Objective: To investigate the role of STAT3 phosphorylation in ferroptosis regulation and its impact on cisplatin resistance mechanisms in human osteosarcoma cells． Methods: Human osteosarcoma HOS cells and cispl- atin-resistant HOS / DDP cells were treated with cisplatin ( 0. 5，1，2，4，8，16，32 mg / L) ，the ferroptosis inducer Erastin ( Era) ( 2 μmol / L) ，and / or the ferroptosis inhibitor Ferrostatin-1 ( Fer1) ( 10 μmol / L) ．Cell viability and proliferation were assessed using the Cell Counting Kit-8 ( CCK-8) and colony formation assays，and cell migration was evaluated via a scratch assay．Reactive oxygen species ( ROS) ，intracellular ferrous iron levels，mitochondrial membrane potential，mitochondrial function，malondialdehyde ( MDA) levels，and the reduced glutathione / oxi- dized glutathione ( GSH / GSSG) ratio were measured using commercial kits．The mRNA expression of ferroptosis- related genes was analyzed by quantitative reverse transcription polymerase chain reaction ( RT-qPCR) ．The protein levels of glutathione peroxidase 4 ( GPX4) ，solute carrier family 7 member 11 ( SLC7A11) ，phosphorylated STAT3 ( p-STAT3) ，and total STAT3 were determined by Western blot． Results: With cisplatin treatment，HOS cells ex- hibited decreased cell viability，mitochondrial membrane potential，and GSH / GSSG ratio ( P＜0. 01) ，along with elevated levels of ROS，ferrous ion，and MDA content ( P ＜0. 01) ． The protein levels of GPX4 ( P ＜0. 01) ， SLC7A11，and p-STAT3 also decreased ( P ＜0. 05) ．Coadministration with the ferroptosis inhibitor Ferrostatin-1 ( Fer-1) reversed these aforementioned effects ( P ＜0. 05) ．In HOS / DDP cells，the mRNA levels of ferroptosis- suppressive genes ( GPX4，FTH1，SLC7A11，and AIFM2) were significantly higher than those in HOS cells ( P < 0. 05) ，whereas the expression of ferroptosis-promoting genes ( ACSL4 and PTGS2) was significantly lower ( P < 0. 05) ．The cisplatin-induced reductions in cell viability and mitochondrial membrane potential，as well as the in- creases in ROS，ferrous ion，and MDA levels，were less pronounced in HOS / DDP cells than in HOS cells．The SLC7A11 protein level showed no significant change． However，combined treatment with the ferroptosis inducer Erastin ( Era) resulted in significant decreases in viability，mitochondrial membrane potential，and the GSH / GSSG ratio in HOS / DDP cells ( P＜0. 05) ．Furthermore，the protein levels of p-STAT3，GPX4，and SLC7A11 were also markedly reduced ( P＜0. 05) ． Conclusion The activation of ferroptosis mediated by p-STAT3 enhances cisplatin sensitivity in HOS / DDP cells．

Objective To explore the effect and potential mechanisms of melatonin combined with gemcitabine on the chemosensitivity of human pancreatic cancer cell line PANC-1.Methods Human pancreatic cancer cell line PANC-1 was trea-ted with gemcitabine alone or in combination with melatonin.Cell viability was assessed using CCK-8.Effect of melatonin and gem-citabine alone or in combination on the clonogenic capacity of PANC-1 cells were observed through colony formation experiments.Scratch assays and transwell experiments were conducted to evaluate cell migration ability.Reactive oxygen species(ROS)and mitochondrial membrane point JC-1 assay kit were used to determine reactive oxygen species synthesis and membrane potential levels.Intracellular Fe2+level was measured using ferrous ion fluorescent probe.The protein expression levels of LC3,P62,GPX4 and SLC7A11 in different treatment groups were detected by immunofluorescence and Western blotting.Results CCK-8 results