Development of genipin crosslinked gelatin matrices on surface interaction:Enhan

Qiang-Song Wang,Gui-Fang Wang,Hai-Yun Zhang,Fa-Quan Zhao,Yuan-Lu Cui,

1 Tianjin Key Laboratory of Biomedical Materials,Institute of Biomedical Engineering,Chinese Academy of Medical Science &Peking Union Medical College,Tianjin 300192,China

2 State Key Laboratory of Component-based Chinese Medicine,Research Center of Traditional Chinese Medicine,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,

China

Keywords:Genipin Gelatin matrices Anti-inflammation Biocompatibility NF-κB

ABSTRACT The inflammation can be stimulated by the surgical implantation and biomaterial presence through the foreign body via bio-interface.Macrophages play a key role in the interaction of host tissue to implant surfaces.In present study,the immuno-inflammatory responses of genipin crosslinked gelatin matrices(GCGM)to macrophages in vitro and the host tissue surfaces in rats were investigated.The results showed that the mechanical properties,swelling and degradation of gelatin matrices were improved by the crosslinking of genipin at physiological conditions.The macrophage on the surface of GCGM could avoid to be activated.The interaction of macrophage and GCGM suggested that GCGM could reduce the inflammatory response with downregulating the production and mRNA expression of pro-inflammatory cytokines.The anti-inflammatory effect of GCGM was demonstrated to be related to inhibit nuclear factor kappa-B (NF-κB) signaling pathway.Furthermore,gelatin matrices crosslinked with genipin could decrease the acute and chronic inflammatory response interacting with host tissue surfaces to enhance the biocompatibility in rats.These results showed that GCGM could avoid to active macrophages and were endowed with anti-inflammatory properties,suggesting the significant potential for clinical success with the development of immunomodulatory biomaterials.

1.Introduction

The field of tissue engineering,such as the implantation of scaffold materials,aims to develop strategies to replace the nonfunctional tissue and promote the tissue for the restoration of intrinsic function [1].Inflammation is an essential component of wound healing for the implanted biomaterials.The biomaterial implantation can also induce inflammatory response through the foreign body via bio-interface.The host tissue reacts to the implant via an inflammatory response with recruitment of macrophages,neutrophils,and lymphocytes to implantation[2].Acute inflammation is the first initial response to implant with macrophage releasing cytokines and chemokines,such as tumor necrosis factor alpha(TNF-α),interleukin (IL)-1β and IL-6 and interferon (INF)-γ [3],which lasts from just minutes to days.Moreover,the generated INF-γ can induce the production of TNF-α,IL-1β,and IL-6 [4].Chronic inflammation is a simultaneous state with acute inflammation,fibrosis and repair with presence of macrophages,fibroblasts,and connective tissue remodeling [5].Therefore,the biocompatibility and inflammatory response should be focused on the design of implanted biomaterials for tissue engineering.NF-κB is a nuclear transcription factor which regulates the expression of cytokines in various autoimmune diseases and inflammation [6].The classical NF-κB signaling pathways is representative of IκB degradation dependent pathway [7].NF-κB exists in lots of cells as dimers with inactive in the cytoplasm of cells associated with the NF-κB inhibitory protein(IκB).Activation of the κB kinase complex can lead to phosphorylation,ubiquitination and degradation of IκB proteins.Then,the separated NF-κB dimers translocate into the nucleus and bind specific DNA sequences to improve transcription of target genes.

Gelatin is a hydrolyzed product from collagen,which is a mixture of proteins obtained from different animal bodies,such as skin,tendon and cartilage.As the excellent biocompatibility and biodegradability [8,9],gelatin is extensively applied in the pharmaceutical and medical fields,such as drug delivery [10] and soft-tissue engineering[11].Gelatin hydrogel can be formedwith a conformational change from a random coil to a triple helix below 35 °C [12,13].The secondary bonding structure is broken leading to low mechanical properties at the temperature above 35 °C,which were limited at physiological temperatures [14].In order to increase its stability and mechanical properties,gelatin generally is crosslinked by chemical crosslinkers such as carbodiimides and glutaraldehyde [15,16].However,there are undesirable side effects after chemical crosslinking,such as higher cytotoxicity,decreasing the superb non-immunogenic property of gelatin[17,18].Natural crosslinking agents with lower cytotoxicity are paid more attention for the modification of biomaterials.

