Adhesion Of L929 Cells Research Articles

Stimuli-responsive biphenyl-tripeptide supramolecular hydrogels as biomimetic extracellular matrix scaffolds for cartilage tissue engineering

Supramolecular hydrogel composed of aromatic short peptide gelator was an attractive biomaterial owing to its simple and convenient synthetic route, nano-fibrillar microstructure resembling natural collagen fibers and intelligent response to external stimulus. Herein, stimuli-responsive biphenyl-tripeptide supramolecular hydrogels was prepared to simulate extracellular matrix scaffolds by temperature switch, ion induction and pH switch. The amino acid arrangement substantially affected gelation behavior, only BPAA-βAFF and BPAA-FFβA could form nanostructured supramolecular hydrogels with 8-10 nm nanotubes or nanofibers by potential intermolecular hydrogen bond interactions and π-π stacking. The minimum gelation concentration (MGC) and maximum storage modulus were 0.4 mM (0.023 wt%) and around 8.2 KPa. The two supramolecular hydrogels could support adhesion and proliferation of L929 cells. Moreover, the BPAA-βAFF hydrogel promoted proliferation and ECM secretion of chondrocytes in vitro, and facilitate the phenotype maintenance of hyaline cartilage. All the results demonstrated that BPAA-βAFF hydrogel hold great potential application prospects in cartilage tissue engineering. Statement of SignificanceDiphenylalanine was served as a core segment conjugating with 4-biphenylacetic acid (BPAA) to produce biphenyl-tripeptide compounds with transforming amino sequence, and multiple external stimuli was applied to study the gelation properties of the aromatic short peptide gelators. “FF” brick (phenylalanine-phenylalanine) was crucial for formation of fibrous supramolecular hydrogels. Meanwhile, the sequence of amino acids arrangement also had an essential effect on the gelation behavior. Optimal BPAA-βAFF with ultra-low minimum gelation concentration (0.4 mM, about 0.023 wt%) and similar microstructure to extracellular matrix (ECM) of nature cartilage tissue could promote the proliferation and ECM secretion of chondrocytes in vitro, and facilitate the formation of hyaline cartilage.

Biocompatible and antibacterial soy protein isolate/quaternized chitosan composite sponges for acute upper gastrointestinal hemostasis.

Innovative biomedical applications have high requirements for biomedical materials. Herein, a series of biocompatible, antibacterial and hemostatic sponges were successfully fabricated for the treatment of acute upper gastrointestinal bleeding (AUGB). Quaternized chitosan (QC) and soy protein isolate (SPI) were chemically cross-linked to obtain porous SPI/QC sponges (named SQS-n, with n = 30, 40, 50 or 60 corresponding to the weight percentage of the QC content). The chemical composition, physical properties and biological activity of SQS-n were investigated. SQS-n could support the adhesion and proliferation of L929 cells while triggering no obvious blood toxicity. Meanwhile, SQS-n exhibited good broad-spectrum antibacterial activity against both gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). The in vivo hemostatic effect of SQS-n was evaluated using three different bleeding models. The results revealed that SQS-50 performed best in reducing blood loss and hemostatic time. The overall hemostatic effect of SQS-50 was comparable to that of a commercial gelatin sponge. The enhanced antibacterial and hemostatic activities of SQS-n were mainly attributed to the QC component. In conclusion, this work developed a QC-functionalized hemostatic sponge that is highly desirable for innovative biomedical applications, such as AUGB.

Conjugation of Polysulfobetaine via Poly(pyrogallol) Coatings for Improving the Antifouling Efficacy of Biomaterials.

Antifouling treatment is critical to certain biomedical devices for their functions and patients’ life. Facial, versatile, and universal coating methods to conjugate antifouling materials on a wide variety of biomaterials are beneficial for the fabrication of low-fouling biomedical devices. We developed a simple one-step coating method for surface conjugation of zwitterionic poly(sulfobetaine) via deposition of self-polymerized pyrogallol (PG). Poly(pyrogallol) could deposit copolymers of sulfobetaine methacrylate and aminoethyl methacrylate (pSBAE) on various biomaterials. pSBAE coatings inhibited as high as 99.8% of the adhesion of L929 cells and reduced protein adsorption significantly. The resistance against L929 cell adhesion was increased with increasing coating time and was positively correlated with the surface hydrophilicity and film thickness. Such a coating was robust to resist harsh sterilization conditions and stable for long-term storage in phosphate-buffered saline. We expect that the simple low-fouling pSBAE coating is applicable to the manufacture of medical devices.

