Search

Although poly(β-amino esters) (PAEs) have been widely applied in nonviral gene transfection, drug delivery systems, and regenerative medicine, the multifunctional modification of PAEs and bio-orthogonal strategies of PAE-based hydrogel functionalization is still a challenge. Herein, a strategy of poly(β-amino ester)-based hydrogel functionalization was developed via bio-orthogonal reactions in this study. Acrylate-terminated poly(β-amino esters) containing alkyne groups were synthesized by Michael addition reaction. Alkyne groups on poly(β-amino esters) could conjugate bioactive molecules with azide of K(N

Magnesium alloy is considered as one of the ideal cardiovascular stent materials owing to its good mechanical properties and biodegradability. However, the in vivo rapid degradation rate and the insufficient biocompatibility restrict its clinical applications. In this study, the magnesium alloy (AZ31B) was modified by combining the surface chemical treatment and in-situ self-assembly of 16-phosphonyl-hexadecanoic acid, followed by the immobilization of chitosan-functionalized graphene oxide (GOCS). Heparin (Hep) and GOCS were alternatively immobilized on the GOCS-modified surface through layer by layer (LBL) to construct the GOCS/Hep bioactive multilayer coating, and the corrosion resistance and biocompatibility were extensively explored. The results showed that the GOCS/Hep bioactive multilayer coating can endow magnesium alloys with an excellent in vitro corrosion resistance. The GOCS/Hep multilayer coating can significantly reduce the hemolysis rate and the platelet adhesion and activation, resulting in an excellent blood compatibility. In addition, the multilayer coating can not only enhance the adhesion and proliferation of the endothelial cells, but also promote the vascular endothelial growth factor (VEGF) and nitric oxide (NO) expression of the attached endothelial cells on the surfaces. Therefore, the method of the present study can be used to simultaneously control the corrosion resistance and improve the biocompatibility of the magnesium alloys, which is expected to promote the application of magnesium alloys in biomaterials or medical devices, especially cardiovascular stent., Graphical abstract Image 1, Highlights • The multilayer coating of GOCS and heparin was constructed on magnesium surface. • The coating can obviously improve the corrosion resistance of magnesium alloys. • The coating can enhance the hemocompatibility and endothelial cell growth behaviors.

Hydrogels have promising application in tissue regeneration due to their excellent physicochemical and biocompatible properties, whereas anisotropic micropatterns are been proven to directionally induce cell alignment and accelerate cell migration. However, an effect of biofunctionalized anisotropic hydrogel micropatterns on nerve regeneration has rarely been reported. In this study, the anisotropic polyacrylamide (PAM) hydrogel micropatterns with aligned ridge/groove structures were first prepared via in situ free radical polymerization and micromolding, and then biofunctionalized using YIGSR peptide for better promoting cell growth. The morphology, swelling ratio, wettability, mechanical properties, and stability of the prepared hydrogel were characterized. The successful immobilization of YIGSR peptide on the PAM hydrogel was monitored using FTIR, immunofluorescence staining, and ELISA. The effects on adhesion, directional growth, and biological function of Schwann cells were evaluated. The results displayed that the anisotropic PAM hydrogel micropatterns with inner porous structure possessed good stability, swelling, and mechanical properties. The YIGSR peptide could be well immobilized on hydrogel micropatterns with a percentage of 62.6%. The biofunctionalized anisotropic hydrogel micropatterns could effectively regulate the orientation growth of Schwann cells, and obviously up-regulate BDNF (40%) and β-actin (50%) expression compared with single hydrogel micropatterns, without negatively affecting the normal secretion of neurotropic factors by Schwann cells. To the best of our knowledge, this is the first time to study the construction and effect of biofunctionalized anisotropic hydrogel micropatterns on nerve regeneration, which may provide an experimental and theoretical basis for the design and development of artificial implants for nerve regeneration application.

Biofunctionalization of artificial nerve implants by incorporation of specific bioactive factors has greatly enhanced the success of grafting procedures for peripheral nerve regeneration. However, most studies on novel biofunctionalized implants have emphasized the promotion of neuronal and axonal repair over vascularization, a process critical for long-term functional restoration. We constructed a dual-biofunctionalized chitosan/collagen composite scaffold with Ile-Lys-Val-Ala-Val (IKVAV) and vascular endothelial growth factor (VEGF) by combining solution blending, in situ lyophilization, and surface biomodification. Immobilization of VEGF and IKVAV on the scaffolds was confirmed both qualitatively by staining and quantitatively by ELISA. Various single- and dual-biofunctionalized scaffolds were compared for the promotion of endothelial cell (EC) and Schwann cell (SC) proliferation as well as the induction of angiogenic and neuroregeneration-associated genes by these cells in culture. The efficacy of these scaffolds for vascularization was evaluated by implantation in chicken embryos, while functional repair capacity in vivo was assessed in rats subjected to a 10 mm sciatic nerve injury. Dual-biofunctionalized scaffolds supported robust EC and SC proliferation and upregulated the expression levels of multiple genes and proteins related to neuroregeneration and vascularization. Dual-biofunctionalized scaffolds demonstrated superior vascularization induction in embryos and greater promotion of vascularization, myelination, and functional recovery in rats. These findings support the clinical potential of VEGF/IKVAV dual-biofunctionalized chitosan/collagen composite scaffolds for facilitating peripheral nerve regeneration, making it an attractive candidate for repairing critical nerve defect. The study may provide a critical experimental and theoretical basis for the development and design of new artificial nerve implants with excellent biological performance.