Genipin,the aglycon of geniposide from the fruit of Gardenia jasminoides ELLIS,can react spontaneously with proteins or amines.As the low cytotoxicity and good biocompatibility of crosslinked biomaterials,genipin is widely used as the natural crosslinker for tissue engineering[19].Our previous study suggested that the gardenia blue pigments resulting from the crosslinking of genipin and glycine or tyrosine exhibited excellent anti-inflammatory effects via NF-κB inactivation [20] and antidepressant-like effects [21].Furthermore,the genipin crosslinked 3-D scaffolds showed better biocompatibility for liver tissue engineering [22].

In this study,genipin is introduced to improve the stability of natural gelatin matrices.We propose a hypothesis that antiinflammatory effect of genipin is reserved after crosslinking with gelatin matrices,suggesting the significant potential for immunomodulatory biomaterials.The anti-inflammatory effect of GCGM is investigated in vitro and in vivo.The possible mechanisms underlying the anti-inflammatory effects of GCGM are proved through regulating the NF-κB signal pathway(Fig.1).These results suggest that GCGM can decrease the acute and chronic inflammatory response in vitro and in vivo,indicating that the antiinflammatory properties of genipin is reserved after crosslinking with gelatin matrices materials.

2.Materials and Method

2.1.Materials

Type B gelatin from bovine skin was bought from Sigma-Aldrich Co.(St.Louis,MO,USA).Genipin was bought from Wako (Osaka,Japan).Murine IFN-γ was purchased from PeproTech Co.(Rocky Hill,NJ,USA).IL-6,IL-1β and TNF-α Mouse ELISA kits were obtained from Invitrogen Co.(Carlsbad,CA,USA).BCA Protein Assay Kit was obtained from Pierce (Rockford,IL,USA).Mouse Cytokine Array Panel A Array kit was purchased from R&D Systems,Inc.(USA).Improm-II Reverse Transcription System was purchased from Promega Co.(Madison,WI,USA).Fast Start Universal SYBR Green Master (ROX) kit was purchased from Roche (Mannheim,Germany).P-IκB-α,IκB-α,IKK-α,IKK-β,P65,P50 antibodies and peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Danvers,MA,USA),and β-actin antibody was purchased from Sigma-Aldrich Co.(St.Louis,MO,USA).

RAW 264.7 murine macrophages cell line was obtained from Cell Culture Center of Chinese Academy of Medical Sciences (Beijing,China),and maintained in DMEM supplemented with heat inactivated fetal bovine plasma (HI-FBS) at 37 °C in a humidified incubator containing 5% CO2.The animal experiment procedures were approved by the Animal Ethics Committee of Chinese Academy of Medical Science &Peking Union Medical College.

2.2.Preparation and characterization of GCGM

Gelatin matrices were prepared with 6%gelatin solution on the petri dishes (diameter=60 mm) at room temperature and transferred matrices to gelled at 4 °C.GCGM were obtained by gelatin matrices crosslinking with genipin solution at different concentrations(0.5%,1%and 2.0%,by mass)for 48 h.The samples were dehydrated with absolute ethyl alcohol at 4 °C for 12 h and at room temperature for 12 h,then air dried.The samples were sterilized using60Coγ irradiation with 25 kGy dose.

The surface morphologies of both the gelatin matrices and GCGM are visualized by scanning electron microscopy.Differential scanning calorimetry (DSC) curves of samples are recorded on a differential scanning calorimeter.Fourier Transform Infrared Spectrophotometry (FT-IR) spectra of samples is recorded using FT-IR spectroscopy with the wave number range 4000–400 cm-1using KBr pellets.In the mechanical properties measurement,test samples are prepared with dumbbell shape first and placed in an environment with humidity of 60%for 24 h.Stress–strain curves of test samples then are determined using the electronic tensile machine at a constant speed of 10 mm.min-1.

2.3.The swelling and in vitro degradation of GCGM

GCGM were weighted in air-dried conditions.Then the matrices are immersed in PBS solution for different time point.Wet samples are wiped with filter paper to remove excess liquid and re-weighted.The amount of adsorbed water was calculated as following:where Wxand W0are the mass of the wet and the air-dried samples as shown in Eq.(1),respectively.