Facile fabrication of soy protein isolate-functionalized nanofibers with enhanced biocompatibility and hemostatic effect on full-thickness skin injury.

Extensive full-thickness skin defect lacks self-healing ability. Tissue engineering wound dressing is considered as the most promising approach to promote wound healing. In this study, a series of biocompatible and hemostatic nanofiber dressings were fabricated. Soy protein isolate (SPI) and poly(L-lactic acid) (PLLA) solutions were mixed in certain proportions for high-voltage electrospinning. The obtained products were coded as SPNF-n (n = 100, 80, 60 and 40, corresponding to the weight percentage of PLLA solution). We found that SPNF-n (n = 100, 80, 60 and 40) could facilitate the adhesion and spread of L929 cells. In particular, SPNF-80 was capable of promoting fibroblast proliferation and diminishing inflammation. Compared with the neat PLLA film (SPNF-100), the biosafety and hemostatic effect of SPNF-80 got significantly improved. The hemostatic effect of SPNF-80 was comparable with that of a commercial gelatin sponge. In vivo wound healing assay demonstrated that SPNF-80 could accelerate the wound healing process by enhancing vascularization, re-epithelization and collagen formation. In conclusion, our results reveal that SPNF-n has good biocompatibility and hemostatic effect, and exhibits great application potential in wound healing.

Preparation of Poly(ether-ether-ketone)/Nanohydroxyapatite Composites with Improved Mechanical Performance and Biointerfacial Affinity.

Poly(ether-ether-ketone) (PEEK) displays promising potential in hard tissue repair and orthopedic surgery due to its adaptable mechanical performance, good chemical resistance, and bioinertness. However, the low biointerfacial affinity of pure PEEK implants and the decrease of mechanical strength after processing greatly limit their clinical applications. In this work, the influences on mechanical performance and biointerfacial affinity of the PEEK/nanohydroxyapatite (nHA) composites are systematically investigated. Results show that the mechanical performance of PEEK/nHA composites was improved by adjusting the nHA content. The maximum values of the tensile, compressive, bending, and impact strength of the composites were increased by approximately 16.2, 25, 54, and 21%, respectively, when compared with that of pure PEEK. Studies in vitro show that PEEK/nHA composites display good cytocompatibility and promote the biomimic formation of HA, adhesion, and proliferation of L929 cells on the surface. Studies in vivo demonstrate that, compared to the pure PEEK, PEEK/nHA composites exhibit higher biointerfacial affinity, including the adhesion and encapsulation of muscle tissues on the surface of the implants and the suppression of inflammatory reaction around the implants. Our findings could pave the way for extensive applications of PEEK/nHA composites in hard tissue repair, particularly orthopedic surgery.

Versatile Cell-Specific Ligand Arrangement System onto Desired Compartments of Biodegradable Matrices for Site-Selective Cell Adhesion Using DNA Tags.

A promising approach for the regeneration of tissues or organs with three-dimensional hierarchical structures is the preparation of scaffold-cell complexes that mimic these hierarchical structures. This requires an effective technique for immobilizing cell-specific ligands at arbitrarily chosen positions on matrices. Here, we report a versatile system for arranging cell-specific ligands onto desired compartments of biodegradable matrices for site-selective cell arrangement. We utilized the specific binding abilities of specific DNAs, immobilizing them as tags to arrange cell-recognition ligands at desired areas of the matrices by specific binding with cell-recognition ligand-DNA conjugates. We synthesized poly(l-lactide) (PLLA), a biodegradable polymer, with an oligo-DNA (trimer of deoxyguanosine: dG3) attached via a poly(ethylene glycol) (PEG) spacer to generate dG3-PEG-b-PLLA. The peptides Arg-Gly-Asp-Ser (RGDS) and Arg-Glu-Asp-Val (REDV) were chosen as cell-recognition ligands and were attached to an adapter DNA (aDNA), which can specifically bind to the dG3 moiety through G-quadruplex formation. The obtained dG3-PEG-b-PLLA was deposited on a small spot of the PLLA film, and the aDNA-RGDS or aDNA-REDV conjugate was added on the film to immobilize these ligands at the spot. We confirmed the specific adhesion of L929 cells (a mouse fibroblast cell line) and human umbilical vein endothelial cells (HUVECs) on the small areas coated with dG3-PEG-b-PLLA in the presence of aDNA-RGDS and aDNA-REDV, respectively, even after applying shear stress by flowing medium across the spot. Cell-specific attachment of the target cells was effectively achieved in a spatially controlled manner. This technique has the potential for the construction of cell-scaffold complexes that mimic the hierarchical structures of natural organs and may represent a breakthrough in realizing regenerative medicine and tissue engineering of complex organs.

