3D Bioprinting Chondroconductive Scaffolds Based On Photo-crosslinkable Xanthan For Customized Cartilage Tissue Engineering

OBJECTIVE:Recently,millions of people suffering from articular cartilage defects worldwide from Teenagers to adults,limited endogenous regeneration and poor integration with grafts makes the repair of articular cartilage defects still a huge challenge.Three-dimensional(3D)bioprinting has been developed as one of the most promising and advanced fabrication methods in the field of cartilage regeneration.3D bioprinting involves living cell encapsulated combined adequate materials resulted in complex structures for cartilage defect repairs in the field of cartilage regeneration.However,the availability of biocompatible,biodegradable,printable biomaterials is very limited.Here we developed a photo-crosslinkable and printable xanthan gum(XG)based material serve as bio-ink and incorporated with bone mesenchymal stroma cells(BMSCs)for cell growth,chondrogenesis and repair of cartilage defact.XG was modified with methacrylic anhydride(MA)to be xanthan gum methacryloyl(XGMA).METHODS:XGMA was prepared by reaction of Xanthan gum with methacrylic anhydride to obtain XGMA0.5 and XGMA2 with low or high degrees of MA.XG as control,XGMA was characterized for its chemical structure by 1HNMR.The XGMA hydrogel precursors with different concentrations were prepared to determine the XGMA concentration with good shape,high resolution and low air pressure.The physical and chemical properties of the printed scaffolds were determined,The Scanning electron microscopy(SEM)was performed to observe the morphology of 3D bioprinting XGMA constructs.The compression modulus of the printed constructs were measured using a compression strength tester.To evaluate the degradation and swelling ratio of XGMA,XGMA were soaked in PBS(pH 7.4).XGMA2 exhibited the best mechanical performance.Basis on the results,XGMA2 scaffold was chosen for further investigation.,The experiment was divided into alginate,collagen,XG-Fe3+and XGMA2 in vitro.MSCs were isolated from the femoral marrow of of 3-day-old New Zealand rabbits.Intracellular reactive oxygen species were detected by DCFH-DA fluorescence probe After incubation for 24 hours in the medium containing 10ng/ml IL-6.The mass of DNA and GAG content was determined according to literature after 7 or 14 days of printed and cultured in the medium containing lOng/L TGF-β.The expression of cartilage specific genes Sox9,Col2A1 and aggrecan,fibrosis marker genesCol1A1,hypertrophic markers gene Col10A1 was evaluated by qRT-PCR.collagen type Ⅱ level was detected by immunohistochemistry,After three months of the treatment,the animals were sacrificed and the repaired articular cartilage was harvested.The cartilage regeneration evaluation was based on the International Cartilage Repair Society(ICRS),Serial sections were stained with standard hematoxylin-eosin staining(HE),Safranin O/Fast green,and immunohistochemistry(collagen Ⅱ).RESULTS:The 1HNMR results revealed that the methacryloyl groups was successfully conjugated onto XG.it is possible to approximate a percentage of degree of substitution(DS),the average DS increased from XGMA0.5(4.3%)to XGMA2(15.3%).To select optimal concentration of XGMA for 3D-printing,2%,3%and 4%XGMA were printed.4%XGMA bioink was determined for 3D printing due to perfect viscosity to keep its shape and high fidelity during printing and post-printing.Physiochemical characteristic shows difference between the groups.It was found that the reversibled cross-linking between XG and Fe3+,while to photo-crosslinging groups which have no found.The results showed that three types of scaffolds presented apparent difference in swelling ratio,and swelling ratio of XG-Fe3+was obviously higher compare to XGMA0.5 and XGMA2.SEM images demonstrated that XGMA0.5,XGMA2 were porous network structure,while there were no pores observed on the surface of XG-Fe3+.Higher photo-crosslinkable ability in XGMA2 increase crosslinking density.The higher the cross-linking density,the smaller the pore size.The results of mechanical property analysis displayed an increasing