showed that the viability of PANC-1 cells was inhibited by gemcitabine alone after 48 h and 72 h of treatment in a time-and dose-dependent manner.The cell viability of gemcitabine combined with melatonin group was significantly lower than that of gemcitabine group,and the cell viability decreased with the increase of melatonin concentration.Scratch assays,transwell experiments,and plate colony formation assay results demonstrated that the proliferation and migration of cells in the gemcitabine combined with the me-latonin group were significantly inhibited compared with the gemcitabine group.The levels of reactive oxygen species and Fe2+in PANC-1 in gemcitabine combined with the melatonin group were higher than those in the gemcitabine group,and the mitochondri-al membrane potential was significantly decreased(P＜0.01).Western blotting and immunofluorescence results showed that the ra-tio of autophagy-related protein LC3-Ⅱ/LC3-Ⅰ in gemcitabine combined with the melatonin group was lower than that in the gem-citabine group,and the expression of P62 was up-regulated,and the expression of anti-iron death-related protein GPX4 and SLC7A11 was significantly inhibited(P＜0.05),suggesting that melatonin combined with gemcitabine can inhibit autophagy and promote ferroptosis in PANC-1 cells.Conclusion Melatonin enhances the chemosensitivity of pancreatic cancer cell PANC-1 to gemcitabine by inhibiting autophagy and promoting ferroptosis of tumor cells.

Objective:To investigate the application of virtual reality technology combined with case-based learning in forward surgical team (FST) basic skill teaching for undergraduates.Methods:A total of 42 undergraduates who received clinical practice in The Second Affiliated Hospital of Navy Medical University from January 2020 to January 2021 were selected as research subjects, and they were randomly divided into experimental group (virtual reality technology combined with case-based learning for FST basic skill teaching) and control group (traditional teaching methods for FST basic skill teaching). A questionnaire survey and assessments were performed to evaluate the effectiveness of teaching, and SPSS 23.0 was used to perform the t-test, the chi-square test, or the Fisher's exact test. Results:The questionnaire survey showed that there were no significant differences between the two groups in the degree of overall satisfaction with teaching, comprehension and practice in learning, and post-learning memory, and compared with the control group, the experimental group had significantly higher scores of improvement in theoretical knowledge (4.33±0.26 vs. 4.17±0.21, P<0.05), improvement in skill operation (4.32±0.22 vs. 4.12±0.27, P<0.05), improvement in the ability to analyze and solve practical problems (4.04±0.37 vs. 3.69±0.38, P<0.05), learning interest and enthusiasm (4.34±0.28 vs. 3.92±0.43, P<0.05), learning attention (4.21±0.35 vs. 3.81±0.34, P<0.05), and learning interaction (4.18±0.29 vs. 4.01±0.21, P<0.05). The results of assessments showed that compared with the control group, the experimental group had a significantly higher total score (85.96±5.35 vs. 77.03±7.29, P<0.05) and significantly better scores of theoretical knowledge (28.25±4.74 vs. 25.01±5.37, P<0.05) and skill operation (57.47±4.96 vs. 51.99±8.03, P<0.05). Conclusions:Virtual reality technology combined with case-based learning has unique advantages in FST basic skill teaching for undergraduates, and related studies and application research can be conducted in the future.