Thermoresponsive Poly(2-propyl-2-oxazoline) Surfaces of Glass for Nonenzymatic Cell Harvesting.

As one of the nonenzymatic cell-harvesting technologies, a thermal-responsive surface based on poly(2-oxazoline)s has achieved initial success in supporting the adhesion and thermal-induced detachment of animal cells. However, because of the laborious preparation procedure, this technique was only limited to research purposes. In this work, through using poly(glycidyl methacrylate) (PGMA) as the anchor layer, poly(2-propyl-2-oxazoline)s (PPOx) were grafted onto glass wafers through a facile two-step coating and annealing procedure for nonenzymatic cell harvesting. In the first step, the piranha solution-activated glass wafers were immersed into the chloroform solution of PGMA and then annealed for a given period of time to immobilize PGMA onto the glass wafers through the bonding between epoxy groups and hydroxyl groups. In the second step, the PGMA-coated glass wafers were further immersed into the chloroform solution of carboxyl-functionalized PPOx. After annealing, PPOx were immobilized onto the PGMA layer through the bonding between carboxyl groups and the residual epoxy groups. Atomic force microscopy, X-ray photoelectron spectroscopy, and ellipsometry were used to characterize the modified glass wafers. The results of cytocompatibility evaluation showed that the PPOx-coated glass wafers were almost nontoxic and were able to support the adhesion and proliferation of L929 cells well. By lowering the temperature to 8 °C, L929 and Vero cells were successfully detached from the PPOx-coated glass wafers without any enzymatic treatment. Further cultivation has demonstrated that the cooling procedure had little effect on cell viability, and the cells still retained good viability after harvesting.

Nonadherent Zwitterionic Composite Nanofibrous Membrane with a Halloysite Nanocarrier for Sustained Wound Anti-Infection and Cutaneous Regeneration.

Wound dressing synechia and sustained postoperative bacterial infection would cause serious secondary damage to nascent cutaneous tissue and impede normal regeneration of injured wound. Endowing wound dressings with nonadherent capability and long-lasting antibacterial property could optimize the postoperative wound healing conditions and promote wound tissue neogenesis, which have important clinical application value and demand. In this study, novel nanocarrier-embedded zwitterionic composite nanofibrous membranes are fabricated using the co-electrospinning/photo-cross-linking method for the purpose of painless removal and eliminating long-lasting antibacterial infection during postoperative wound therapy. The prepared membranes possess good biocompatibility, excellent antibiofouling ability against both bacteria and plasma proteins, and platelet and L929 cell adhesion. Furthermore, in vitro and in vivo antibacterial evaluations exhibit that the composite nanofibrous membranes with a sustained drug release profile could effectively inhibit bacterial proliferation for at least 16 days. Additionally, in vivo wound regeneration assessment indicates that the obtained membranes could better enhance skin regeneration than the commercial 3M Tegaderm film, which highlights the application prospect of such novel zwitterionic composite nanofibrous membranes for sustained postoperative wound anti-infection and cutaneous regeneration.

Novel porous three-dimensional nanofibrous scaffolds for accelerating wound healing

Too small pore size of electrospinning wound dressings limits cells of adjacent tissue to grow into the wound. Herein, a new aqueous phase fiber reassembly technology was addressed to fabricate polycaprolactone-polyethylene glycol-polycaprolactone (PCL-PEG-PCL) 3D nanofibrous scaffolds (TNSs) as a promising candidate for accelerating wound healing. The TNSs showed larger pore size with higher porosity compared with PCL-PEG-PCL 2D mats. The micron-sized hierarchical porous structure of TNSs enhanced water storage capacity, resulting in excellent water absorption. In addition, likely due to the rough surface and loose pore structure, the TNSs could effectively enhance L929 cell adhesion, proliferation and migration behaviors compared to that of 2D mats. Finally, in vivo tests supported the results of cellular experiments, TNSs could significantly promote the wound healing compared with 2D mats.

Poly(DL-Lactide-co-ε-Caprolactone)/Poly(Acrylic Acid) Composite Implant for Controlled Delivery of Cationic Drugs.

