OBJECTIVE: To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. METHODS: The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs ( n=7) and GGTA1 gene-edited pigs ( n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. RESULTS: IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves ( P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference ( P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs ( P<0.05). CONCLUSION The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.
Animals ; Transplantation, Heterologous ; Galactosyltransferases/genetics* ; Sciatic Nerve/immunology* ; Swine ; Tissue Engineering/methods* ; Humans ; Graft Rejection/prevention & control* ; Gene Editing ; Mice ; Mice, Nude ; Heterografts/immunology* ; Animals, Genetically Modified ; Tissue Scaffolds ; Decellularized Extracellular Matrix

Objective To summarize the experience and practical value of living donor kidney harvesting in Bama miniature pigs with six gene modified. Methods The left kidney of Bama miniature pigs with six gene modified was obtained by living donor kidney harvesting technique. First, the ureter was occluded, and then the inferior vena cava and abdominal aorta were freed. During the harvesting process, the ureter, renal vein and renal artery were exposed and freed in sequence. The vascular forceps were used at the abdominal aorta and inferior vena cava, and the renal artery and vein were immediately perfused with 4℃ renal preservation solution, and stored in ice normal saline for subsequent transplantation. Simultaneously, the donor abdominal aorta and inferior vena cava gap were sutured. The operation time, blood loss, warm and cold ischemia time, postoperative complications and the survival of donors and recipients were recorded. Results The left kidney of the genetically modified pig was successfully harvested. Intraoperative bleeding was 5 mL, warm ischemia time was 45 s, and cold ischemia time was 2.5 h. Neither donor nor recipient pig received blood transfusion, and urinary function of the kidney transplanted into the recipient was recovered. The donor survived for more than 8 months after the left kidney was resected. Conclusions Living donor kidney harvesting is safe and reliable in genetically modified pigs. Branch blood vessels could be processed during kidney harvesting, which shortens the process of kidney repair and the time of cold ischemia. Living donor kidney harvesting contributes to subsequent survival of donors and other scientific researches.

Objective To investigate the differences and the immunocompatibility of wild-type (WT), four-gene modified (TKO/hCD55) and six-gene modified (TKO/hCD55/hCD46/hTBM) pig erythrocytes with human serum. Methods The blood samples were collected from 20 volunteers with different blood groups. WT, TKO/hCD55, TKO/hCD55/hCD46/hTBM pig erythrocytes, ABO-compatible (ABO-C) and ABO-incompatible (ABO-I) human erythrocytes were exposed to human serum of different blood groups, respectively. The blood agglutination and antigen-antibody binding levels (IgG, IgM) and complement-dependent cytotoxicity were detected. The immunocompatibility of two types of genetically modified pig erythrocytes with human serum was evaluated. Results No significant blood agglutination was observed in the ABO-C group. The blood agglutination levels in the WT and ABO-I groups were higher than those in the TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups (all P<0.001). The level of erythrocyte lysis in the WT group was higher than those in the ABO-C, TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups. The level of erythrocyte lysis in the ABO-I group was higher than those in the TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups (both P<0.01). The pig erythrocyte binding level with IgM and IgG in the TKO/hCD55 group was lower than those in the WT and ABO-I groups. The pig erythrocyte binding level with IgG and IgM in the TKO/hCD55/hCD46/hTBM group was lower than that in the WT group and pig erythrocyte binding level with IgG was lower than that in the ABO-I group (all P<0.05). Conclusions The immunocompatibility of genetically modified pig erythrocytes is better than that of wild-type pigs and close to that of ABO-C pigs. Humanized pig erythrocytes may be considered as a blood source when blood sources are extremely scarce.