Objective To evaluate the passage stability of HEK293 cells in suspension culture by using population doubling level(PDL)and verify the method,in order to provide experimental basis for the industrial production and culture of this cells. Methods The working seed lot of HEK293 cells were subcultured continuously for 60 d,one generation every 2 d,and the cell stability was evaluated when PDL increased to 10-60. The calculation of related research results showed that when HEK293 cells were cultured to 2 500 L,the PDL of each generation should be controlled within 2 ± 0. 2. The working seed lot of HEK293 cells were cultured in different stages of shaking flask(125,500,1 000 and 3 000 mL,four generations),cell expansion system(CES)(25,25 and 50 L,three generations)and bioreactor(100,500 and 500,three generations). The PDL of each generation was controlled within 2 ± 0. 2. Totally three batches of cells were cultured and analyzed for the indicators such as cell density,viability,agglomeration rate and diameter. After culture for 72 h in a 500 L bioreactor,the HEK293cells were inoculated with the working seed lot of adenovirus at a MOI of 5-10,cultured for 2 d,then the virus liquid was harvested and detected for the number of virus particles. Results HEK293 cells in the working seed lot in serial passage maintained high cell density and viability when the PDL reached 60. When PDL was controlled in the range of 2 ± 0. 2,the density of three batches of HEK293 cells in the shaking flask,CES and bioreactor was all greater than 2. 0 × 10~6cells/mL,the viability was all greater than 96%,and the cell diameter was about 17 μm. The agglomeration rates were all lower than 35%. The three batches of HEK293 cells cultured in the 500 L bioreactor were inoculated with virus for 2 d,and the number of virus particles reached 11. 68 × 1010,12. 55 × 10~(10)and 9. 38 × 10~(10)vp/mL,respectively. Conclusion It is feasible to evaluate the stability of passage of HEK293 cells by PDL,which can reflect the growth status of passage cells more scientifically.

Primary central nervous system lymphoma(PCNSL)is an aggressive extranodal non-Hodgkin's lymphoma involving cerebrospinal fluid,medulla spinalis,intraocular structures,cranial nerves,and pia.It is an infiltrating malignant tumor with no involvement outside the central nervous system.PCNSL is sensitive to radiotherapy and chemotherapy,and treatment includes high-dose methotrexate-based remission induction,surgery and consolidation therapy with whole-brain radiotherapy.In recent years,some new drugs,including Bruton tyrosine kinase inhibitor,programmed cell death protein-1 inhibitor,lenalidomide,rituximab,pemetrexed,chimeric antigen receptor T-cell immunotherapy and selective inhibitor of nuclear export,have entered clinical trials or use stage and shown good therapeutic effect.This article reviews the traditional treatment programs of PCNSL patients and summarizes the latest progress of treatment.

Objective To investigate the value of CT radiomics combined with CT and preoperative pathological features for predicting postoperative early recurrence(ER)of local advanced esophageal squamous cell carcinoma(LAESCC).Methods Data of 334 patients with LAESCC were retrospectively analyzed.The patients were divided into training set(n=234)and verification set(n=100)at the ratio of 7:3 and were followed up to observe ER(recurrence within 12 months after surgery)or not.Univariate and multivariate logistic regression were used to analyze clinical,CT and preoperative pathological features of LAESCC in patients with or without ER in training set.The independent risk factors of ER were screened,and a CT-preoperative pathology model was constructed.Based on venous phase CT in training set,the radiomics features of lesions were extracted and screened to establish radiomics model,and finally a combined model was established based on radiomics model and the independent risk factors.Receiver operating characteristic(ROC)curves were drawn,and the area under the curve(AUC)was calculated to evaluate the diagnostic efficacy of each model.Results Among 334 cases,168 were found with but 166 without ER.In training set,117 cases were found with while the rest 117 without ER,while in verification set,51 were found with but 49 without ER.The length of lesions,cT stage and cN stage shown on CT and tumor differentiation degree displayed with preoperative pathology were all independent risk factors for ER of LAESCC(all P＜0.05).The AUC of CT-preoperative pathology model in training set and validation set was 0.759 and 0.783,respectively.Ten best radiomics features of LAESCC were selected,and AUC of the established radiomics model in training set and validation set was 0.770 and 0.730,respectively.The AUC of combined model in training and validation set was 0.838 and 0.826,respectively.The AUC of CT radiomics combined with CT and preoperative pathological features in training set was higher than that of CT-preoperative pathologymodel and radiomics model(both P＜0.01).Conclusion CT radiomics combined with CT and preoperative pathological features could effectively predict postoperative ER of LAESCC.