Poly(DL-lactide-co-ε-caprolactone)/poly(acrylic acid) implantable composite reservoirs for cationic drugs are synthesized by sequentially applying photoirradiation and liquid phase inversion. The chemical composition and microstructure of reservoirs are characterized with Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and scanning electron microscopy (SEM), respectively. Drug loading and release properties are investigated using methylene blue as the drug model. Biocompatibility of reservoirs is examined through a series of in vitro tests and an in vivo experiment of subcutaneous implantation in Dark Agouti rats. Reservoirs show good ion-exchange capacity, high water content, and fast reversible swelling with retained geometry. Results of drug loading and release reveal excellent loading efficiency and diffusion-controlled release during 2 weeks. Biocompatibility tests in vitro demonstrate the lack of implant proinflammatory potential and hindered adhesion of L929 cells on the implant surface. Implants exhibit low acute toxicity and elicit a normal acute foreign body reaction that reaches the early stages of fibrous capsule formation after 7 days.

Polyurethane/poly(d,l-lactic acid) scaffolds based on supercritical fluid technology for biomedical applications: Studies with L929 cells

Biomaterials can be applied in tissue engineering as scaffolds that resemble the extracellular matrix functioning as a temporary structure for cell proliferation and reconstruction of new organs and tissues. To evaluate the potential use of scaffolds as a biomaterial, this work proposes the development and characterization of polyurethane (PU), poly(D,L-lactic acid) (PDLLA) and polyurethane/poly(d,l-lactic acid) (PU/PDLLA) scaffolds produced by gas foaming technique. The neat polymers and the blends were characterized, in film form, by gel permeation chromatography (GPC), thermogravimetry (TG), differential scanning calorimetry (DSC) and field emission gun scanning electron microscopy (FEG-SEM). After supercritical fluid technology, in scaffolds form, the samples were characterized by FEG-SEM, pore size, density, cytotoxicity and cell adhesion. For film characterization the PU/PDLLA sample presented intermediate characteristics compared to the neat polymers, exhibiting the behavior of both polymers in the sample without phase separation in the FEG-SEM micrograph and bimodal molar weight distribution by GPC. The scaffolds showed interconnectivity and pore size of 141 μm ± 108 μm for PUsc and 52 μm ± 32 μm for PDLLAsc. The PU/PDLLAsc exhibited a bimodal structure in which the PU in the mixture revealed pores of 75 μm ± 57 μm, while for PDLLA, the pore size was 19 μm ± 12 μm. In vitro tests confirmed the adhesion of L929 cells to PUsc, PDLLAsc and PU/PDLLAsc, showing no cytotoxic effect. Finally, it can be concluded that it is possible to produce PU, PDLLA and PU/PDLLA scaffolds by supercritical fluid, which may be applied as biomaterials.

An RGD-Containing Peptide Derived from Wild Silkworm Silk Fibroin Promotes Cell Adhesion and Spreading.

Arginine-Glycine-Aspartate (RGD) tripeptide can promote cell adhesion when present in the amino acid of proteins such as fibronectin. In order to demonstrate the bioactivity of an RGD-containing silk protein, a gene encoding the RGD motif-containing peptide GSGAGGRGDGGYGSGSS (–RGD–) derived from nonmulberry silk was designed and cloned, then multimerised and inserted into a commercial pGEX expression vector for recombinant expression of (–RGD–)n peptides. Herein, we focus on two glutathione-S-transferase (GST)-tagged fusion proteins, GST–(–RGD–)4 and GST–(–RGD–)8, which were expressed in Escherichia coli BL21, purified by GST affinity chromatography, and analyzed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). Target peptides (–RGD–)4 and (–RGD–)8 (6.03 and 11.5 kDa) were cleaved from the GST-tag by thrombin digestion, as verified with MS and SDS-PAGE. Isoelectric point analysis confirmed that target peptides were expressed and released in accordance with the original design. Target peptides self-assembled into a mainly α-helical structure, as determined by circular dichroism spectroscopy. Furthermore, (–RGD–)4 and (–RGD–)8 modified mulberry silk fibroin films were more effective for rapid cell adhesion, spreading and proliferative activity of L929 cells than some chemically synthesized RGD peptides modified and mulberry silk lacking the RGD motif.

Three-Dimensionally Printed Silk-Sericin-Based Hydrogel Scaffold: A Promising Visualized Dressing Material for Real-Time Monitoring of Wounds.

