1.Background data of SD rats in embryo-fetal development toxicity study
Manman ZHAO ; Zihe LIANG ; Xiaomeng LIU ; Ying YANG ; Chao WANG ; Tingting ZHAO ; Xingchao GENG ; Xiaobing ZHOU ; Sanlong WANG
Chinese Journal of Pharmacology and Toxicology 2024;38(7):526-532
OBJECTIVE To set up normal ranges for indexes in embryo-fetal development toxicity studies in Sprague-Dawley(SD)rats and to establish a background database to provide reference for the embryo-fetal development toxicity evaluation of drugs.METHODS The data on embryonic develop-ment and fetal growth from embryo-fetal development toxicity studies(11 items)conducted by our center between 2013 and 2022 was statistically analyzed,involving 205 pregnant rats and 3037 fetuses in total,with the mean and standard deviation,coefficient of variation and 95%confidence interval calculated.The indexes included body mass,body mass gain and food consumption during pregnancy,pregnancy outcomes(pregnancy rate,average corpora lutea,average Implant sites,average live conceptuses,live conceptuse rate,resorption rate and dead conceptuse rate),fetal growth and development(fetal mass,placental mass and sex ratio),appearance abnormality rate,visceral abnormality rate,and skeletal abnormality rate.RESULTS The mass of pregnant rats trended up during gestation,with significant increases in the late period.Food consumption increased along with gestation.Caesarean section was conducted on gestation day 20,and the pregnancy rate was 93.2%.The average corpora lutea,Implant sites and live conceptuses were 18.0±3.2,15.9±2.8 and 14.8±3.0,respectively.The live conceptuse rate was 93.4%while the total dead embryo rate was 6.6%.The average mass of fetuses and placenta were respectively 3.6±0.3 and(0.6±0.3)g,and the fetal sex ratio(male/female)was 0.94.The incidence of fetal appearance abnormalities was about 0.2%,and that of soft tissue abnormalities was approximately 0.8%.The rate of skeletal abnormalities was about 1.2%,with higher incidence of non-ossification and incomplete ossification mostly identified on sternum and hyoid bone.The numbers of ossifications of metacarpal bones,metatarsal bones and sacrococcygeal vertebrae were 7.0±0.7,8.0±0.1 and 7.4±0.5,respectively.The rate of ossification of sternumⅠtoⅣwas higher,with an average of about 98.6%-99.9%.The ossification rates of sternum Ⅴ and Ⅵ were(68.0±28.4)%and(82.8±23.9)%.CONCLUSION The background database of indexes in the embryo-fetal development toxicity study on SD rats is established for our GLP laboratory,which provides reference for reproductive toxicity studies.
2.Efficacy and safety of mitoxantrone hydrochloride liposome injection in treatment of peripheral T-cell lymphomas: a multicenter, non-interventional, ambispective cohort, real-world study (MOMENT)
Huiqiang HUANG ; Zhiming LI ; Lihong LIU ; Liang HUANG ; Jie JIN ; Hongyan TONG ; Hui ZHOU ; Zengjun LI ; Zhenqian HUANG ; Wenbin QIAN ; Kaiyang DING ; Quande LIN ; Ming HOU ; Yunhong HUANG ; Jingbo WANG ; Pengcheng HE ; Xiuhua SUN ; Xiaobo WANG ; Zunmin ZHU ; Yao LIU ; Jinhai REN ; Huijing WU ; Liling ZHANG ; Hao ZHANG ; Liangquan GENG ; Jian GE ; Ou BAI ; Liping SU ; Guangxun GAO ; Xin LI ; Yanli YANG ; Yijian CHEN ; Aichun LIU ; Xin WANG ; Yi WANG ; Liqun ZOU ; Xiaobing HUANG ; Dongping HUANG ; Shujuan WEN ; Donglu ZHAO ; Jun MA
Journal of Leukemia & Lymphoma 2023;32(8):457-464
Objective:To evaluate the efficacy and safety of mitoxantrone hydrochloride liposome injection in the treatment of peripheral T-cell lymphoma (PTCL) in a real-world setting.Methods:This was a real-world ambispective cohort study (MOMENT study) (Chinese clinical trial registry number: ChiCTR2200062067). Clinical data were collected from 198 patients who received mitoxantrone hydrochloride liposome injection as monotherapy or combination therapy at 37 hospitals from January 2022 to January 2023, including 166 patients in the retrospective cohort and 32 patients in the prospective cohort; 10 patients in the treatment-na?ve group and 188 patients in the relapsed/refractory group. Clinical characteristics, efficacy and adverse