Objective:To prepare the whole bladder acellular matrix (BAM) using the self-designed perfusion decellularization system, and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells (ADSCs).Methods:This study was conducted from October 2020 to April 2021. The self-designed perfusion decellularization system was used, and four different decellularization protocols (group A, group B, group C and group D) were formulated, according to the flow direction of the perfusate and the action time of different decellularization solutions. Among them, the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B. The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D. In groups A and C, 1% Triton X-100 was treated for 6 h, and 1% sodium dodecyl sulfate (SDS) was treated for 2 h. In groups B and D, 1% Triton X-100 was treated for 7 h, and 1% sodium dodecyl sulfate (SDS) was treated for 1 h. In addition, the tissue in the normal bladder group was directly obtained from the natural bladder tissue, which did not require perfusion, cryopreservation and thawing. The fast and efficient decellularization protocol was screened out through HE, DAPI, Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold. The prepared BAM was used as the scaffold material, and the ADSCs were used as the seeding cells to construct the tissue engineering bladder. HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results:HE and DAPI staining showed that there was no obvious nuclear residue in the group C. Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C. There was no significant difference in bladder wall thickness between the group C and the normal bladder group [(975.44±158.62)μm vs.(1 064.49±168.52)μm, P > 0.05]. The DNA content in the group C [(43.59 ±4.59) ng/mg] was lower than that in the normal bladder group, group A, group B and group D [(532.50±26.69), (135.17±6.99), (182.49±13.69) and(84.00±4.38)ng/mg], and the difference was statistically significant ( P<0.05). The collagen content [(10.98 ± 0.29)μg/mg] and glycosaminoglycan content [(2.30±0.18)μg/mg] in group C were not significantly different with those in the normal bladder group [(11.69±0.49) and (2.36±0.09)μg/mg, P>0.05]. Scanning electron microscopy showed that a large number of pore structures could be observed on the surface of the prepared BAM in groups A-D and were facilitated to cell adhesion. The isolated and cultured ADSCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29, and the negative expression of CD45 and CD106. Live/dead staining and CCK-8 detection confirmed that the prepared BAM in the group C had no cytotoxicity. HE and DAPI staining showed that a large number of ADSCs were distributed on the surface and inside of the tissue engineering bladder. Conclusions:The whole bladder shape BAM prepared by the self-designed perfusion decellularization system could be used as the scaffold material for bladder tissue engineering, and the constructed tissue engineering bladder could be used for bladder repair and reconstruction.

Objective:To investigate the predictive model construction of anastomotic thickening character after radical surgery of esophageal cancer based on computed tomogralphy(CT) radiomics and its application value.Methods:The retrospective cohort study was conducted. The clinicopathological data of 202 patients with esophageal squamous cell carcinoma (ESCC) who were admitted to The First Affiliated Hospital of Zhengzhou University from January 2013 to June 2021 were collected. There were 147 males and 55 females, aged (63±8) years. Based on random number table, 202 patients were assigned into training dataset and validation dataset at a ratio of 7:3, including 141 cases and 61 cases respectively. Patients underwent radical resection of ESCC and enhanced CT examination. Observation indicators: (1) influencing factor analysis of malignant anas-tomotic thickening; (2) construction and evaluation of predictive model; (3) performance comparison of 3 predictive models. The normality of continuous variables was tested by Kolmogorov-Smirnov method. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was analyzed using the t test. Measurement data with skewed distribution were represented as M( Q1, Q3), and comparison between groups was analyzed using the Mann-Whintney U test. Count data were represented as absolute numbers, and comparison between groups was analyzed using the chi-square test or Fisher's exact probability. The consistency between subjective CT features by two doctors and measured CT numeric variables was analyzed by Kappa test and intraclass correlation coefficient (ICC), with Kappa >0.6 and ICC >0.6 as good consistency. Univariate analysis was conducted by corresponding statistic methods. Multivariate analysis was conducted by Logistics stepwise regression model. The receiver operating characteristic (ROC) curve was drawn, and area under curve (AUC), Delong test, decision curve were used to evaluate the diagnostic efficiency and clinical applicability of model. Results:(1) Influencing factor analysis of malignant anastomotic thickening. Of the 202 ESCC patients, 97 cases had malignant anastomotic thickening and 105 cases had inflammatory anastomotic thickening. The consistency between subjective CT features by two doctors and measured CT numeric variables showed Kappa and ICC values >0.6. Results of multivariate analysis showed that the maximum thickness of anastomosis and CT enhancement pattern were independent influencing factors for malignant anastomotic thickening[ hazard ratio=1.46, 3.09, 95% confidence interval ( CI) as 1.26-1.71,1.18-8.12, P<0.05]. (2) Construction and evaluation of predictive model. ① Clinical predictive model. The maximum thickness of anasto-mosis and CT enhancement pattern were used to construct a clinical predictive model. ROC curve of the clinical predictive model showed an AUC, accuracy, sensitivity, specificity as 0.86 (95% CI as 0.80-0.92),0.77, 0.77, 0.80 for the training dataset, and 0.78 (95% CI as 0.65-0.89), 0.77, 0.77, 0.80 for the validation dataset, respectively. Results of Delong test showed no significant difference in AUC between the training dataset and validation dataset ( Z=1.22, P>0.05). ② Radiomics predictive model. A total of 854 radiomics features were extracted and 2 radiomics features (wavelet-LL_first order_ Maximum and original_shape_VoxelVolume) were finally screened out to construct a radiomics predictive model. ROC curve of the radiomics predictive model showed an AUC, accuracy, sensitivity, specificity as 0.87 (95% CI as 0.81-0.93), 0.80, 0.75, 0.86 for the training dataset, and 0.73 (95% CI as 0.63-0.83), 0.80, 0.76, 0.94 for the validation dataset, respectively. Results of Delong test showed no significant difference in AUC between the training dataset and validation dataset ( Z=-0.25, P>0.05). ③ Combined predictive model. Results of multivariate analysis and radiomics features were used to construct a combined predictive model. ROC curve of the combined predictive model showed an AUC, accuracy, sensitivity, specificity as 0.93 (95% CI as 0.89-0.97),0.84, 0.90, 0.84 for the training dataset, and 0.79 (95% CI as 0.70-0.88), 0.89, 0.86, 0.91 for the validation dataset, respectively. Results of Delong test showed no significant difference in AUC between the training dataset and validation dataset ( Z=0.22, P>0.05). (3) Performance comparison of 3 predictive models. Results of Hosmer-Lemeshow goodness-of-fit test showed that the clinical predictive model, radiomics predictive model and combined predictive model had a good fitting degree ( χ2=4.88, 7.95, 4.85, P>0.05). Delong test showed a significant difference in AUC between the combined predictive model and clinical predictive model, also between the combined predictive model and radiomics predictive model ( Z=2.88, 2.51, P<0.05 ). There was no significant difference in AUC between the clinical predictive model and radiomics predictive model ( Z=-0.32, P>0.05). The calibration curve showed a good predictive performance in the combined predictive model. The decision curve showed a higher distinguishing performance for anastomotic thickening character in the combined predictive model than in the clinical predictive model or radiomics predictive model. Conclusions:The maximum thickness of anastomosis and CT enhancement pattern are independent influencing factors for malignant anastomotic thickening. Radiomics predictive model can distinguish the benign from malignant thickening of anastomosis. Combined predictive model has the best diagnostic efficacy.