Fig.1.The schematic illustration of GCGM on surface interaction:enhancing the biocompatibility by attenuating sterile inflammation.

The in vitro degradability of GCGM is analyzed by immersing the matrices materials in PBS solution and incubating at 37 °C for different periods of time.After degradation,the test samples first are lyophilized for 24 h and then weighed.The mass loss ratio for each test sample is calculated as follows:where W0is its initial mass and Wtis the mass of test samples after degradation as shown in Eq.(2).

2.4.Cell morphology and viability on GCGM

RAW 264.7 cells are seeded on tissue culture plates(TCPs),gelatin matrices,and GCGM for 24 h.Then,the fluorescence of cells on plates with GCGM,control gelatin matrices and TCPs is obtained by inverted fluorescence microscope (Nikon,Japan).Atto 565 Phalloidin,DAPI is used to stain the cytoskeleton and cell nuclei,respectively.MTT was used to detect the cell viability.

2.5.The immune response of macrophage on GCGM

RAW 264.7 cells are seeded on TCPs,control gelatin matrices and GCGM.After treating with INF-γ (100 unit per ml) for 24 h,the nitrite accumulated in culture medium is measured by Griess reagent system.Intracellular NO production is evaluated with a fluorescent NO derivative.Visual images are generated by inverted fluorescence microscope (Nikon,Japan).The production of TNF-α and IL-6 are detected by ELISA kits.

2.6.Cytokine protein array analyses

RAW 264.7 cells are seeded on TCPs,control gelatin matrices and GCGM.After treating with INF-γ(100 unit per ml)for 24 h,different proteins,cytokines,and chemokines in culture medium are analyzed with a Proteome profiler array from R&D Systems.The array allows to detect 40 proteins.Blots are developed by Horseradish peroxidase substrate (Millipore Corporation,USA) and data are captured by exposure to Kodak BioMax Light films.Films are scanned and the densitometry is analyzed by the Image-Pro Plus version 6.0.

2.7.The mRNA expression of COX-2,iNOS,TNF-α,IL-6,IL-1β on GCGM

Total RNA from RAW 264.7 cells on different matrices is isolated using Trizol total RNA extraction kit.RNA is reversely transcribed using ImProm-II Reverse Transcription System cDNA synthesis kit.The real-time RT-PCR oligonucleotide primers of mouse iNOS,COX-2,IL-6,TNF-α,IL-1β and β-actin are shown in Table S1.The reactions are performed by Fast Start Universal SYBR Green Master(ROX) kit.The amplification is performed using a 7500 Real-Time PCR System.The fold increase or decrease is analyzed after normalizing to a house-keeping gene by 2-ΔΔCTmethod.

2.8.NF-κB signaling pathway

The cells are stimulated with INF-γ for 30 min,and collected by centrifugation.Then,the cells are resuspended in extraction buffer of protein extraction kit (Sangon,China).Then protein (40 mg) is electro-blotted onto a PVDF membrane following separation on a 10%SDS-polyacrylamide gel electrophoresis.Then the PVDF membrane is incubated with blocking solution for 2 h,and incubated overnight with anti-IKK α,anti-IKK β,anti-PIK α,anti-IK α,anti-P50,anti-P65 antibody at 4 °C.PVDF membranes are washed four times with TTBS and then incubated with HRP-conjugated secondary antibody for 1 h.The blots are washed and developed by Horseradish peroxidase substrate.Furthermore,nuclear extracts are prepared with the manufacture’s instruction.The quantitative DNA-binding activity of NF-κB p50 and p65 is detected with universal EZ-TFA transcription factor assay kit.

2.9.The inflammatory response of GCGM in rats

2.9.1.Acute inflammatory reaction of host tissues surrounding GCGM in rats

Four incisions on the back of each rat are made under aseptic condition.The incisions (1 cm long each),are made through the skin up to the underlying muscular tissue layers.The sterilized gelatin matrices,polyethylene (PE),GCGM are placed on incisions of rats.The diameter of each circular test sample is approximately 6 mm.The paired wounds are treated with same gelatin matrices,PE,GCGM in one rat,respectively.After closure of the incisions,the rats are kept separately and marked in cages allowing identification.Then the surrounding host tissues are harvested after 24 h to investigate the mRNA expressions of cytokine,protein (IL-6,TNF-α,IL-1β,iNOS,COX-2) and the productions of cytokines (IL-6,TNF-α and IL-1β).The real-time RT-PCR oligonucleotide primers of Rat iNOS,COX-2,IL-6,IL-1β and GAPDH are shown in Table S2.