A wound dressing which can be convenient for real-time monitoring of wounds is particularly attractive and user-friendly. In this study, a nature-originated silk-sericin-based (SS-based) transparent hydrogel scaffold was prepared and evaluated for the visualization of wound care. The scaffold was fabricated from a hybrid interpenetrating-network (IPN) hydrogel composed of SS and methacrylic-anhydride-modified gelatin (GelMA) by 3D printing. The scaffold transformed into a highly transparent hydrogel upon swelling in PBS, and thus, anything underneath could be easily read. The scaffold had a high degree of swelling and presented a regularly macroporous structure with pores around 400 μm × 400 μm, which can help maintain the moist and apinoid environment for wound healing. Meanwhile, the scaffolds were conducive to adhesion and proliferation of L929 cells. A coculture of HaCaT and HSF cells on the scaffold showed centralized proliferation of the two cells in distributed layers, respectively, denoting a promising comfortable environment for re-epithelialization. Moreover, in vivo studies demonstrated that the scaffold showed no excessive inflammatory reaction. In short, this work presented an SS-based transparent hydrogel scaffold with steerable physical properties and excellent biocompatibility through 3D printing, pioneering promising applications in the visualization of wound care and drug delivery.

A novel pathway for in situ synthesis of modified gelatin microspheres by silane coupling agents as a bioactive platform

ABSTRACTThis study investigated the fabrication and modification of gelatin microspheres via single emulsion method and silane coupling agents, respectively. Herein, the influence of oil phase and washing procedure on morphology and uniformity of microspheres was evaluated. The results of field emission scanning electron microscopy indicated a positive effect of olive oil on the production of uniform and approximately mono‐size spheres while monodispersity increased after samples were washed under ultrasonication. Investigation of crosslinking degree demonstrated that GPTMS could not show efficiency as high as glutaraldehyde, but the lack of toxicity and inducing bioactive behavior to the microspheres has made them appropriate for bone applications. Moreover, the stability of spheres after 14 days in phosphate buffered saline proved the ability of (3‐glycidyloxypropyl)trimethoxysilane to crosslink gelatin. The presence of silane coupling agents in prepared spheres caused the biomimetic formation of hydroxyapatite after a 14‐day immersion in simulated body fluid as observed by field emission scanning electron microscopy‐energy‐dispersive X‐ray spectroscopy, changes in pH, Fourier transform infrared spectroscopy, X‐ray diffraction, and atomic force microscopy. Adhesion, spreading, and proliferation of L929 cells on the hybrid microspheres corroborated the potential of gelatin spheres for biomedical application. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46739.

Physicochemical modifications of laser treated PES membrane and their effects on platelet and L929 cell adhesion

The degree of the biocompatibility of polymeric materials used as biomaterial can be controlled by surface modification for various biomedical engineering applications. This paper reports UV laser-assisted modification of polyethersulfone membrane with respect to the fabrication of polymer devices for cell culture applications. For this purpose physicochemical changes on the membrane surface were investigated as a function of laser and process parameters. The membrane was irradiated with nanosecond UV pulses at 193 and 308 nm. The change in surface chemistry was characterized by FTIR spectroscopy and contact angle measurements. In order to test the influence of these changes for biological applications, the modified membranes were studied for L929 cell culture assay and platelet adhesion.

Novel enzymatic crosslinked hydrogels that mimic extracellular matrix for skin wound healing

Skin defects are an important and pressing clinical problem. The use of hydrogels as a regenerative scaffold presents a promising approach to cure skin defects by inducing dermal reconstruction. Although synthetic material hydrogels display good mechanical properties, their poor biocompatibility restricts their application. To develop a good dermal substitute, we have successfully prepared hydrogels that mimic the extracellular matrix of the human body and can be used for tissue engineering skin scaffolds. The hydrogels were synthesized by adding hyaluronic acid (HA) and carboxylated chitosan (CCS) to human-like collagen (HLC) that can be used to tissue engineering skin scaffolds and using transglutaminase as a crosslinking agent. The mechanical characteristics of the hydrogels were explored by cyclic compressive mechanical tests and a uniaxial tension protocol. The compressive stress of HLC/HA/CCS (GEL4) hydrogel reached 0.2136 ± 0.034 MPa when the compressive strain reached 60%. The tensile strain of GEL4 was 126.99 ± 2.38%. The hydrogels significantly promoted adhesion, proliferation and migration of L929 cells, demonstrating the good biocompatibility of the hydrogels. For in vivo analysis, we constructed a full-thickness skin defect model and demonstrated that the hydrogels could effectively prevent invasion of the wound by outside bacteria and certificate that they are beneficial in promoting wound healing over pathologic healing. Subcutaneous implantation experiments revealed that the degradation period of the hydrogels with HA and CCS is suitable for the healing cycle of skin tissue, and the inflammatory reaction could be reduced to a very short time, indicating the good histocompatibility of the hydrogels. Therefore, the hydrogels are favourable, soft and porous materials that demonstrate good potential for skin repair, drug delivery, cartilage treatment and other tissue engineering applications.