BACKGROUND:Compared with traditional two-dimensional culture,three-dimensional microtissue culture can show greater advantages.However,more favorable cultivation methods in three-dimensional culture still need to be further explored. OBJECTIVE:To evaluate the cell behavior of microtissue and its ability to promote cartilage formation under two three-dimensional culture methods. METHODS:Cartilage-derived microcarriers were prepared by chemical decellularization and tissue crushing.DNA quantification and nuclear staining were used to verify the success of decellularization,and histological staining was used to observe the matrix retention before and after decellularization.The microcarriers were characterized by scanning electron microscopy and CCK-8 assay.Cartilage-derived microtissues were constructed by combining cartilage-derived microcarriers with human adipose mesenchymal stem cells through three-dimensional static culture and three-dimensional dynamic culture methods.The cell viability and chondrogenic ability of the two groups of microtissues were detected by scanning electron microscopy,live and dead staining,and RT-qPCR. RESULTS AND CONCLUSION:(1)Cartilage-derived microcarriers were successfully prepared.Compared with before decellularization,the DNA content significantly decreased after decellularization(P<0.001).Scanning electron microscope observation showed that the surface of the microcarrier was surrounded by collagen,maintaining the characteristics of the natural extracellular matrix of cartilage cells.CCK-8 assay indicated that microcarriers had no cytotoxicity and could promote cell proliferation.(2)Scanning electron microscopy and live and dead staining results showed that compared with the three-dimensional static group,the three-dimensional dynamic group had a more extended morphology of microtissue cells,and extensive connections between cells and cells,between cells and matrix,and between matrix.(3)The results of RT-qPCR showed that the expressions of SOX9,proteoglycan,and type Ⅱ collagen in microtissues of both groups were increased at 7 or 14 days.The relative expression levels of each gene in the three-dimensional dynamic group were significantly higher than those in the three-dimensional static group at 14 days(P<0.05).At 21 days,the three-dimensional static group had significantly higher gene expression compared with the three-diomensional dynamic group(P<0.001).(4)The results showed that compared with three-dimensional static culture microtissue,three-dimensional dynamic culture microtissue could achieve higher expression of chondrogen-related genes in a shorter time,showing better cell viability and chondrogenic ability.

Objective To evaluate the preservation effect of normothermic mechanical perfusion with red blood cells from humanized genetically modified pig on severed human limbs.Methods Severed human limbs were perfused with red blood cells from humanized genetically modified pigs for 6 h.Perfusion solution was taken every hour to measure the oxygen partial pressure,Na+,K+,Ca2+,pH value,glucose,lactic acid and creatine kinase levels.Superficial flexor muscle was sampled to detect the changes of tumor necrosis factor(TNF)-α,interleukin(IL)-2 and IL-1 levels.At 0 and 6 h after perfusion,the superficial flexor muscles of the forearm were taken for pathological examination.Intercellular space and glycogen consumption of skeletal muscles were observed.An appropriate amount of forearm vessels was collected every 2 h to detect the apoptosis of vascular endothelial cells.X-ray angiography was performed before perfusion and 6 h after perfusion to observe the filling degree of finger-tip peripheral vessels.Results The oxygen partial pressure was observed in the normal range throughout the perfusion.Na+concentration peaked at 1 h,reaching 138.7 mmol/L,and then fluctuated within the normal range.K+level peaked at 2 h up to 6.08 mmol/L,then decreased and fluctuated within the normal range.Ca2+concentration reached the peak at 4 h,up to 1.03 mmol/L.Glucose level was gradually decreased at the beginning of perfusion,reaching the lowest value of 17.7 mmol/L at 2 h after perfusion,and then maintained a dynamic balance.The pH value was decreased to 7.28 at 6 h after perfusion.The lactic acid level was increased to 9.6 mmol/L at 1 h after perfusion,and then gradually decreased.The creatine kinase level was increased at the start of perfusion,reached the peak at 2 h up to 20 030 U/L,then decreased and remained stable at the end of perfusion.At the end of perfusion,the morphology of muscle fibers was normal,the gap among muscle fibers was expanded slightly,and the glycogen of skeletal muscles was not significantly accumulated.At 0 h perfusion,the number of apoptotic cells in vascular endothelial cells was large,which was declined at 6 h perfusion.Evident vascular filling was observed at 0 h,and the filling degree of some finger-tip vessels was decreased at 6 h.Conclusions Normothermic mechanical perfusion of severed human limbs with red blood cells from humanized genetically modified pigs may continuously and stably supply energy and oxygen,adjust the ion pH balance of perfusion solution,maintain normal cellular metabolism and exerts certain protective effect upon severed human limbs.