2.9.2.Chronic inflammatory reaction of host tissues surrounding GCGM in rats

The sterilized gelatin matrices,polyethylene (PE),GCGM are transplanted into rats,and the host tissues surrounding the different matrices are harvested after 1,4 and 12 weeks.The samples are fixed in 10%paraformaldehyde solution and prepared for the histological examination.The fixed samples are embedded in paraffin and sectioned into a thickness of 5 μm,then stained with hematoxylin and eosin (H&E).The stained sections of samples are observed using light microscopy.

2.10.Statistical analysis

The statistically significant differences between various experimental groups are performed with Origin Pro 8.5 software by the Student’s t-test or one-way ANOVA.Data are expressed as means ± S.D.excluding values given in animal experiment with mean± SEM.The value of p <0.05 or p <0.01 is considered statistically significant.

3.Results and Discussion

3.1.Characterization,swelling and degradation of GCGM

Gelatin has been safe used in pharmaceuticals and food products for a long history,which is also considered as a generally regarded as safe (GRAS) material by the United States Food and Drug Administration(FDA)[23].However,at physiological temperatures (37 °C),the gelatin hydrogels without crosslinking show a low shape stability and poor mechanical strength,leading to limit their biomedical applications.Genipin as a natural crosslinker is wide used to crosslink biomaterials as the low cytotoxicity,which can crosslink with the amino group of gelatin to enhance the stability (Fig.2A).The morphology of gelatin matrices and GCGM is shown in Fig.1B.The superficial structure of GCGM is smooth and not changed compared with the gelatin matrices (Fig.2C).The DSC data show that the characteristic peaks of genipin disappear and new peaks emerge in the thermogram after genipin crosslinking with gelatin,which indicate the interaction between genipin and gelatin (Fig.S1A).The characteristic peaks of gelatin(1630 cm-1for amide I and 1551 cm-1for amide II) shift after crosslinking with genipin.It suggests that partially amide group on gelatin transformed into heterocyclic of genipin(Fig.S1B).Furthermore,the mechanical properties of the gelatin matrices and GCGM are investigated.The GCGM (2%) have the highest ultimate tensile strength value and strain at fracture than the gelatin matrices (Table S3).

Fig.2.The morphology,swelling and in vitro degradation of GCGM.(A) The crosslinking reaction of genipin with gelatin.(B) The photograph of gelatin matrices (GM) and GCGM (GCGM).(C) SEM micrographs of GM) and GCGM.(D) Swelling rate of different crosslinking density of GCGM.(E) The in vitro degradation of GCGM at different time points.

Based on the theory of equilibrium swelling,swelling ratio is concerned with the effective crosslinking density,which is evaluated by swelling measurement [24].Gelatin is soluble and gelatin matrices can be induced rapid swelling in physiological solution(about 700% after 1 h).The degree of swelling can be reduced by treating with different genipin concentrations (Fig.2D).The biodegradation rate of GCGM in vitro is investigated with immersing in PBS solution and incubating at 37 °C.As a result,gelatin matrices without crosslinking are completely dissolved after 1 days of soaking.The degradation trend is attenuated with the increasing concentration of genipin.It is noted that there is a slow degradation of GCGM less than 10% in first 30 soaking days (Fig.2E).The degradation rates of the 0.5% GCGM increase to 32.4%,but the 1%and 2% GCGM show little degradation rates at 80 soaking days.Even to 120 soaking days,the degradation rate is only 16.1% in 2% GCGM.These results indicate that gelatin matrices crosslinked by genipin can not only enhance the mechanical strength,but reduce the swelling ratio and biodegradation rate,which can be used as direct implant biomaterials or modifying materials with satisfactory stability.

3.2.Cell morphology and viability on GCGM

As shown in Fig.3A,the cell morphology on gelatin matrices and genipin crosslinked matrices is not changed significantly than TCPs.This indicates that the genipin crosslinked matrices possessed good biocompatibility and avoid to stimulate the macrophages.The cytotoxicity is further investigated with MTT assay.The cell viability is not inhibited with genipin (0.5%–2%) crosslinked gelatin matrices (Fig.S2).