events were summarized, and the overall survival (OS) and progression-free survival (PFS) were analyzed.Results:All 198 patients were treated with mitoxantrone hydrochloride liposome injection for a median of 3 cycles (range 1-7 cycles); 28 cases were treated with mitoxantrone hydrochloride liposome injection as monotherapy, and 170 cases were treated with the combination regimen. Among 188 relapsed/refractory patients, 45 cases (23.9%) were in complete remission (CR), 82 cases (43.6%) were in partial remission (PR), and 28 cases (14.9%) were in disease stabilization (SD), and 33 cases (17.6%) were in disease progression (PD), with an objective remission rate (ORR) of 67.6% (127/188). Among 10 treatment-na?ve patients, 4 cases (40.0%) were in CR, 5 cases (50.0%) were in PR, and 1 case (10.0%) was in PD, with an ORR of 90.0% (9/10). The median follow-up time was 2.9 months (95% CI 2.4-3.7 months), and the median PFS and OS of patients in relapsed/refractory and treatment-na?ve groups were not reached. In relapsed/refractory patients, the difference in ORR between patients with different number of treatment lines of mitoxantrone hydrochloride liposome injection [ORR of the second-line, the third-line and ≥the forth-line treatment was 74.4% (67/90), 73.9% (34/46) and 50.0% (26/52)] was statistically significant ( P = 0.008). Of the 198 PTCL patients, 182 cases (91.9%) experienced at least 1 time of treatment-related adverse events, and the incidence rate of ≥grade 3 adverse events was 66.7% (132/198), which was mainly characterized by hematologic adverse events. The ≥ grade 3 hematologic adverse events mainly included decreased lymphocyte count, decreased neutrophil count, decreased white blood cell count, and anemia; non-hematologic adverse events were mostly grade 1-2, mainly including pigmentation disorders and upper respiratory tract infection. Conclusions:The use of mitoxantrone hydrochloride liposome injection-containing regimen in the treatment of PTCL has definite efficacy and is well tolerated, and it is a new therapeutic option for PTCL patients.
3.Continuation, reduction, or withdrawal of tofacitinib in patients with rheumatoid arthritis achieving sustained disease control: a multicenter, open-label, randomized controlled trial.
Mengyan WANG ; Yu XUE ; Fang DU ; Lili MA ; Liang-Jing LU ; Lindi JIANG ; Yi-Li TAO ; Chengde YANG ; Hui SHI ; Honglei LIU ; Xiaobing CHENG ; Junna YE ; Yutong SU ; Dongbao ZHAO ; Sheng-Ming DAI ; Jialin TENG ; Qiongyi HU
Chinese Medical Journal 2023;136(3):331-340
BACKGROUND:
Rheumatoid arthritis (RA), a chronic systemic autoimmune disease, is characterized by synovitis and progressive damage to the bone and cartilage of the joints, leading to disability and reduced quality of life. This study was a randomized clinical trial comparing the outcomes between withdrawal and dose reduction of tofacitinib in patients with RA who achieved sustained disease control.
METHODS:
The study was designed as a multicenter, open-label, randomized controlled trial. Eligible patients who were taking tofacitinib (5 mg twice daily) and had achieved sustained RA remission or low disease activity (disease activity score in 28 joints [DAS28] ≤3.2) for at least 3 months were enrolled at six centers in Shanghai, China. Patients were randomly assigned (1:1:1) to one of three treatment groups: continuation of tofacitinib (5 mg twice daily); reduction in tofacitinib dose (5 mg daily); and withdrawal of tofacitinib. Efficacy and safety were assessed up to 6 months.
RESULTS:
Overall, 122 eligible patients were enrolled, with 41 in the continuation group, 42 in the dose-reduction group, and 39 in the withdrawal group. After 6 months, the percentage of patients with a DAS28-erythrocyte sedimentation rate (ESR) of <3.2 was significantly lower in the withdrawal group than that in the reduction and continuation groups (20.5%, 64.3%, and 95.1%, respectively; P < 0.0001 for both comparisons). The average flare-free time was 5.8 months for the continuation group, 4.7 months for the dose reduction group, and 2.4 months for the withdrawal group.