Limb dismemberment injuries are common in clinical practice,and safe and effective protection of the dismembered limb is the key to successful limb replantation.Normothermic machine perfusion has made significant breakthrough in the field of organ transplantation,which may maintain the active function of organs and tissues for a long period of time and prolong the preservation time.These findings have been validated in large animal models and clinical trials.Meantime,this technology is expected to provide novel reference for the preservation and functional recovery of severed limbs.Therefore,this paper reviews the problems of static cold preservation in the preservation of disarticulated limbs,the development history of mechanical perfusion,the current status of clinical application of ambient mechanical perfusion of disarticulated limbs as well as the problems to be solved,and looks forward to the direction of its development and the prospect of its clinical application,with a view to promoting the wide application of this technology in the clinic.

Although blood banks based on human blood can provide blood transfusions for the wounded timely and effec-tively,scientific research has never given up on finding new blood sources due to the restrictions of human blood sources.With the application of transgenic technology and the successful breeding of gene-edited pigs,gene-edited pig blood as a po-tential source of clinical transfusion has attracted wide attention.Now there are preclinical studies showing the feasibility of transfusing gene-edited pig red blood cells into primates.This paper discusses the related research and future development of xenogeneic transfusion of porcine red blood cells by gene editing.

Approach-avoidance conflict(AAC)refers to the internal conflict that individuals experi-ence when faced with conflicting approach or avoidance thoughts.It reveals some characteristics of mental disorders,such as anxiety,depression,and addiction represented by excessive tendencies of approach or avoidance.The function of the cortico-limbic-striatal system influences behavioral choices at the neural level during the onset of AAC,and the development of related behavioral paradigms that can better represent AAC behaviors is critical to evaluating the efficacy of drugs and guiding the development of new drugs.This paper summarizes the neural mechanisms,behavioral paradigms,and applications in behavioral pharmacology related to AAC behaviors from the perspective of psychopharmacology with a view to providing new perspectives and methods for the diagnosis and treatment of related neuro-psychiatric disorders.