Objective:To compare the prognosis and complications of muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC) patients undergoing radical cystectomy (RC) followed by ileal neobladder.Methods:The clinical data of 103 patients who underwent orthotopic ileal neobladder in Jiangsu Province Hospital from April 2010 to October 2021 were retrospectively analyzed. There were 51 MIBC patients and 52 NMIBC patients. In the MIBC group, there were 49 males and 2 females, aged (58.1 ± 8.9) years, with American Society of Anesthesiologists (ASA) score of 1-2 in 48 cases and 3 in 3 cases. Open radical cystectomy (ORC) was performed in 2 cases, laparoscopic (LRC) in 34 cases and robot-assisted radical cystectomy (RARC) in 15 cases. In the NMIBC group, there were 49 males and 3 females, aged (55.7 ± 9.9) years, ASA score of 1-2 in 51 cases and ASA score of 3 in 1 case. LRC was performed in 41 cases, and RARC in 11 cases. There were no statistically differences between the two groups in above indicators ( P>0.05). The Clavien-Dindo grading system (CCS) was used to assess the complications, defining CCS Ⅰ-Ⅱ as mild complications and CCS Ⅲ-Ⅴ as severe complications. According to their relationship to the neobladder, complications were be classified as neobladder-related and non-neobladder-related complications. The occurrence of complications and the prognosis of neobladder between MIBC and NMIBC were compared. Results:The average operation time of the MIBC group and NMIBC group were (421.2 ± 119.7) min vs. (439.8 ± 106.2) min. The blood loss were 400 (300, 700) ml vs. 400 (300, 625) ml. The frequency of lymph nodes removed were (14.9 ± 8.3) vs. (14.8 ± 8.5). The postoperative defecation time were 5 (4, 6) d vs. 5 (3, 6) d. And the postoperative hospital stay were 20 (15, 28) d vs. 22 (19, 28) d. There were no statistically differences between the two groups in above indicators ( P>0.05). The MIBC group had a significantly lower rate of pelvic lymph node metastasis [17.6% (9/51) vs. 0(0/52), P=0.001] and tumor thrombosis [23.5% (12/51) vs. 5.8% (3/51), P=0.011] than the MIBC group. Moreover, the NMIBC group had a considerably superior 5-year overall survival (OS) (97.6% vs. 70.2%, P=0.035). The proportion of pads needed in the daytime of the MIBC group and NMIBC group were 14.6% (7/46) vs. 6.7% (3/45). The frequency of urination were (2.0 ± 0.7) h vs. (2.4 ± 0.7) h. Furthermore, The proportion of pads needed at night were 47.9% (23/48) vs. 53.3% (24/45). The frequency of nocturnal urination were 3.1±1.5 vs. 2.3 ± 1.7. And the number of pads needed at night were all 1 (0, 1) pad. The daytime and nighttime incontinence rate were 25.0% (12/48) and 62.5% (30/48) respectively in MIBC, compared to 11.1% (5/45) and 62.2% (28/45) respectively in NMIBC. And the proportion of erectile function retention were 15.8% (6/38) vs. 25.0% (10/40). There were no statistically significant differences in the prognosis of neobladder function between the two groups ( P>0.05). Furthermore, the proportions of mild complications in the MIBC group and NMIBC group were [41.2% (21/51) vs. 51.9 (27/52)]. The proportions of severe complications were [21.6% (11/51) vs. 19.2% (27/52)]. The proportions of neobladder-related complications were [27.5% (14/51) vs. 25.0% (13/52)]. And the proportions of non-neobladder-related complications were [39.2% (20/51) vs. 25.0% (13/52)]. There were no statistically significant differences in the complications between the two groups ( P>0.05). Conclusions:There was no statistically significant difference in functional prognosis and complications of neobladder between MIBC group and NMIBC group, and NMIBC had a better oncologic prognosis.