CONCLUSION:
Withdrawal of tofacitinib in patients with RA with stable disease control resulted in a rapid and significant loss of efficacy, while standard or reduced doses of tofacitinib maintained a favorable state.
TRIAL REGISTRATION
Chictr.org, ChiCTR2000039799.
Humans
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Quality of Life
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China
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Arthritis, Rheumatoid/drug therapy*
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Piperidines/therapeutic use*
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Treatment Outcome
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Antirheumatic Agents/therapeutic use*
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Pyrroles/therapeutic use*
4.Perioperative blood loss in different approaches by percutaneous endoscopic discectomy
Zhihua WU ; Jiahui HE ; Huantong CHENG ; Shaohao LIN ; Zhilin GE ; Jianchao CUI ; De LIANG ; Xiaobing JIANG
Journal of Xi'an Jiaotong University(Medical Sciences) 2022;43(1):133-137
【Objective】 To compare the perioperative blood loss between interlaminar and transforaminal approaches by percutaneous endoscopic discectomy in order to provide more reference for guiding the proper choice of surgical methods clinically. 【Methods】 We retrospectively analyzed the clinical data of 160 patients who underwent percutaneous endoscopic lumbar discectomy from June 2019 to November 2020, with 80 patients in interlaminar approach group and 80 in transforaminal approach group. The blood loss was calculated according to Gross formula. 【Results】 The perioperative total blood loss (mL), hidden blood loss (mL) and hemoglobin loss (g/L) were significantly lower in interlaminar approach group than in transforaminal approach group (119.73±179.26 vs. 158.6±190.65, 109.73±179.53 vs. 148.78±190.19, 3.76±8.12 vs. 4.31±7.62) (P<0.05). However, there was no significant difference in visible blood loss between the two groups. 【Conclusion】 The perioperative hidden blood loss accounts for a large proportion in percutaneous endoscopic lumbar discectomy. In addition, the interlaminar approach causes less blood loss than the transforaminal approach.
5.The pivotal role of autophagy in bone cells:bone-related cell activity and bone metabolism
Youqiang SUN ; Chao MA ; Mengmeng LIANG ; Pengfei XIN ; Hua ZHANG ; Xiaobing XIANG
Chinese Journal of Tissue Engineering Research 2022;26(2):276-282
BACKGROUND:The importance of autophagy for maintaining cellular homeostasis and stress response has long been recognized.As a way for cells to selectively clear their damaged organelles to achieve the recycling of cellular components,autophagy has a pivotal role in bone metabolism.OBJECTIVE:To review the role and possible mechanisms of autophagy in regulating bone-related cell activity and function among bone marrow mesenchymal stem cells,osteoblasts,osteocytes,and osteoclasts.METHODS:PubMed was searched for studies related to autophagy using the keywords of "autophagy;bone marrow mesenchymal stem cells;osteoblasts;osteocytes;osteoclasts."RESULTS AND CONCLUSION:We finally included 84 papers.Autophagy plays an important role in bone metabolism.Autophagy is involved in maintaining the balance between mineralization and absorption,and then maintaining bone homeostasis.An appropriate autophagy inducer may also benefit bone remodeling.Abnormal autophagy can lead to disorders of bone balance,leading to diseases such as osteoporosis.We may prevent or treat bone-related diseases by regulating the level of autophagy as its function in maintaining the balance of mineralization and resorption in bone homeostasis.