3.3.The immune response of macrophage on GCGM

Macrophages play a vital role in host defense through the production of pro-inflammatory mediators,chemokines and cytokines in connection with the immune response,which can recruit more macrophages to attempt to remove and degrade foreign substances by phagocytosis in the body [25].If the foreign substances are large,macrophages undergo cell fusion to form multinucleated foreign body giant cells (FBGC),which are able to secrete proteinases and other lysosomal enzymes to degrade larger foreign substances[26].IFN-γ,which is endogenic and different ectogenic lipopolysaccharide(LPS),has long been recognized as a representative pro-inflammatory cytokine playing an important role in inflammation and autoimmune disease.Once inflammation is initiated,produced IFN-γ subsequently works via a variety of molecules and pathways of immune system to amplify the inflammatory process [27].In order to increase the cascade reaction of inflammation,endogenic IFN-γ is introduced in this study.As shown in Fig.3B,compared with TCPs,the gelatin matrices increase the NO production,which is the reason of foreign body irritation from gelatin matrices.IFN-γ stimulation also increases higher NO production than gelatin matrices.GCGM (0.5%,1% and 2%) can decrease the NO production significantly(P <0.01).Moreover,compared with gelatin matrices in the presence of INF-γ,there is little intracellular NO production (Fig.3C).TNF-α,IL-6 are known to be pro-inflammatory cytokines that possess a multitude of biological activities for acute or chronic inflammatory diseases [28].After treating with the presence of INF-γ,the secretion of TNF-α and IL-6 are increased significantly than TCPs.GCGM(0.5%,1% and 2%) can inhibit the secretion of IL-6 significantly,and the secretion of TNF-α is inhibited in 2%GCGM(Fig.3D and E).

Since GCGM are found to potently inhibit the pro-inflammatory mediators,we further investigate the mRNA expression of iNOS,TNF-α,IL-6 on GCGM.Furthermore,the other two inflammatory mediators,COX-2 and IL-1β are also investigated.As shown in Fig.4A-D,the mRNA expression of iNOS is inhibited significantly by GCGM compared with INF-γ induced gelatin matrices(P <0.01).Additionally,we find that the effect of GCGM on mRNA expression of iNOS is coincident with that on NO production.Therefore,the decrease in NO production is related to the effect of GCGM on down-regulating the mRNA expression of iNOS.The mRNA expression of IL-6 is inhibited significantly by GCGM compared with INF-γ induced gelatin matrices(P<0.01).Furthermore,the mRNA expression of TNF-α,IL-1β and COX-2 is inhibited significantly by GCGM compared with INF-γ induced gelatin matrices(P <0.01).These results indicate that the crosslinking of genipin can reduce inflammatory response by decreasing the production of pro-inflammatory mediators and cytokines,which are in accordance with the previous study [20].The anti-inflammatory effect of genipin is reserved even if the crosslinking occurred,which suggest the promising application on modification and functionalization of implant biomaterials.

3.4.Effect of GCGM on multiple cytokines

In inflammatory process,multiple cytokines secreted in macrophages can be activated.About 15 kinds of cytokines are responded to INF-γ stimulation (Fig.4F).The cluster heatmap is a graphical representation of data where the individual values contained in a matrix are represented as colors to achieve data visualization[29].As shown in Fig.4G,the cluster analysis results of samples from different groups are exhibited.Multiple cytokines such as BLC,G-CSF,IL-1α,IL-1ra,IP-10,KC,MIP-1β,RANTES,TIMP-1,IL-6,TNF-α are up-regulated after INF-γ stimulation,and GCGM can downregulate the expression of above cytokines compared with INF-γ induced gelatin matrices.For other cytokines,such as Sicam-1,MIP alpha,MIP beta,MMIP-2,JE,there is no significance between GCGM and INF-γ induced gelatin matrices.The GCGM can modulate the production of several cytokines which are involved in inflammatory process.