Development of Antifouling Hyperbranched Polyglycerol Layers on Hydroxyl Poly-p-xylylene Coatings.

Antifouling surfaces that are resistant to protein adsorption and cell adhesion are desirable for many biomedical devices, such as diagnostic devices, biosensors, and implants. In this study, we developed an antifouling hyperbranched polyglycerol (hPG) surface on hydroxyl poly-p-xylylene (PPX-OH). PPX-OH was deposited via chemical vapor deposition (CVD), and an hPG film was then developed via the ring-opening reaction of glycidol. The hPG film greatly reduced the adhesion of L929 cells and platelets as well as protein adsorption. The addition of alkenyl groups in the hPG layer allows the conjugation of biomolecules, such as peptides and biotin, and elicits specific biological interactions. Since the CVD deposition of PPX-OH could be applied to most types of materials, our approach makes it possible to decorate an antifouling hPG film on most types of materials. Our method could be applied to biosensors, diagnostics, and biomedical devices in the future.

Polyurethane-based bioadhesive synthesized from polyols derived from castor oil (Ricinus communis) and low concentration of chitosan

Abstract

Biologically active and biomimetic dual gelatin scaffolds for tissue engineering

We have designed, developed and optimized Genipin cross-linked 3D gelatin scaffolds that were biologically active and biomimetic, show a dual activity both for growth factor and cell delivery. Type B gelatin powder was dissolved in DI water. 100mg of genipin was dissolved in 10ml of DI water. Three genipin concentrations were prepared: 0.1%, 0.2% and 0.3% (w/v). Solutions were mixed at 40°C and under stirring and then left crosslinking for 72h. Scaffolds were obtained by punching 8 mm-cylinders into ethanol 70% solution for 10min and then freeze-drying. Scaffolds were biologically, biomechanically and morphologically evaluated. Cell adhesion and morphology of D1-Mesenchymal stem cells (MSCs) and L-929 fibroblast was studied. Vascular endothelial grwoth factor (VEGF) and Sonic hedgehog (SHH) were used as model proteins. Swelling ratio increased and younǵs module decreased along with the concentration of genipin. All scaffolds were biocompatible according to the toxicity test. MSC and L-929 cell adhesion improved in 0.2% of genipin, obtaining better results with MSCs. VEGF and SHH were released from the gels. This preliminary study suggest that the biologically active and dual gelatin scaffolds may be used for tissue engineering approaches like bone regeneration.

Elastic chitosan/chondroitin sulfate multilayer membranes

Freestanding multilayered films were obtained using layer-by-layer (LbL) technology from the assembly of natural polyelectrolytes, namely chitosan (CHT) and chondroitin sulfate (CS). The morphology and the transparency of the membranes were evaluated. The influence of genipin (1 and 2 mg ml−1), a naturally-derived crosslinker agent, was also investigated in the control of the mechanical properties of the CHT/CS membranes. The water uptake ability can be tailored by changing the crosslinker concentration that also controls the Young’s modulus and ultimate tensile strength. The maximum extension tends to decrease upon crosslinking with the highest genipin concentration, compromising the elastic properties of CHT/CS membranes: nevertheless, when using a lower genipin concentration, the ultimate tensile stress is similar to the non-crosslinked one, but exhibits a significantly higher modulus. Moreover, the crosslinked multilayer membranes exhibited shape memory properties, through a simple hydration action. The in vitro biological assays showed better L929 cell adhesion and proliferation when using the crosslinked membranes and confirmed the non-cytotoxicity of the developed CHT/CS membranes. Within this research work, we were able to construct freestanding biomimetic multilayer structures with tailored swelling, mechanical and biological properties that could find applicability in a variety of biomedical applications.