6.Guideline for postoperative rehabilitation treatment following vertebral augmentation for osteoporotic vertebral compression fracture (version 2022)
Zhengwei XU ; Dingjun HAO ; Liming CHENG ; Baorong HE ; Bohua CHEN ; Chen CHEN ; Fei CHE ; Jian CHEN ; Qixin CHEN ; Liangjie DU ; Shunwu FAN ; Zhong FANG ; Shiqing FENG ; Yanzheng GAO ; Haishan GUAN ; Zhong GUAN ; Hua JIANG ; Weimin JIANG ; Dianming JIANG ; Jun JIANG ; Yue JIANG ; Lijun HE ; Yuan HE ; Bo LI ; Tao LI ; Jianjun LI ; Xigong LI ; Yijian LIANG ; Bin LIN ; Bin LIU ; Bo LIU ; Yong LIU ; Zhibin LIU ; Xuhua LU ; Chao MA ; Lie QIAN ; Renfu QUAN ; Hongxun SANG ; Haibo SHEN ; Jun SHU ; Honghui SUN ; Tiansheng SUN ; Jun TAN ; Mingxing TANG ; Sheng TAO ; Honglin TENG ; Yun TIAN ; Jiwei TIAN ; Qiang WANG ; Xinwei WANG ; Jianhuang WU ; Peigen XIE ; Weihong XU ; Bin YAN ; Yong YANG ; Guoyong YIN ; Xiaobing YU ; Yuhong ZENG ; Guoqing ZHANG ; Xiaobo ZHANG ; Jie ZHAO ; Yue ZHU
Chinese Journal of Trauma 2022;38(11):961-972
Osteoporotic vertebral compression fracture (OVCF) can lead to lower back pain and may be even accompanied by scoliosis, neurological dysfunction and other complications, which will affect the daily activities and life quality of patients. Vertebral augmentation is an effective treatment method for OVCF, but it cannot correct unbalance of bone metabolism or improve the osteoporotic status, causing complications like lower back pain, limited spinal activities and vertebral refracture. The post-operative systematic and standardized rehabilitation treatments can improve curative effect and therapeutic efficacy of anti-osteoporosis, reduce risk of vertebral refracture, increase patient compliance and improve quality of life. Since there still lack relevant clinical treatment guidelines for postoperative rehabilitation treatments following vertebral augmentation for OVCF, the current treatments are varied with uneven therapeutic effect. In order to standardize the postoperative rehabilitation treatment, the Spine Trauma Group of the Orthopedic Branch of Chinese Medical Doctor Association organized relevant experts to refer to relevant literature and develop the "Guideline for postoperative rehabilitation treatment following vertebral augmentation for osteoporotic vertebral compression fracture (2022 version)" based on the clinical guidelines published by the American Academy of Orthopedic Surgeons (AAOS) as well as on the principles of scientificity, practicality and advancement. The guideline provided evidence-based recommendations on 10 important issues related to postoperative rehabilitation treatments of OVCF.
7.Difficult and complicated oral ulceration: an expert consensus guideline for diagnosis.
Xin ZENG ; Xin JIN ; Liang ZHONG ; Gang ZHOU ; Ming ZHONG ; Wenmei WANG ; Yuan FAN ; Qing LIU ; Xiangmin QI ; Xiaobing GUAN ; Zhimin YAN ; Xuemin SHEN ; Yingfang WU ; Lijie FAN ; Zhi WANG ; Yuan HE ; Hongxia DAN ; Jiantang YANG ; Hui WANG ; Dongjuan LIU ; Hui FENG ; Kai JIAO ; Qianming CHEN
International Journal of Oral Science 2022;14(1):28-28
The complexity of oral ulcerations poses considerable diagnostic and therapeutic challenges to oral specialists. The expert consensus was conducted to summarize the diagnostic work-up for difficult and complicated oral ulcers, based on factors such as detailed clinical medical history inquiry, histopathological examination, and ulceration-related systemic diseases screening. Not only it can provide a standardized procedure of oral ulceration, but also it can improve the diagnostic efficiency, in order to avoid misdiagnosis and missed diagnosis.