3.5.NF-κB signaling pathway

The productions of pro-inflammatory cytokines by macrophages exposed to implants are regulated by intracellular systems[30],such as NF-κB signaling pathway.The interfacial tissues around implants are significantly impacts by this NF-κB-directed protein expression [31].Suppression of NF-κB activity can be achieved by different strategies:(i) upstream activating signal of NF-κB;(ii) the activity of IKKα/β kinase;(iii) protein stability of IκB;(iv) protein functions of NF-κB,such as nuclear translocation or DNA binding ability.In this study,GCGM can not only inhibit the expression of IKK-α and IKK-β,but block the expression of IκB-α degradation and IκB-α phosphorylation.The activation of NF-κB is critically required for the activations of IL-1β,TNF-α,IL-6,iNOS and COX-2,DNA-binding activity of NF-κB is detected (Fig.5).Since IKK-α and-β are upstream kinases of IκB in the NF-κB signal pathway,the effects of GCGM on IKK-α,-β activation are also detected (Fig.5A).Compared with INF-γ induced gelatin matrices,GCGM downregulate the expression of IKK-α and IKK-β(Fig.5 B(a and b)).We further investigate the expression of IκB-α degradation and IκB-α phosphorylation.The IκB-α degradation is significantly blocked by GCGM,which is related to IκB-α phosphorylation,which is reduced by GCGM significantly compared with INF-γ induced gelatin matrices (Fig.5B (c and d)).Furthermore,the expression of NF-κB subunits p50 and p65 is investigated,and the results suggest that GCGM can downregulate the expression of NF-κB subunits p50 and p65(Fig.4B(e and f)).A NF-κB DNA binding assay is carried out using nuclear extracts to analyze the DNAbinding activity of NF-κB subunits p50 and p65 quantificationally.The tendency is coincident with the WB results (Fig.5 C and D).Taken together,the above findings show that GCGM suppress IL-1β,TNF-α,IL-6,iNOS and COX-2 expressions at least in part via NF-κB dependent mechanism.

Fig.3.The cell morphology change and immune response of macrophage (RAW 264.7 cells) on GCGM.(A) Atto 565 Phalloidin (Red fluorescence) is used to dye cytomembrane,and DAPI(Blue fluorescence)is used to dye cell nucleus.(B)The NO content in culture medium is analyzed by the Griess reagent method;(C)Intracellular NO production is evaluated with DAF-FM diacetate;The production of IL-6(D)and TNF-α(E)is detected by ELISA.# P<0.05,## P<0.01 compared with TCPs,*P<0.05,**P<0.01(n=6) compared with GCGM+INF-γ.

Fig.4.The mRNA expression of iNOS(A),COX-2(B),TNF-α(C),IL-6(D),IL-1β(E)and multiple cytokines productions on TCPs,GM,GM+IFN-γ,GMGC+IFN-γ(F).*P<0.05,**P<0.01(n=6)compared with GM+IFN-γ.Numbers on membranes mark the following targets:“1′′ BLC;“2”G-CSF;“3”sICAM-1;“4”IL-1α;“5”IL-1ra;“6”IL-6;“7”IP-10;“8”KC;“9”JE;“10”MIP-1α;“11”MIP-1β;“12”MIP-2;“13”RANTES;“14”TIMP-1;“15”TNF-α;(G)Hierarchical clustering of 15 cytokines expression profiling on TCPs,GM,GM+IFN-γ,GMGC+IFN-γ.

Fig.5.Effects of GCGM on NF-κB pathway.(A) The degradation of IKK-α,IKK-β,IκB-α,phosphorylation of IκB-α,P65 and P50.The density analysis of IKK-α (B),IKK-β (C),IκB-α(D),phosphorylation of IκB-α(E),P65(F),P50(G),and the DNA-binding activity of NF-κB subunits p50(H)and p65(I).# P<0.05,## P<0.01 vs.TCPs;*P<0.05,**P<0.01 vs.GCGM+INFγ.

Fig.6.The levels of IL-1β(A),TNF-α(B),IL-6(C)and the RNA expression of iNOS(D),COX-2(E),IL-1β(F)and IL-6(G)in host tissues surrounding GCGM.# P<0.05,## P<0.01 vs.TCPs;*P<0.05,**P<0.01 vs.GCGM.