In the present study, a new approach for fabricating micropatterned polycaprolactone (PCL) scaffolds with ridge/groove structure on the surface was developed by combining use of electrospinning and micromolding method. A series of physicochemical properties, including morphology, wettability, component, crystal pattern and mechanical properties, of prepared PCL scaffolds were characterization, respectively. Stability of the micropatterned PCL scaffolds was measured using phosphate buffer solution immersion for a certain period. Then, the regulating effects of the micropatterned PCL scaffolds on attachment, orientation and normal biological function of Schwann cells were evaluated. And the protein adsorption behavior in various PCL scaffolds was also detected. The results showed that the micropatterned PCL scaffolds demonstrated a porous micro/nano complex structure with enhanced hydrophobicity and mechanical properties as a function of electrospun flow-rate of PCL solution. The micropatterned PCL scaffolds possessed good stability and could effectively regulate the attachment and orientation of Schwann cells at the early stage after cell culture. Importantly, the electrospun flow-rate of PCL solution was found to play an important role in scaffold properties, cell behavior and protein adsorption. The micropatterned scaffolds with a flow-rate of PCL solution at 0.12 mL h-1 demonstrated the better regulation on Schwann cells attachment and alignment without negatively affect the normal biological function of the cells. To the best of our knowledge, this is the first report of combining use of electrospinning and micromolding method for preparing artificial nerve implants. The study is anticipated to have potential application in peripheral nerve and other tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3123-3134, 2018.

Various hydrogels made from natural or synthetic polymers have been widely used in biologic tissues, drug delivery, and artificial implants due to their good biocompatibility, indicating a promising perspective in regenerative medicine. In the present study, a composite hydrogel named polyacrylamide/graphene oxide/gelatin/sodium alginate (PAM/GO/Gel/SA) for accelerating peripheral nerve regeneration was fabricated through in situ free radical polymerization for the first time. A series of physicochemical properties including morphology, porosity, swelling behaviors, component, mechanical properties, and in vitro degradation behavior of the prepared composite hydrogel were characterized. The effects of the composite hydrogel on Schwann cells growth were evaluated and the related molecular mechanism was further penetrated. The results showed that the prepared PAM/GO/Gel/SA composite hydrogels displayed different color appearance as the function of component variations. The surface morphology, components, swelling ratio, mechanical properties, and porosity were all changed with the concentration alteration of each ingredient, while no obvious degradation behavior was observed, indicating a controllable physicochemical property. The culture of cells exhibited that the composite hydrogels could well support the attachment and proliferation of Schwann cells. The gene expression levels of Sox10, GAP43, and myelin basic protein (MBP) in PGG0.5 SYR1 and PGG1 SYR0.5 were higher than those of NC. This study may provide important theoretical and experimental basis for the design and development of hydrogel scaffolds for nerve tissue engineering application. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1951-1964, 2018.

To further improve the property of promoting peripheral nerve regeneration of chitosan materials, CaTiO3 nanoparticles with various concentrations were synthesized in chitosan (CS) solution and formed to porous CS/CaTiO3 hybrid scaffolds. The properties including morphology, wettability, porosity, crystallization intensity and surface charges were characterized, respectively. The influence of the porous CS/CaTiO3 hybrid scaffolds on Schwann cells growth was evaluated. The results showed that the CaTiO3 hybridized CS scaffolds possessed homogeneous nanoparticles distribution with concentration-dependent effect. The hybridization of CaTiO3 nanoparticles could increase the hydrophobicity while reduce the porosity and surface charge density of the porous CS/CaTiO3 hybrid scaffolds The crystal structure of the hybridized scaffolds was mainly the orthorhombic structure of the calcium titanate accompanied by the amorphous phase of chitosan. Culture of Schwann cells indicated that the CS/CaTiO3 hybrid scaffolds with a suitable concentration of CaTiO3 nanoparticles could obviously promote the attachment, proliferation and biological function maintenance of Schwann cells, thus showing potentially great significance towards application in peripheral nerve regeneration.

Artificial chitosan scaffolds have been widely investigated for peripheral nerve regeneration. However, the effect was not as good as that of autologous grafts and therefore could not meet the clinical requirement. In the present study, the nerve growth factor (NGF) loaded heparin/chitosan scaffolds were fabricated via electrostatic interaction for further improving nerve regeneration. The physicochemical properties including morphology, wettability and composition were measured. The heparin immobilization, NGF loading and release were quantitatively and qualitatively characterized, respectively. The effect of NGF loaded heparin/chitosan scaffolds on nerve regeneration was evaluated by Schwann cells culture for different periods. The results showed that the heparin immobilization and NGF loading did not cause the change of bulk properties of chitosan scaffolds except for morphology and wettability. The pre-immobilization of heparin in chitosan scaffolds could enhance the stability of subsequently loaded NGF. The NGF loaded heparin/chitosan scaffolds could obviously improve the attachment and proliferation of Schwann cells in vitro. More importantly, the NGF loaded heparin/chitosan scaffolds could effectively promote the morphology development of Schwann cells. The study may provide a useful experimental basis to design and develop artificial implants for peripheral nerve regeneration and other tissue regeneration.