Consensus
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Humans
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Oral Ulcer/therapy*
8.Application of three-dimensional bioprinting ink containing platelet-rich plasma derived from human umbilical cord blood in the treatment of full-thickness skin defects in nude mice
Wei SONG ; Zhao LI ; Shijun ZHU ; Chao ZHANG ; Bin YAO ; Yi KONG ; Liting LIANG ; Xiaobing FU ; Sha HUANG
Chinese Journal of Burns 2022;38(10):905-913
Objective:To investigate the printability and cytocompatibility of sodium alginate-gelatin (AG) bioink containing platelet-rich plasma derived from human umbilical cord blood (HUCB-PRP), named HUCB-PRP-AG bioink, and the effect of the three-dimensionally printed tissue with the bioink on full-thickness skin defect wounds in nude mice.Methods:The method of experimental research was used. HUCB-PRP-AG bioinks with 2.5%, 5.0%, and 10.0% of HUCB-PRP by volume were prepared and named 1P-AG, 2P-AG, and 4P-AG, respectively. The appearances of AG, 1P-AG, 2P-AG, and 4P-AG at room temperature were observed, and their viscosity and storage/loss modulus were measured by a rotational rheometer. The above four bioinks were used for three-dimensional bioprinting respectively, and the appearances of the printed tissue were observed (the printed tissue was subsequently cross-linked and used). The four kinds of bioprinted tissue were respectively co-cultured with human umbilical vein endothelial cells (HUVECs) in Transwell chambers with HUVEC special medium for 24 h, and the cell proliferation level was detected by cell counting kit 8 ( n=3). The four kinds of bioprinted tissue were respectively cultured in Dulbecco's modified eagle medium for 12, 24, and 48 h, which were dried and weighed, and the degradation rate was calculated ( n=3). The expression of vascular endothelial growth factor (VEGF) in the culture supernatant of 1P-AG, 2P-AG, or 4P-AG cultured in phosphate buffer solution at 0.5, 24.0, and 48.0 h was detected by enzyme-linked immunosorbent assay ( n=5). Sixteen female BALB/c-NU nude mice aged 6-8 weeks were selected to establish a full-thickness skin defect wound model on the back and were divided into conventional control group with wounds being covered with medical hydrocolloid dressing alone, HUCB-PRP group with additional HUCB-PRP dripping to the wounds, AG group additionally covered with AG printed tissue, and 4P-AG group additionally covered with 4P-AG printed tissue, respectively (with 4 nude mice in each group). The wound healing of 3 nude mice in each group was observed on post injury day (PID) 4, 8, and 14, and the wound healing rate was calculated. The wound tissue of the remaining nude mouse in each group was collected on PID 8, the histopathological changes were observed after hematoxylin and eosin staining, and the CD31-positive new blood vessels were observed after immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, least significant difference test, and Bonferroni correction. Results:At room temperature, AG, 1P-AG, 2P-AG, and 4P-AG were semi-transparent liquid, and AG was light yellow, while 1P-AG, 2P-AG, and 4P-AG were light red but the color successively deepened. The viscosity of AG, 1P-AG, 2P-AG, and 4P-AG decreased with the increase of shear rate at the temperature of 10 ℃ and shear rate of 0.1-10.0 s -1; the storage moduli of the four bioinks were greater than the loss moduli at the temperature of 10 ℃ and angular frequency range of 1-100 rad/s. Both the resolution and morphology of the printed tissue of four bioinks were similar. The proliferation levels of HUVECs co-cultured with 1P-AG, 2P-AG, and 4P-AG printed tissue for 24 h were 0.885±0.030, 1.126±0.032, and 1.156±0.045, respectively, which were significantly higher than 0.712±0.019 of HUVECs co-cultured with AG printed tissue ( P<0.01). The proliferation levels of HUVECs co-cultured with 2P-AG and 4P-AG printed tissue for 24 h were significantly higher than the level of HUVECs co-cultured with 1P-AG printed tissue ( P<0.01). The degradation rates of 1P-AG, 2P-AG, and 4P-AG printed tissue were significantly higher than those of AG printed tissue at 12, 24, and 48 h of culture ( P<0.01). The degradation rates of 2P-AG and 4P-AG printed tissue at 24 and 48 h of culture were significantly higher than those of 1P-AG printed tissue ( P<0.01). The degradation rate of 4P-AG printed tissue at 12 h of culture was significantly higher than that of 1P-AG printed tissue ( P<0.01), and the degradation rates of 4P-AG printed tissue at 24 and 48 h of culture were significantly higher than those of 2P-AG printed tissue ( P<0.01). At 0.5, 24.0, and 48.0 h of culture, the expressions of VEGF in the culture supernatant of 2P-AG printed tissue were significantly higher than those of 1P-AG printed tissue ( P<0.01), and the expressions of VEGF in the culture supernatant of 1P-AG and 2P-AG printed tissue were significantly lower than those of 4P-AG printed tissue ( P<0.01). The wounds of nude mice in conventional control group