Preparation Of γ-polyglutamic Acid/chitosan Composite Hydrogel And Detection Of Chondrocyte Compatibility

Hydrogels are hydrophilic polymer materials with a three-dimensional（3D）network structure formed by various cross-linking techniques,and have the ability to absorb and retain large amounts of water in the interstices of their structures.Because of its special properties,such as high water content,three-dimensional network structure,easy loading of growth factors,unique molecular structure and excellent biocompatibility,etc.,it has received extensive attention in various application fields,such as tissue scaffolds,cartilage repair,drug release,wound dressings,and wearable devices.The main research contents of this paper were as follows:First,two kinds of raw materials with good compatibility were selected:γ-polyglutamic a-cid（γ-PGA）and decarboxylated chitosan（CS）.Theγ-PGA/CS composite hydrogels were prepared by the cross-linking method of 1-ethyl-（3-dimethylaminopropyl）carbodiimide hydrochloride/N-hydrox-ysuccinimide（EDC/NHS）,and the cell scaffold material was prepared by freeze-drying method.Secondly,the inverted vial method was used to determine the critical gel-forming concentration of the composite hydrogel asγ-PGA/CS=3:1.Then,in order to explore the ionic complexation,pore size,mechanical properties,density and porosity,swelling and degradation ofγ-PGA/CS composite hydrogels,various experimental techniques and methods were used,such as attenuated total reflection-flourier transform infrared（ATR-FTIR）,X-ray diffraction（XRD）,scanning electron microscopy（SEM）,rotational rheometer,density and porosity,swelling and in vitro degradation.The results of the ATR-FTIR showed that different concentrations ofγ-PGA/CS composite hydrogels were peaked at 1205 cm^-1,but not observed inγ-PGA and CS.It was speculated that it may be caused by the interaction of ionic complexes betweenγ-PGA and CS.XRD results showed that two strong crystalline peaks appeared in the XRD diffractogram of CS,and no significant crystalline peaks appeared in the XRD diffractograms ofγ-PGA or of theγ-PGA/CS hydrogel.Hence,we deduced that the crystalline structure within chitosan was disrupted after the formation of the ionic complex interactions withγ-PGA.From the results of the SEM,each group ofγ-PGA/CS composite hydrogels formed a uniform porous structure,showing a distinct three-dimensional network.The pore size was between 85.5±4.0μm and 142.3±7.8μm,gradually decreased with increasing concentration.The results of shear rheological studies showed that the storage modulus（G’）remained basically unchanged before the 400%strain force,presumably due to the stable three-dimensional structure of theγ-PGA/CS composite hydrogel.In the range of 0.1～100 rad/s,the storage modulus（G’）were always greater than the loss modulus（G’’）,indicating that the hydrogel had good rigidity and toughness.The results of density and porosity studies showed that the concentration ofγ-PGA:CS=3:1 hydrogel had the smallest density and the largest porosity.The concentration ofγ-PGA:CS=12:1 hydrogel had the largest density and the smallest porosity.The results of swelling and in vitro degradation studies showed that the composite hydrogel exhibited obvious swelling properties and reached equilibrium within 4 hours.After one week,most concentrations of hydrogels remained above 65%of their original weight,indicating that theγ-PGA/CS composite hydrogel scaffold had the ability to keep water without loss under normal temperature conditions.At last,in order to verify the cytocompatibility of theγ-PGA/CS composite hydrogels,the γ-PGA/CS composite hydrogel were co-cultured with 293T cells and chondrocytes,respectively.The cytocompatibility was investigated by means of CCK-8 method,live and dead cell staining and excitation confocal microscopy.The results of the CCK-8 method showed that the absorbance increased with time.It was indicated that theγ-PGA/CS composite hydrogel could promote the proliferation of two kinds of cells.From live/dead cell staining results,both types of cells cultured in the hydrogel extracts of each group had good cell viability.The results of the excitation confocal 3D scanning study showed that the two types of cells were uniformly distributed in the composite hydrogel scaffold material,and the cell mass increased with time.It was indicated that cells could effectively proliferate and adhere in 3D composite hydrogels.Then,real-time quantitative PCR was used to verify whether the composite hydrogel could maintain the chondrocyte phenotype or not.The results of real-time fluorescence quantitative PCR study showed that the expression levels of four genes,Rho A,Co L2a,COMP,and SOX9 inγ-PGA:CS=4:1 and 6:1 hydrogels were higher than those in the control group.It was indicated that theγ-PGA/CS composite hydrogel had a certain promoting effect in maintaining the phenotype of chondrocytes in the synthesis of extracellular matrix.Among them,the effect of the hydrogel with the concentration ofγ-PGA:CS=6:1 was more significant.Finally,the degradation behavior of the composite hydrogel in mice was explored by embeddingγ-PGA/CS composite hydrogel in C57BL mice.The results of in vivo degradation experiments showed that the subcutaneously embeddedγ-PGA/CS composite hydrogel scaffold had been basically completely degraded at 20 days.The surrounding tissue was smooth without obvious redness and swelling,and no other complications were found during the whole process.It was proved that theγ-PGA/CS composite hydrogel scaffold also had good in vivo compatibility.In this study,theγ-PGA/CS composite hydrogel scaffold prepared by ionic interaction with good mechanical properties.The cell compatibility test confirmed that it had good compatibility,and the in vivo degradation test showed that it had good compatibility.Those properties provided a certain theoretical basis for the research and development ofγ-PGA/CS composite hydrogel scaffold as a biomedical material.