Mechanical property is an important factor of cellular microenvironment for neural tissue regeneration. In this study, polyacrylamide (PAM) hydrogels with systematically varying elastic modulus were prepared using in situ radical polymerization. We found that the hydrogel was biocompatible, and the length of dorsal root ganglion (DRG)'s axon and cell density were optimal on the hydrogels with elastic modulus of 5.1 kPa (among hydrogels with elastic modulus between 3.6 kPa and 16.5 kPa). These DRGs also exhibited highest gene and protein expression of proliferation marker Epha4, Ntn4, Sema3D and differentiation marker Unc5B. Our study revealed the mechanism of how material stiffness affects DRG proliferation and differentiation. It will also provide theoretical basis and evidence for the design and development of nerve graft with better repair performance.

The hydrogels have been widely used in various tissue engineering fields. Its excellent threedimension network structure and hydrophilicity could be used as cell culture scaffold for tissue regeneration. Graphene oxide (GO) related hydrogels have attracted more and more attention in recent years. However, rare report referring to the application of GO related hydrogel in peripheral nerve regeneration. In this study a composite hydrogel consisting of polyacrylamide, silk fibroin and graphene oxide was prepared. The materials properties were characterized and Schwann cells were cultured onto hydrogels in vitro to evaluate the influence on cell growth. The results showed that the composite hydrogel possessed excellent three-dimensional network structure, the hydrophilicity, wettability and porosity. Moreover, the mechanical strength of the hydrogels could be obviously enhanced. The morphological characterization of Schwann cells cultured on the composite hydrogels showed mononeuritis than single polyacrylamide hydrogel, indicating a better growth environment for cells. The results indicated that the polyacrylamide/silk fibroin/graphene oxide composite hydrogel with suitable content of each component could significantly affect the mechanical properties of the hydrogel and further the morphology of Schwann cells. The study provides a theoretical basis for design and potential application of hydrogels in regenerative medicine and tissue engineering.

Recently, chemotherapy-based polymeric nanoparticles have been extensively investigated for solid tumor treatment. Tumor targeted nanoparticles demonstrated great potential for improved accumulation in the tumor tissue, superior anticancer activity and reduced side effects. Thus, inulin-ibuprofen polymer was synthesized by esterification between inulin and ibuprofen, and RGD targeted epirubicin (EPB) loaded nanoparticles were prepared by the self-assembly of inulin-ibuprofen polymer and in situ encapsulation of EPB. RGD conjugated EPB loaded nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). The EPB release from the nanoparticles showed pH-dependent profile and accelerated by the decreased pH value, which would favor the effective drug delivery in vivo. Intracellular uptake analysis suggested that RGD conjugated nanoparticles could be easily internalized by the cancer cells. In vitro cytotoxicity revealed that RGD conjugated EPB loaded nanoparticles exhibited the better antitumor efficacy compared with non-conjugated nanoparticles. More importantly, RGD conjugated EPB loaded nanoparticles showed superior anticancer effects and reduced toxicity than free EPB and non-conjugated nanoparticles by in vivo antitumor activity, EPB biodistribution and histology analysis.

Various hydrogel materials have been developed for improving the regeneration of peripheral nerve. Among which the graphene related hydrogels with excellent mechanical properties have attracted great attention. However, the effect of these hydrogels on peripheral nerve regeneration is still unclear. In the present study, the graphene oxide/polyacrylamide (GO/PAM) composite hydrogels were fabricated by in-situ free radical polymerization. The morphology, wettability, composition, swelling ratio, mechanical property and degradation behavior of the prepared GO/PAM composite hydrogels were separately characterized. The effect of GO/PAM hydrogel on the attachment and proliferation of Schwann cells was evaluated. Moreover, the release of biofactors by Schwann cells and adsorption of matrix proteins were further measured. The results showed that the color of the hydrogel became darker with the increased GO concentration, while the surface pore structure also displayed large variation when GO concentration was increased. The hydrophobicity and mechanical properties of hydrogel were increased with the ascending GO concentration. In addition, the variation of GO concentration displayed no obvious influence on the degradation of the composite hydrogel in different medium. The GO/PAM composite hydrogel with 0.4% GO (G0.4) could effectively enhance the attachment and proliferation of Schwann cells. Furthermore, the cells on G0.4 hydrogel displayed higher biofactors release and larger matrix adsorption than other samples. The results demonstrated that GO with suitable concentration in PAM hydrogel could effectively promote Schwann cell growth. The study may provide an important experimental basis for the design and development of new nerve grafts with potential application for peripheral nerve regeneration.