and HUCB-PRP group were dry and smaller on PID 8 compared with those on PID 4, and the wounds of nude mice in HUCB-PRP group were smaller with no scabs on PID 14 compared with those in conventional control group. The printed tissue on the wound of nude mice in AG and 4P-AG groups was significantly degraded with no obvious exudation being observed on the wounds on PID 4, the wounds were significantly epithelialized and smaller on PID 8, and there was no scab on the wound on PID 14. The wounds of nude mice in 4P-AG group were completely epithelialized on PID 14. Compared with those in conventional control group, the wound healing rate of nude mice in AG group was significantly decreased on PID 4 ( P<0.05), and the wound healing rates of nude mice in HUCB-PRP group and 4P-AG group at all time points after injury and in AG group on PID 8 and 14 were significantly increased ( P<0.01). Compared with those in HUCB-PRP group, the wound healing rates of nude mice were significantly decreased on PID 4 and 8 in AG group and on PID 4 in 4P-AG group ( P<0.01), while the wound healing rates of nude mice were significantly increased on PID 14 in AG group and on PID 8 and 14 in 4P-AG group ( P<0.01). The wound healing rate of nude mice in 4P-AG group was significantly higher than that in AG group at all time points after injury ( P<0.01). On PID 8, a large number of inflammatory cells infiltration, a small amount of new microvessels, and a small amount of CD31-positive new blood vessels were observed in the wounds of nude mice in conventional control group; a large number of inflammatory cells infiltration, abundant new microvessels, and quite a lot CD31-positive new blood vessels were observed in the wounds of nude mice in HUCB-PRP group; light inflammatory inflammation, a small amount of new microvessels, and a small amount of CD31-positive new blood vessels were observed in the wounds of nude mice in AG group; light inflammatory inflammation, a large number of new microvessels, and a large number of CD31-positive new blood vessels were observed in the wounds of nude mice in 4P-AG group. Conclusions:HUCB-PRP-AG bioink has good printability and cytocompatibility, and its three-dimensionally printed tissue can promote vascularization of full-thickness skin defect wounds in nude mice and accelerate wound healing.
9.Preliminary study on screening and diagnosis of fetal cleft palate by two-dimensional combined with three-dimensional ultrasound in early pregnancy
Guangzhi HE ; Zhou LI ; Lianying LIANG ; Weiping KONG ; Gengzhou FANG ; Fangmei ZHANG ; Bo LI ; Xiaobing LIANG
Chinese Journal of Ultrasonography 2021;30(4):324-330
Objective:To explore the feasibility of ultrasound screening and diagnosis of fetal cleft palate in early pregnancy, analyze and summarize the imaging technology and image characteristics of two-dimensional and three-dimensional ultrasound in normal fetus and cleft palate fetus.Methods:A total of 10 519 pregnant women participated in the early pregnancy were included from January 2016 to June 2020 in Shenzhen Hospital, University of Chinese Academy of Sciences. The palatal line on the standard section of fetal nuchal translucency (NT) measurement was used as a screening marker for routine observation. For fetuses with abnormal palatine line, posterior nasal triangle of coronal plane and axial plane of maxillary alveolar arch of two-dimensional ultrasound were added as the diagnostic sections, and three-dimensional volume data of fetal face were collected, and three dimensional multimodal imaging technology was used to analyze the volume data off-line to determine or exclude fetal severe cleft palate. All fetuses were followed up during the second trimester for deformity scanning and post natal (or induced labor) assessment.Results:Of the 10 519 fetuses, the standard NT plane was obtained and the palatal line was observed in 10 204 cases(97.01%), with normal palatal line in 10 169 cases.In 35 suspected cases, 13 cases were confirmed cleft lip and palate by two and three dimensions ultrasound, and were confirmed by induced labor. There were 7 cases in unilateral side, 3 cases in bilateral, 2 cases in median cleft lip and palate, 1 cases in irregular cleft lip and palate, and no false positive results were reported. Twenty-two suspicious cases were excluded by increasing the two-dimensional sectional and three-dimensional volumetric off-line analysis, and screening after the second trimester and after birth. There was 1 case of missed diagnosis of simple cleft palate.Conclusions:Palatal line is a good screening marker for fetal cleft palate in early pregnancy. For fetuses with abnormal palatine line, the adding of posterior nasal triangle and the axial plane of maxillary alveolar arch, and combining three-dimensional volume data for off-line analysis can determine or exclude severe cleft palate. This study is of great significance for early screening and diagnosis of severe fetal cleft palate, prenatal genetic counseling and prevention birth defect.