Objective:This study aimed to investigate the physical properties, biocompatibility, and 3D printing performance of a novel hybrid bioink composed of gelatin methacrylated (GelMA) and chitin nanocrystal (ChiNC).Methods:The study was conducted from May 2021 to December 2022, four different bioinks were prepared by adding varying amounts of ChiNC to GelMA bioink. The GelMA concentration in all four bioinks was 100 mg/ml, while the ChiNC concentrations were 0 mg/ml (no ChiNC added), 5 mg/ml, 10 mg/ml, and 20 mg/ml, respectively, named as GC0, GC5, GC10, and GC20 bioinks. The cross-sectional morphology of the hydrogels formed after photocuring the four bioinks was observed using scanning electron microscopy, and the porosity was calculated. Weighing the hydrogels before and after swelling, and then calculate the equilibrium swelling rate. HUVECs were seeded on the surfaces of the hydrogels prepared from the four bioinks and cultured in medium. Cell proliferation was assessed using CCK-8 assays at 1d, 3d, and 7d to compare the proliferation rates of cells on the four hydrogels. HUVECs were added to the four bioinks, and grid-like scaffolds were printed and cultured in medium. Live-Dead staining was performed at 1d and 7d to observe cell viability. Compare the printing effect of bioinks by observing its forming continuous threads properties during extrusion. Finally, tissue-engineered bladder patches simulating the mucosal layer, submucosal layer, and muscular layer anatomical structures of the bladder wall were 3D bioprinted using the optimized bioink composition, and the stability and fidelity of the printed structures were observed to further validate the feasibility of printing multi-layered complex structures with the bioink.Results:Scanning electron microscopy revealed that the porosity of the GC0, GC5, GC10, and GC20 hydrogels were (51.43±6.23)%, (51.85±6.47)%, (50.55±4.59)%, and (42.49±2.20)%, respectively. The differences in porosity between the GC0 group and the other three groups were not statistically significant ( P=0.9994, P=0.9948, P=0.1200). The equilibrium swelling ratio of the other three groups [(8.81±0.41), (7.95±0.19), (7.71±0.14)] was significantly lower than that of the GC0 group (9.37 ± 0.49), and the differences were statistically significant ( P=0.0457, P<0.01, P<0.01). CCK-8 assay showed no significant difference in absorbance value between the GC10 group (0.360±0.009) and the GC0 group (0.357±0.007), GC5 group (0.350±0.012), and GC20 group (0.345±0.018) on the first day ( P=0.9332, P=0.5464, P=0.4937). However, on the third day, the absorbance value of the GC10 group (0.755±0.012) was significantly higher than that of the GC0 group (0.634±0.010), GC5 group (0.704±0.009), and GC20 group (0.653±0.015) ( P<0.01, P=0.0033, P=0.0002). On the seventh day, the absorbance value of the GC10 group (1.001±0.031) was significantly higher than that of the GC0 group (0.846±0.026), GC5 group (0.930±0.043), and GC20 group (0.841±0.024)( P=0.0012, P=0.1390, P=0.0010). The addition of human umbilical vein endothelial cells (HUVECs) into the four groups of hydrogels enabled the printing of grid-like scaffolds, and Live-Dead staining was performed on day 1 and day 7. The cell viability of HUVECs in the four groups on day 1 was (90.13±1.63)%, (90.6±2.45)%, (92.58±2.15)%, and (91.40±3.17)%, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.9869, P=0.3093, P=0.8008). On day 7, the cell viability was (89.97±3.10)%, (92.18±2.21)%, (92.05±2.25)%, and (90.12±1.97)% for the four groups, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.3965, P=0.4511, P=0.9995). Bioink extrusion test showed that the GC0 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 25℃, while the GC10 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 27℃. Printing tissue engineered bladder patches simulating the anatomical structure of the bladder mucosal layer, submucosal layer, and muscular layer using GC10 bioink, and the printed patches were stable, without collapse, and had high fidelity. Conclusions:Adding ChiNC to GelMA promotes cell adhesion, proliferation, and expands the printing window of GelMA bioink. The biocompatibility of the mixed bioink prepared by adding 10 mg/ml ChiNC in GelMA is good, capable of printing tissue-engineered bladder patches that mimic the anatomical structure of natural bladder walls.