Injectable hydrogels have potential applications for regenerating the injured spinal cord, but their performance needs to be further improved. In this study, an injectable silk fibroin/polydopamine (SF/PDA) composite hydrogel was fabricated via first Schiff base reaction between the amino groups of silk fibroin and the quinone structure of oxidized dopamine (DA), and then self-polymerization of free DA with SF-conjugated DA. The physicochemical properties of the hydrogel were characterized. The cytotoxicity and biocompatibility were evaluated using L929 fibroblasts and hippocampal neurons, respectively, and the expression levels of neurites-related proteins were analyzed by western blot. Finally, the effect of hydrogel on SCI repair was initially evaluated by in vivo animal experiment. With the increase of DA concentration, the hydrogels color became darker, the swelling rate, water uptake and the porosity decreased. Meanwhile, the Young's modulus continued to decrease when the DA concentration was increased from 0.5 mg/mL to 10 mg/mL, whereas rheological test showed the good injectability of the prepared hydrogels. NMR, FTIR and XPS confirmed that dopamine could combine with silk fibroin via Schiff base reaction and then polymerize to form the SF/PDA composite hydrogel. The hydrogels showed no cytotoxicity and the hippocampal neurons on hydrogel with 2 mg/mL DA displayed the largest axon length, the highest cell density and expression level of neuritis-related proteins. Further on, immunohistochemical staining showed initially that the prepared hydrogel possessed the capability of promoting SCI repair. In summary, the developed injectable SF/PDA hydrogel here may contribute to the potential application for promoting functional recovery of SCI, and provide important experimental and theoretical basis for developing SCI implants.

Peripheral nerve regeneration is a clinical challenge that tremendously affected the patients’ quality of life. Recently, anisotropic microtopology has shown promise in treating peripheral nerve regeneration. However, the systematically investigation of how micropatterning influence Schwann cells (SCs), including morphology, biofunction and gene expression etc, has never been investigated. Herein, we fabricated the chitosan micropatterning with four different ridge/groove (RG) and subsequently cultured the SCs on those samples. As a result, the SCs showed obvious orientationally growth to micropatterning and 30/30 μm size showed the best regulation effect. And in the meantime, 30/30 μm size did not affect the DNA synthesis phase of SC cells (G2 and S phase) and significantly down-regulated both the relative protein level of N- cadherin and β-catenin from SCs. More importantly, the anisotropic micropatterning could significantly up-regulate the expression of several key genes of SCs related to neuronal plasticity and axon regeneration (smad6), cytoskeleton development (β-actin) and myelination (MPZ). Overall, the results demonstrated that the anisotropic chitosan micropatterning with 30/30 μm size can regulate SCs’ orientation well with a tendency to myelination; and the systemic evaluation provide mechanical understanding, which acts as important references for future design of anisotropic biomaterials for peripheral nerve regeneration.

To penetrate the effect of protein gradient micropattern on peripheral nerve regeneration, the hierarchically aligned gradient collagen micropattern was prepared by micromoulding method and the influence on Schwann cells growth behavior was studied. The morphology, wettability, stability and component variation of the micropatterns were firstly characterized. Then, Schwann cells were cultured and the related mechanism was penetrated. The results showed that the gradient collagen micropattern could be well fabricated. The surface wettability varied with the change of collagen concentration, and the prepared gradient micropattern showed a good stability after PBS immersion for 15 days. The results of Schwann cells culture and morphological index analysis displayed that the prepared gradient collagen micropatten could well regulate the orientation growth of Schwann cells, while a much better cell alignment growth was obtained on the gradient micropattern with higher collagen concentration and wider pattern size. PCR and WB showed that the micropattern structure could effectively up-regulate the key specific genes for axon regeneration and myelination process. Overall, the study provides a systematic and facile method for understanding the effect of various sized micropatterns on cell behavior, which may have a great significance for the development of artificial implants for tissue engineering and regenerative medicine.

Chitosan has been well known for promoting peripheral nerve regeneration, however, its effect is still not as good as that of autografts. In this study, the feasibility of using negatively charged heparin and positively charged γ-aminopropyltriethoxysilane (APTE) treatment as biocompatible modification of lyophilized porous chitosanscaffolds was evaluated. The morphology of the prepared chitosan scaffolds as a function of treatment with different charged molecules showed no significant differences, while a skin-like surface was observed for the scaffolds modified with high APTE concentration and heparin. The quantitative and qualitative characterization of heparin and amino densities by Toluidine Blue O (TBO) and Acid Orange (AO) assays confirmed the successful immobilization of heparin and APTE on the chitosan scaffolds. The measurement of surface charge densities indicated that the scaffolds treated with APTE showed increased charge densities while heparin decreased the cationic charge density. Moreover, the fabricated charge processed chitosan scaffolds were stable after immersion in phosphate buffer saline for more than ten days. Further on, the chitosan scaffolds processed with 2 mg/mL heparin did facilitate the attachment, proliferation and maintain the biological function of Schwann cells in vitro . The study demonstrates that chitosan scaffolds treated with suitable heparin concentration provides an effective selection for biomaterials surface modification and shows great potential for the application in peripheral nerve regeneration.