10.Preliminary investigation on the wound healing effect of three-dimensional bioprinting ink containing human adipose-derived protein complexes
Chao ZHANG ; Zhao LI ; Wei SONG ; Bin YAO ; Mengde ZHANG ; Liting LIANG ; Yufeng JIANG ; Xiaobing FU ; Sha HUANG
Chinese Journal of Burns 2021;37(11):1011-1023
Objective:To investigate the effects of human adipose-derived protein complex (ADPC) on the proliferation and migration ability of human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs), and the repairing effects of ADPC-containing three-dimensional (3D) bioprinting ink (Bioink) in full-thickness skin defect wounds of nude mice.Methods:The experimental research method was used. Discarded subcutaneous adipose tissue from 3 female patients with chronic wounds (aged 29-34 years) admitted to PLA General Hospital for abdominal flap transfer from October 2020 to March 2021 and discarded liposuction adipose tissue from 3 healthy female (aged 24-36 years) for abdominal liposuction during the same period were collected to prepare normal ADPC (nADPC) and liposuction-derived ADPC (lADPC), respectively. The protein concentration of the two kinds of ADPC was measured by bicinchoninic acid method, and the extraction efficiency of them was calculated. The sample numbers were 3. HSFs and HUVECs in logarithmic growth phase were taken for the subsequent experiments. HSFs and HUVECs were divided into phosphate buffered saline (PBS) control group, 4 μg/mL nADPC group, 20 μg/mL nADPC group, 100 μg/mL nADPC group, and 200 μg/mL nADPC group according to the random number table (the same grouping method below), with 5 wells in each group. Cells in PBS control group were cultured with PBS, and the cells in the 4 remaining groups were cultured with the corresponding final mass concentration of nADPC. After 24 h of conventional culture, the cell proliferation viability was detected by cell counting kit 8 method. HSFs and HUVECs were taken and divided into PBS control group, nADPC alone group, lADPC alone group, tumor necrosis factor-α (TNF-α) alone group, TNF-α+nADPC group, and TNF-α+lADPC group. Cells in PBS control group and TNF-α alone group were added with PBS. nADPC or lADPC was added to the cells in nADPC alone group, lADPC alone group, TNF-α+nADPC group, and TNF-α+lADPC group with a final mass concentration of 100 μg/mL, respectively. TNF-α with a final mass concentration of 20 ng/mL was added to the cells in TNF-α alone group, TNF-α+nADPC group, and TNF-α+lADPC group. The cell migration rate was calculated after the scratch test at 24 h after scratching ( n=3), and the cell proliferation viability was detected after 24 h of culture as above ( n=5). Gelatin-alginate composite Bioink (Bioink AG) was taken. Bioink AG containing 100 μg/mL lADPC (lADPC-Bioink AG) was prepared. The morphology of the two at room temperature and after condensation was observed. The morphology after 3D bioprinting and cross-linking was observed. The low-temperature gel formation time was recorded when detecting rheological properties using rheometer ( n=3). Twenty BALB/c-NU female nude mice of 8-10 weeks old were taken to establish the full-thickness skin defect wounds on the back, and then they were divided into routine dressing change group, lADPC alone group, Bioink AG alone group, and lADPC-Bioink AG group, with 5 nude mice in each group. The wounds of nude mice in routine dressing change group were covered with hydrocolloid dressings and performed with routine dressing changes only, while the wounds of nude mice in the remaining 3 groups were treated with lADPC, Bioink AG, and lADPC-Bioink AG accordingly in addition. General observation was performed from treatment day (TD) 0, and the wound healing rate was calculated on TD 2, 6, and 10. On TD 10, histopathological observation of wounds was performed with hematoxylin eosin staining. Data were statistically analyzed with independent samples t test, one-way analysis of variance, analysis of variance for repeated measurement, Student-Newman-Keuls q test, and least significant difference t test. Results:The protein concentration and extraction efficiency of lADPC were respectively (1.306±0.011) mg/mL and (11.1±1.5)%, which were