trend of compressive modulus in XGMA0.5,XGMA2 scaffolds.The compressive Modulus of XG-Fe3+,XGMA0.5 and XGMA2 Hydrogel is 726 KPa,52 KPa and 187 KPa,respectively after incubation for 0 hour.The compressive Modulus of XG-Fe3+,XGMA0.5 and XGMA2 Hydrogel is 1.7,41 and 152 KPa,respectively after incubation for 24 hours.The degradation ration of XGMAs showed that the weight loss of XGMA0.5,XGMA2 were 75%,46%immersed in PBS solution for 60 days,respectively.DCFH-DA fluorescence probe showed that the lowest fluorescence intensity for XG-Fe3+and XGMA2,suggesting that they had the strongest antioxidant capacity.Cell viability was determined using Live/Dead assays cultured on days 7,14.As shown MSCs in Alginate,XG-Fe3+,XGMA2,collagen hydrogel exhibited good for cell growing and there were no statistical difference among the groups.The chondrogenic differentiation of MSCs on 3D bioprinting XGMA2 scaffold was evaluated by qRT-PCR,the results showed that the expression of cartilage specific genes Sox9,Col2A1 and ACAN significantly up-regulated in XGMA2,which increased 5.5,78.7 and 3.7 folds compared with control group at days 7,as well as increased 7.7,194.3 and 6.6 folds at days 14.Meanwhile,mRNA expression of Col1A1(fibrosis marker)and Col10A1(hypertrophic markers)were significantly suppressed in XGMA2.Protein of collagen Ⅱ and GAG levels also confirmed that XGMA2 drastically increased compared to XG-Fe3+and collagen group.To evaluate the cartilage repair capacity in vivo,there were no synovial hyperplasia and inflammation were assessed after 3 months by gross examination of knee joints.We still could find defects in the non-treated group.White neo-tissue was appeared but small fissures were observed in the XG-Fe3+ group.Moreover,the defects in the XGMA2 group were improved by filling of cartilage-like tissue.We measured the ICRS scores from macroscopic observations and the XGMA2 group revealed the highest scores.The scores were decreased in the order of XGMA2,collagen hydrogel,XG-Fe3+and non-treated group.Histological analysis shown the neo-tissue was primarily composed of fibrous tissue with a loose and detached interface in the non-treated group.However,the tissues repaired using Collagen and XGMA2 were hyaline cartilage with round cells in the lacuna and were well integrated with the surrounding tissues.Specifically,the XGMA2 engineered cartilage exhibited a more uniform and compact tissue with more round cells in the lacuna than the other groups.The tissues repaired using XG-Fe3+ were fibrous cartilage.These results were also confirmed by the histological scores.The mean score of the defect treated using the XGMA2 was greater than that treated using the XG-Fe3+scaffold,which was greater than when the defect was left untreated.The scores in the XGMA2,collagen hydrogel,XG-Fe3+and non-treated group were determined to be 31.7,29.3,20,and 4.7,respectively.Immunohistochemistry assays were detected to access the protein expression of collagen type Ⅱ in neo-tissues.There was mainly negative staining in the repaired area of the control group,and weakly positive staining was appeared in the XG-Fe3+,In the Collagen group,the upper part of the defect was weakly positive staining,and the middle and lower part was positive stainingand collagen groups.In addition,more positive staining in the repaired area of XGMA2 was showed,which was no obviously different than the native cartilage.CONCLUSION:The high viscosity of Xanthan gum solution makes it printable,Its shear-thinning behavior greatly reduces the amount of shear force that cells have to endure through the needle during the printing process.This study shows that XG based material serve as bio-ink and incorporated with BMSCs for chondrogenic differentiation and repair defects in vitro and in vivo for cartilage tissue engineering by 3D bioprinting.XGMAs was readily tunable pore size,mechanical properties by adjusting the varying degrees of MA to meet a variety of applications in tissue engineering.Therefore,xanthan-based biomaterials have many potential applications in cartilage tissue engineering and will have further applications in biomedicine.