The adsorption behavior of heparin and fibronectin was studied by quartz crystal microbalance with dissipation (QCM-D), and the interaction between heparin and fibronectin was evaluated using immunochemistry and isothermal titration calorimetry (ITC) measurement. The results showed that there was competitive adsorption between heparin and fibronectin, and the preadsorption of fibronectin could prevent subsequent heparin adsorption to some extent, and the adsorbed Hep/Fn complex on the surface was in a rigid form. The bioactivity of heparin and fibronectin could be affected by the bulk concentration of each, and both heparin and fibronectin in Hep/Fn complex formed under pH 4 condition displayed larger bioactivity than that formed under pH 7 condition. Moreover, the fibronectin showed more exposed cell-binding sites at the pH value lower than physiological condition. The results of ITC further suggested that the interaction between heparin and fibronectin under pH 4 was stronger than under pH 7, and the complex was also more stable. The study brings forth the detailed interaction between heparin and fibronectin, which will be helpful for better understanding the interaction mechanism of the two biomolecules. The results may be potentially useful for the development of new generation of cardiovascular biomaterials.

Various hydrogels have been used for repairing peripheral nerve injury; however, the silk fibroin (SF)-based hydrogels in peripheral nerve regeneration are still rarely reported. In this study, the SF/pAM hydrogels with different SF concentrations and ethanol treatment time were developed by solution blending and in situ radical polymerization. The physiochemical properties of composite hydrogels were measured, the cytotoxicity of hydrogels was evaluated by L929 fibroblasts, and the effect on peripheral nerve regeneration was evaluated via Schwann cells culture in vitro. The results showed that the physiochemical properties of SF/pAM hydrogels could be changed by varying SF concentration and ethanol treatment time, and the mechanical property was enhanced with increasing SF concentration, while the presence of SF in pAM hydrogels and ethanol treatment does not affect hydrogels structure in per se. All the composite hydrogels displayed no obvious cytotoxicity, while the SF/pAM composite hydrogels with 10% SF and 60-min ethanol treatment could obviously accelerate the attachment and proliferation of Schwann cells. Therefore, the SF/pAM composite hydrogels possessed the beneficial properties required for in situ cell scaffolding and may have potential application in peripheral nerve regeneration.

In this study, a facile process for efficient and scalable fabrication of biomaterials conduits with seamless sidewall and longitudinally aligned structure on luminal inwall by combining use of lyophilization and micromolding method was firstly developed and reported. The micropatterned chitosan conduits possessed better mechanical matching with the normal sciatic nerve. The highly aligned microstructure could accelerate the orientation growth of newborn tissue, whereas the porous sidewall is anticipated to be beneficial for loading biofactors and reducing nutrition leakage or neurite exogenesis. The as prepared chitosan conduits with micropatterned inwall are proven to significantly promote the regeneration of 10 mm-gapped sciatic nerve of rat. Taken together, the study provides an important experiment and application basis for design and development of new generation of artificial implants for peripheral nerve regeneration, which may also substantially extend current knowledge concerning the engineered biomaterials conduits for tissue engineering and regenerative medicine.

Ibuprofen modified inulin was synthesized through a direct esterification linkage in which the in situ activation of the carboxylic acid with N,N'-carbonyldiimidazole was carried out. The critical aggregation concentration of the ibuprofen modified inulin was determined by using pyrene as the fluorescence probe. Methylprednisolone loaded nanoparticles were prepared by the self-assembly of the ibuprofen modified inulin copolymer and methylprednisolone. In vitro release of the methylprednisolone and the cytotoxicity of the methylprednisolone loaded nanoparticles against RSC-96 cells were evaluated. Since the ibuprofen and methylprednisolone could stimulate a significant neurite growth and diminish the human neurological deficits after the spinal cord injury, the methylprednisolone loaded nanoparticles based on the ibuprofen modified inulin copolymer may have a great potential in the synergetic effect treatment for spinal cord injury.

To address the effect of chitosan micropatterning on nerve regeneration, two sizes of parallel microstripes of chitosan are fabricated on the surface of coverslips using a micromodeling method. The morphology of the prepared polydimethylsiloxane stamps and chitosan micropatterning is observed by scanning electron microscopy and the wettability of the prepared micropatterning is evaluated using water contact-angle measurements. Schwann cell (SC) culture is used to evaluate the effect of chitosan micropatterning on cell behavior. The results show that the stripe-like chitosan micropatterning can be successfully fabricated on coverslip surfaces. SCs on 30/30 μm chitosan micropatterning shows the most obvious cell orientation. Moreover, the secretion of nerve growth factor by SCs indicate that the chitosan micropatterning has no negative influence on the normal physiological function of the cells. Thus, the study suggests that chitosan micropatterning can induce and regulate the growth of SCs well, which may have potential application in peripheral nerve regeneration.