3.6.The inflammatory response of GCGM in rats

Acute inflammatory reaction is the main progress of host tissues when medical biomaterials are implanted into body.In the process of acute phase inflammation,lots of mediators are involved in,such as TNF-α,IL-6 and IL-1β.We firstly investigate the productions of pro-inflammatory mediators in host tissues surrounding GCGM.Compared with control group,the IL-1β and TNF-α levels are increased in sham operation (Sham O) group and PE surrounded host tissues,especially in gelatin matrices surrounded host tissues.The IL-1β and TNF-α levels in host tissues surrounding GCGM are decreased significantly compared with gelatin matrices group(P<0.05 or P<0.01)(Fig.6A and B).The IL-6 levels in Sham O group(blank control)and PE(negative control)surrounded host tissues are not changed in acute phase,but are increased significantly in gelatin matrices surrounded host tissues(P<0.01),which are reversed in GCGM groups,especially in 2% GCGM (P <0.01)(Fig.6C).

Fig.7.H&E staining of different groups of gelatin matrices after 1,4 and 12 weeks subcutaneous implantation.(Sham O group,GM,PE films,0.5%GCGM,1%GCGM,and 2%GCGM) (Scale bar:500 μm).

Since GCGM are found to potently inhibit the production of proinflammatory mediators of NO,TNF-α,IL-6,we next investigate the mRNA expressions of iNOS,COX-2,IL-6,IL-1β and TNF-α in host tissuesss surrounding GCGM implanted in rats (Fig.6D-G).NO is recognized as a regulator of inflammatory responses produced by iNOS in activated inflammatory cells.Compared with gelatin matrices group,the mRNA expression of iNOS in host tissues surrounding genipin(1%,2%)crosslinked gelatin matrices is downregulated significantly (P <0.01).Furthermore,in response to foreign body materials,the mRNA expressions of COX-2,IL-6 and IL-1β in host tissues surrounding PE and gelatin matrices are upregulated significantly compared with control group,and GCGM can downregulate the mRNA expressions of COX-2,IL-6 and IL-1β in host tissues surrounding matrices significantly than gelatin matrices(P <0.01).However,the mRNA expression of TNF-α is not downregulated significantly in different groups (data was not shown).These results suggest the GCGM can reduce the acute inflammation response of implanted biomaterials,which are suitable for the implantation and modification of biomaterials in vivo.

As GCGM can reduce the acute inflammatory reaction in vitro and in vivo,next we assess chronic inflammation of the surrounding tissue in rats(Fig.7).Macrophages are professional phagocytes to exert main controlling influences on the wound healing/fibrosis responses.If the implant is large,macrophages can undergo cell fusion to form FBGC.The successful encapsulation of biomaterial device by FBGC can reduce the acute (and chronic) inflammatory reactions with subsequent release of macrophage-derived growth factors leading to the ingress of fibroblasts[32].After 1 week,there are some macrophages on the tissue surrounding the PE films and gelatin matrices,and little macrophages are observed on the tissue surrounding the GCGM,which also maintain a relatively complete shape after 1 week embedment.There is fibrous capsule surrounding the PE films after 4 weeks.The 0.5% GCGM are surrounded by neutrophile granulocyte with much phagocytosis of degradative fragment.The 1% and 2% GCGM maintain a relatively complete shape with little phagocytosis by neutrophile granulocyte and macrophages.After 12 weeks,the tissue surrounding gelatin matrices show a higher degree of fibrosis,and there is still fibrous capsule surrounding the PE films.The 0.5% GCGM is degraded to big fragment which are surrounded by lymphocyte with fibrous encapsulation.The 1% and 2% GCGM show a segmental degradation with fibrous encapsulation and displayed some collagen condensation (2% genipin).The crosslinking of genipin can maintain the mechanical property and shape of gelatin matrices,and emerge satisfactory biocompatibility in short period implantation and long period implantation even if few degradation.

4.Conclusions

In summary,genipin as a natural crosslinker can improve the mechanical properties and reduce the swelling and in vitro degradation of gelatin matrices at physiological conditions.The macrophage can avoided to be activated on the surface of GCGM.The interaction of macrophage and GCGM suggest that GCGM reduce the inflammatory response with downregulating the production and mRNA expression of pro-inflammatory cytokines via inhibiting NF-κB activation.Furthermore,gelatin matrices crosslinked with genipin can decrease inflammatory response interacting to host tissue surfaces and enhance the biocompatibility.These results suggest that the GCGM have the potential for the development of immunomodulatory biomaterials interface for functional tissue engineering scaffold.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81741119) and Fundamental Research Funds for the Central Universities,China (3332020059).