification and cytocompatibility were examined. The results showed that the tensile strength of quercetin-crosslinked ECM was higher than that of GA-crosslinked ECM. After crosslinking with quercetin, the thermal denaturation temperature of ECM was clearly increased. Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any loss of ultimate tensile strength and elasticity. After soaking in D-Hanks solution for 36 days, there was only 11.55% non-crosslinked excess quercetin released and no further release thereafter. Cell culture study shows that no inhibition on proliferation of vascular endothelial cells occurred when the quercetin concentration was lower than 1microg ml(-1). This non-cytotoxic concentration was 100 times higher than that of GA. The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of GA-crosslinked ECM. An in vitro anticalcification experiment showed that quercetin crosslinking could protect ECM from deposition of minerals in simulated body fluid. The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively, which suggests that quercetin might be a new crosslinking reagent for the preparation of bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.