Mulberroside A is a natural polyhydroxylated stilbene compound present at relatively high abundance in the roots and twigs of Morus alba L. It is known for its nephroprotective, hypoglycemic, and antidiabetic effects. Because its metabolite, oxyresveratrol, possessed purported anti-inflammatory and neuroprotective effects, we proposed that mulberroside A may elicit neuroprotective effects that can be used in the treatment of brain ischemic injury. Therefore, we decided to investigate the pharmacological properties of mulberroside A in primary culture of rat cortical neurons after oxygen-glucose deprivation followed by reperfusion (OGD/R), evaluating its ability to counteract the hypoxia-ischemia impairment. The results showed that mulberroside A elicited neuroprotective effects comparable to nimodipine. The mechanistic studies showed that mulberroside A decreased the expressions of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 and inhibited the activation of NALP3, caspase-1, and nuclear factor-κB and the phosphorylation of extracellular signal-regulated protein kinases, the c-Jun N-terminal kinase, and p38, exhibiting anti-inflammatory antiapoptotic effects. Our results also further demonstrate that the proinflammatory cytokines of IL-1β, IL-6, and TNF-α are promising targets for treatment of cerebral ischemic injury. Although further investigation is required for its development, all of these findings led us to speculate that mulberroside A is a candidate for the treatment of ischemic stroke, which would act as a multifactorial neuroprotectant.

The morphology of vascular smooth muscle cells (SMCs) in the normal physiological state depends on cytoskeletal distribution and topology beneath, and presents vertical to the direction of blood flow shear stress (FFSS) although SMCs physiologically are not directly exposed to the shear conditions of blood flow. However, this condition is relevant for arteriosclerotic plaques and the sites of a vascular stent, and little of this condition in vitro has been studied and reported till now. It is unclear what will happen to SMC morphology, phenotype and function when the direction of the blood flow changed. In this paper, the distribution of SMCs in a specific area on Ti surface was regulated by micro-strips of hyaluronic acid (HA). Cell morphology depended on the distribution of the cytoskeleton extending along the micrographic direction. Simulated vascular FFSS was perpendicular or parallel to the direction of the cytoskeleton distribution. Based on investigating the morphology, apoptotic number, phenotypes and functional factors of SMCs, it was obtained that SMCs of vertical groups showed more apoptosis, expressed more contractile types and secreted less TGF-β1 factor compared with SMCs of parallel groups, the number of ECs cultured by medium from SMCs of parallel groups was larger than vertical groups. This study could help to understand the effect of direction change of FFSS on patterned SMC morphology, phenotype and function.

Both coverage and morphology of endothelial cells (ECs) on blood contact biomaterials were involved in the antithrombotic and anti-proliferative process. However, most researches have been focused on the ECs amount on the materials surface, but not the morphology. As of now, little is known about the relationship between the ECs morphology and the cytokine secretion, especially those anticoagulant factors on cardiovascular implants. In this study, three sizes of parallel micro-stripes of high molecular weight hyaluronic acid (HMW-HA) were fabricated on the NaOH-treated titanium (TiOH) surface. These micro-patterns of ridges/grooves were used to regulate the distribution, morphology and cytokine secretion of the ECs. The roughness of bare Ti and TiOH was detected by AFM, and the micro-pattern sizes were imaged by SEM. The chemical bonding of HMW-HA to the TiOH surface was demonstrated by FTIR, and the element distribution was examined by EDX. The morphology index of the ECs was analyzed to evaluate the orientation, elongation and spreading of the ECs on the micro-patterned surfaces and the control, while the cell number and cytokine secretion of the ECs were measured by related kits. The characterization of the ECs demonstrated that the bigger L/B index improved NO, PGI2, Fn release and TM, TFPI, E-Selectin expression as well as reducing vWF secretion. The platelet adhesion test and the whole blood clotting time test proved the increasing anticoagulation property of ECs with their elongation increasing. These results suggest that ECs morphology-related function can be regulated by different sizes of micro-patterns.

Micro-patterning as an effective bio-modification technique is increasingly used in the development of biomaterials with superior mechanical and biological properties. However, as of now, little is known about the simultaneous regulation of endothelial cells (EC) and smooth muscle cells (SMC) by cardiovascular implants. In this study, a co-culture system of EC and SMC was built on titanium surface by the high molecular weight hyaluronic acid (HMW-HA) micro-pattern. Firstly, the micro-pattern sample with a geometry of 25 μm wide HMW-HA ridges, and 25 μm alkali-activated Ti grooves was prepared by microtransfer molding (μTM) for regulating SMC morphology. Secondly, hyaluronidase was used to decompose high molecular weight hyaluronic acid into low molecular weight hyaluronic acid which could promote EC adhesion. Finally, the morphology of the adherent EC was elongated by the SMC micro-pattern. The surface morphology of the patterned Ti was imaged by SEM. The existence of high molecular weight hyaluronic acid on the modified Ti surface was demonstrated by FTIR. The SMC micro-pattern and EC/SMC co-culture system were characterized by immunofluorescence microscopy. The nitric oxide release test and cell retention calculation were used to evaluate EC function on inhibiting hyperplasia and cell shedding, respectively. The results indicate that EC in EC/SMC co-culture system displayed a higher NO release and cell retention compared with EC cultured alone. It can be suggested that the EC/SMC co-culture system possessed superiority to EC cultured alone in inhibiting hyperplasia and cell shedding at least in a short time of 24 h.