significantly lower than (2.039±0.042) mg/mL and (22.2±2.0)% of nADPC ( t=23.83, 6.38, P<0.05 or P<0.01). After 24 h of culture, compared with those in PBS control group, the proliferation viabilities of HSFs ( q=6.943, 6.375, P<0.01) and HUVECs ( q=6.301, 6.496, P<0.01) were significantly decreased in 100 μg/mL nADPC group and 200 μg/mL nADPC group; compared with those in 100 μg/mL nADPC group, the proliferation viabilities of HSFs and HUVECs in 200 μg/mL nADPC group did not change significantly ( P>0.05). At 24 h after scratching, compared with those in PBS control group, the HSF and HUVEC migration rates were significantly lower in nADPC alone group, lADPC alone group, and TNF-α alone group ( q=5.642, 6.645, 11.480, 4.772, 6.298, 10.420, P<0.05 or P<0.01); compared with those in nADPC alone group, there were no significant changes in the HSF and HUVEC migration rates in lADPC alone group ( P>0.05); compared with those in TNF-α alone group, there were no significant changes in the HSF migration rates in TNF-α+nADPC group or TNF-α+lADPC group ( P>0.05), the HUVEC migration rates were significantly higher in TNF-α+nADPC group and TNF-α+lADPC group ( q=8.585, 7.253, P<0.01); compared with those in TNF-α+nADPC group, there were no significant changes in the HSF and HUVEC migration rates in TNF-α+lADPC group ( P>0.05). After 24 h of culture, compared with those in PBS control group, the HSF and HUVEC proliferation viabilities were significantly lower in nADPC alone group, lADPC alone group, and TNF-α alone group ( q=5.803, 5.371, 9.136, 11.580, 9.493, 13.510, P<0.05 or P<0.01); compared with those in nADPC alone group, the HSF and HUVEC proliferation viabilities in lADPC alone group did not change significantly ( P>0.05); compared with those in TNF-α alone group, the HSF ( q=14.990, 10.850, P<0.01) and HUVEC ( q=7.066, 8.942, P<0.01) proliferation viabilities were significantly higher in TNF-α+nADPC group and TNF-α+lADPC group; compared with those in TNF-α+nADPC group, the HSF and HUVEC proliferation viabilities in TNF-α+lADPC group did not change significantly ( P>0.05). At room temperature and in the condensed state, lADPC-Bioink AG had a more slightly turbid appearance than Bioink AG. lADPC-Bioink AG had a similar morphology to Bioink AG after 3D bioprinting and cross-linking. At 10 ℃, the coagulation time of lADPC-Bioink AG was (76.6±0.4) s, which was significantly slower than (74.4±0.6) s of Bioink AG ( t=4.64, P<0.01). On TD 2, the nude mice in routine dressing change group had more wound exudation, while the nude mice in the remaining 3 groups had no significant exudation. On TD 8, the nude mice in lADPC-Bioink AG group had the smallest residual wound area and obvious epithelial coverage. On TD 2, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than that in lADPC alone group ( t=3.59, P<0.05) and similar to the rates in routine dressing change group and Bioink AG alone group ( P>0.05). On TD 6, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than the rates in routine dressing change group, lADPC alone group, and Bioink AG alone group ( t=18.70, 15.70, 3.15, P<0.05 or P<0.01). On TD 10, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than the rates in routine dressing change group and lADPC alone group ( t=12.51, 4.84, P<0.01) but similar to that in Bioink AG alone group ( P>0.05). On TD 10, the wounds of nude mice in lADPC-Bioink AG group had moderate vascularization of the traumatic tissue, adequate epithelialization, and the best healing effect. Conclusions:Liposuction-related operations have little effect on the characterization of ADPC protein concentration and extraction efficiency. LADPC and nADPC have the same biological effects, which can inhibit the proliferation and migration ability of HSFs and HUVECs in non-inflammatory environment and improve the proliferation viabilities of HSFs and HUVECs in inflammatory environment, while improving the migration ability of HUVECs. Adding lADPC to Bioink AG does not significantly affect the physical properties or printing performance of Bioink AG, but can enhance the wound repair effect of full-thickness skin defect wounds in nude mice.

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