Reducing platelet adhesion, controlling platelet distribution and decreasing degree of platelet activation are very important for improving blood compatibility of cardiovascular implants. In this study, the micropattern of heparin and fibronectin mixture (Hep-Fn) embedded within a cell-resistant α-methoxy-poly(ethylene glycol)-ω-succinimidyl carbonate (mPEG-SC) background was fabricated on titanium (Ti) surface. Firstly, the Ti sample was activated by NaOH solution and treated with ethanol solution of (3-aminopropyl)-triethoxysilane (APTE). Secondly, microtransfer molding (μTM) was used to pattern mPEG-SC for resisting protein absorption and platelet adhesion on silanized Ti surface. Finally, Hep-Fn mixture was absorbed on the areas uncovered by mPEG-SC to form a surface for selective adhesion of platelet. Scanning electron microscopy (SEM) showed the surface morphology of the patterned Ti, Fourier transform infrared spectroscopy (FTIR) demonstrated the existence of Hep-Fn and mPEG-SC on the modified Ti surface. The platelet adhesion test was used to evaluate adhesion, activation and distribution of platelets on the bare Ti samples with or without biomolecule micropattern. The results indicated that the patterned samples could effectively reduce platelet adhesion, control platelet distribution and influence platelet activation compared with the non-patterned pure Ti sample. The micropattern sample, P25/10 with a geometry of 25 μm mPEG-SC (ridges) and 10 μm Hep-Fn (grooves), presented better blood compatibility.

One major obstacle in the application of drug delivery systems for cancer chemotherapy is their poor penetration in tumor tissues. Conjugating active ligand moieties to the surface of nanoparticles may be a promising approach for enhancing the tumor accumulation and penetration of nanoparticles. Herein, the cell-penetrating peptide twin-arginine translocation (Tat)-conjugated epirubicin-loaded poly(lactic-glycolic acid) nanoparticles were prepared to achieve deep tumor penetration. The morphology and size of nanoparticles were characterized by scanning electron microscopy and dynamic light scattering, and the biological behaviors of nanoparticles were evaluated. It is demonstrated that Tat-conjugated nanoparticles have a significant improvement in antitumor activity and biodistribution compared with nonconjugated nanoparticles. Importantly, Tat conjugated on the surface of nanoparticles could facilitate the encapsulated drug penetration into deeper tumor tissue. Additionally, Tat-conjugated nanoparticles have good biocompatibility, as demonstrated by hemolytic tests, in vitro cytotoxicity, and histology study. These results suggested that the Tat-conjugated nanoparticles, as a powerful delivery system for chemotherapeutic drug, would have a promising application in human cancer therapy.

Chitosan is found to promote the regeneration of peripheral nerve system in our previous studies, whereas the regeneration speed is not satisfied with clinical request. Micropatterning could promote cell orientation and growth, however, the effect of porous chitosan micropatterning on nerve regeneration is rarely reported. In this study, the porous chitosan micropatterning with surface ridge/groove and inner porosity structure was fabricated using a combination of micromodeling and lyophilization method. The morphology and stability of the prepared chitosan micropatterning were evaluated, the regulation of Schwann cells behavior by chitosan micropatterning was evaluated. The results showed that the chitosan micropatterning displayed stripe-like structure with a clear and complete edge. The micropatterning with 30/30 μm was more stable than 20/20 μm sample. Schwann cells on chitosan micropatterning showed orientation adhesion and began to grow along a certain direction after culture for 2 h, and displayed the minimal orientation angle and the largest length/width ratio on 30/30 μm micropatterning after further culture for 3 d and 5 d, indicating the most obvious cell orientation. Moreover, the secretion of nerve growth factor (NGF) demonstrated that the micropatterned chitosan had no negative influence on the physiological function of Schwann cells. Thus, the results indicate that the porous chitosan micropatterning can regulate Schwann cell growth well, which may have potential application in nerve regeneration. The study provides an important basis for constructing porous nerve conduit with micropatterning structure in the inner wall.

The aim of this study was to evaluate the feasibility of using 3-aminopropyltriethoxysilane (APTE) silanization treatment for modification and biocompatibility of lyophilized chitosan porous scaffolds. The process is beneficial for biomaterial development due to its low toxicity and simplicity. The silanization treatment with low APTE concentration showed no significant influence on the morphology of chitosan scaffolds, while a skin-like surface was observed for the silanized scaffolds treated with high APTE concentration. The porosity and surface amino densities were increased after silanization whereas the swelling ratio was reduced, and the degradation ratio in PBS and anti-acid degradation properties of the silanized chitosan scaffolds were significantly improved. The in vitro Schwann cells culture demonstrated that the silanized scaffolds with 8% APTE could obviously facilitate the attachment and proliferation of Schwann cells, indicating great potential for the application in peripheral nerve regeneration.

To improve the blood compatibility and endothelialization simultaneously and to ensure the long-term effectiveness of the cardiovascular implants, we developed a surface modification method, enabling the coimmobilization of biomolecules to metal surfaces. In the present study, a heparin and fibronectin mixture (Hep/Fn) covalently immobilized on a titanium (Ti) substrate for biocompatibility was investigated. Different systems [N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide, electrostatic] were used for the formation of Hep/Fn layers. Atomic force microscopy (AFM) showed that the roughness of the silanized Ti surface decreased after the immobilization of Hep/Fn. Fourier transform infrared spectroscopy (FTIR), Toluidine Blue O (TBO) test, and immunochemistry assay showed that Hep/Fn mixture was successfully immobilized on Ti surface. Blood compatibility tests (hemolysis rate, APTT, platelet adhesion, fibrinogen conformational change) showed that the coimmobilized films of Hep/Fn mixture reduced blood hemolysis rate, prolonged blood coagulation time, reduced platelets activation and aggregation, and induced less fibrinogen conformational change compared with a bare Ti surface. Endothelial cell (EC) seeding showed more EC with better morphology on pH 4 samples than on pH 7 and EDC/NHS samples, which showed rounded and aggregated cells. Systematic evaluation showed that the pH 4 samples also had much better blood compatibility. All results suggest that the coimmobilized films of Hep/Fn can confer excellent antithrombotic properties and with good endothelialization. We envisage that this method will provide a potential and effective solution for the surface modification of cardiovascular implant materials.

Immobilization of biomolecules with a variety of biological functions has been a promising method to improve the biocompatibility of biomaterials. However, little is known about their inflammatory property and cytotoxicity, which are both key aspects to most biomaterials designed for tissue engineering applications and in vivo implantation. In this in vitro study, heparin/fibronectin complex (Hep/Fn) was coimmobilized onto titanium surface (HF-Ti), which had been proven to have the properties of both anticoagulation and endothelialization in our previous study. Fourier transform infrared (FTIR) spectroscopy and water contact angle measurement were utilized to determine the surface chemical compositions and physical properties. Toluidine Blue O (TBO) and immunochemistry methods were performed to quantify the surface-immobilized heparin and fibronectin. The early inflammatory responses elicited by pristine Ti and HF-Ti were investigated by proinflammatory cytokine secretion of tumor necrosis factor-alpha (TNF-α) released by attached peritoneal macrophages, monocyte chemoattractant protein-1 (MCP-1) and interleukin-1β (IL-1β) released by attached human umbilical vein endothelial cells (ECs), respectively. Scanning electronic microscopy (SEM) and immunofluorescence were employed to investigate the changes in macrophages and ECs morphologies. The incubation period for both cells was 24h and the results showed that HF-Ti revealed a weaker inflammatory response than pristine Ti, which provoked a stronger inflammatory response and higher activation of macrophages. Our data suggest that Hep/Fn coimmobilized biomaterials surface may develop to be a new generation of biomaterials with both biocompatibility and anti-inflammatory properties, especially for used as cardiovascular implants and in tissue engineering applications.

Coimmobilization as a versatile biomodification technique has been widely used in the development of biomimetic materials with superior mechanical and biological properties. In this study, a mixture of heparin and fibronectin (Hep/Fn) was tested for its hemocompatibility after either physisorption or covalent coimmobilization to a titanium (Ti) substrate. The process of substrate activation and film deposition was associated with an increase of roughness; successful immobilization in both cases was demonstrated by FTIR. The immobilized heparin amount was probed by toluidine blue O binding, fibronectin by immunochemistry. Both molecules had slightly higher concentrations on the physisorbed film than after covalent coimmobilization. Plasmatic coagulation activation, tested as activated partial thromboplastin time APTT, and platelet adhesion were significantly improved on the covalently coimmobilized samples than on the physisorbed or blank ones. All these results suggest that the covalent coimmobilization of heparin with fibronectin improved the anticoagulant activity of heparin and caused a favorable blood compatibility. We envisage that this method will provide a potential and effective selection for biomaterials surface modification.

In the present study, bovine serum albumin (BSA) was successfully covalently immobilized on the surface of anatase TiO 2 film by a three-step method, i.e. application of H 3 PO 4 chemisorption to increase surface –OH, which increases the amount of coupling 3-aminopropyl-triethoxylsilane (APTES), thus linking with BSA by imide bond using EDC/NHS/MES. There is no significant –OH group increase on rutile film when using the same method of phosphoric acid treatment, which suggest it is difficult for further chemical modification of the rutile film. After covalent immobilization of BSA on anatase film, an improved hemocompatibility of anti-platelet adhesion and aggregation in vitro could be recognized by LDH and SEM analysis. This study suggests BSA-immobilized anatase surface can serve as hemocompatibility material in vivo.

Peripheral nerve functional recovery after nerve injury generally requires multiple growth factors by synergistic effect. However, the optical combination of multiple synergistic growth factors for axonal regeneration has been scarcely considered up to now. Meanwhile, the use of growth factors in promoting nerve regeneration was limited by its short biological half-life in vivo, its vulnerability to structure disruption or hydrolyzation, leading to loss of bioactivity. Herein, a novel polymeric nanoparticle delivery system composed of heparin and ɛ-poly-L-lysine (PL) was prepared for control release of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF). The nanoparticles were synthesized by polyelectrolyte complexation in aqueous solution at room temperature, followed by cross-linking with biological genipin. The obtained nanoparticles had a spherical shape, with a mean diameter of about 246 nm, and high growth factors encapsulation efficiency as well as good stability. NGF and bFGF were encapsulated in the nanoparticles and showed a continuous and slow release behavior in vitro. The bioactivities of the released growth factors were evaluated, and exhibited the synergistic effect. The controlled release of the dual synergistic growth factors would improve the treatment of peripheral nerve injury to mimic the natural cellular microenvironments.