Objective:To study the in vitro construction of functional and self-renewing cartilage organoids based on cartilage acellular extracellular matrix (ECM) microcarriers.Methods:Fresh porcine articular cartilage was taken. The merely crushed cartilage particles were set as natural cartilage group and ECM microcarriers of appropriate particle size, which were prepared by the acellular method of combining physical centrifugation and chemical extraction, were set as microcarrier group. Cartilage organoids were constructed by loading human umbilical cord mesenchymal stem cells (hUCMSCs) and human chondrocytes (hCho) with a ratio of 3∶1 with microcarriers through a rotating bioreactor. The organoids with different induction times were divided into 0-, 7-, 14-, and 21-day induction groups. The cell residues of the microcarrier group and natural cartilage group were evaluated by 4′, 6-diaminidine 2-phenylindole (DAPI) fluorescence staining and DNA quantitative analysis. The retention of microcarrier components was observed by Safranin O and toluidine blue stainnings, and the collagen and glycosaminoglycan (GAGs) levels in the microcarrier group and the natural cartilage group were determined by colorimetric method and dimethyl-methylene blue (DMMB) method. The microcarriers were further characterized by scanning electron microscopy and energy dispersive spectroscopy. The hUCMSCs cultured with Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with fetal bovine serum (FBS) in a volume fraction of 10% was used as the control group and the hUCMSCs cultured with the microcarrier extract was used as the experimental group. Subgroups of hUCMSCs cultured at 3 time points: 1, 3 and 5 days were set up in the two groups separately. Cell Counting Kit 8 (CCK-8) was used to detect the biocompatibility of the two groups. The cellular activity of the organoids of the 0-, 7-, 14-, and 21-day induction groups was detected by live/dead staining and the self-renewal ability of the cartilage organoids of the 14-day induced group was identified by Ki67 fluorescence staining. The organoids of the 7-, 14-, and 21-day induction groups were detected by RT-PCR in terms of the expression levels of chondrogenesis-related marker aggrecan (ACAN), type II collagen (COL2A1), SRY-related high mobility group-box gene-9 (SOX9), cartilage hypertrophy-and mineralization-related marker type I collagen (COL1A1), Runt-related transcription factor-2 (RUNX2), and osteocalcin (OCN). Colorimetric and DMMB assays were performed to determine the ability of organoids in the 0-, 7-, 14-, and 21-day induction groups to secrete collagen and GAGs.Results:The results of DAPI fluorescent staining showed that the natural cartilage group had a large number of nuclei while the microcarrier group hardly had any nuclei. The DNA content of the microcarrier group was (7.8±1.8)ng/mg, which was significantly lower than that of the natural cartilage group [(526.7±14.7)ng/mg] ( P<0.01). Saffranin O and toluidine blue staining showed that the microcarrier was dark- and uniform-colored and it kept a lot of cartilage ECM components. The collagen and GAGs contents of the microcarrier group were (252.9±1.4)μg/mg and (173.4±0.8)μg/mg, which were significantly lower than those of the natural cartilage group [(311.9±2.2)μg/mg and (241.3±0.7)μg/mg] ( P<0.01). Scanning electron microscopy showed that the surface of the microcarriers had uneven and interleaved collagen fiber network. The results of energy spectrum analysis showed that elements C, O and N were evenly distributed in the microcarriers, indicating that the composition of the microcarrier was uniform. The microcarrier had good biocompatibility and there was no statistical significance in the results of CCK-8 test between the control group and the experimental group after 1 and 3 days of culture ( P>0.05). After 5 days of culture, the A value of the experimental group was 0.53±0.02, which was better than that of the control group (0.44±0.03) ( P<0.05). In the 0-, 7-, 14-, and 21-day induction groups, hUCMSCs and hCho were attached to the surface of the microcarriers, with good cellular activity, and the live/death rates were (70.6±1.1)%, (80.5±0.6)%, (94.5±0.9)%, and (90.8±0.5)% respectively ( P<0.01). There were a large number of Ki67 positive cells in cartilage organoids. RT-PCR showed that the expression levels of ACAN, COL2A1, SOX9, COL1A1, RUNX2 and OCN were 1.00±0.09, 1.00±0.24, 1.00±0.18, 1.00±0.03, 1.00±0.06 and 1.00±0.13 respectively in the 7-day induction group; 4.16±0.28, 5.09±1.25, 5.65±1.05, 0.47±0.01, 1.68±0.02 and 0.21±0.06 respectively in the 14-day induction group; 13.42±0.92, 3.07±0.21, 1.84±1.08, 2.72±0.17, 2.91±0.18 and 3.32±1.20 respectively in the 21-day induction group. Compared with the 7-day induction group, the expression levels of ACAN, COL2A1, SOX9 and RUNX2 in the 14-day group were increased ( P<0.05), but COL1A1 expression level was decreased ( P<0.05), with no significant difference in OCN expression level ( P>0.05). Compared with the 7-day induction group, the expression levels of ACAN, COL1A1 and RUNX2 in the 21-day induction group were significantly increased ( P<0.01), with no significant differences in the expression levels of COL2A1, SOX9 and OCN ( P>0.05). Compared with the 14-day induction group, the expression levels of ACAN, COL1A1, RUNX2 and OCN in the 21-day group were increased ( P<0.05 or 0.01), with no significant difference in the expression level of COL2A1 ( P>0.05), but the expression level of SOX9 was decreased ( P<0.05). The contents of collagen in 0-, 7-, 14-and 21-day induction groups were (219.15±0.48)μg/mg, (264.07±1.58)μg/mg, (270.83±0.84)μg/mg and (280.01±0.48)μg/mg respectively. The GAGs contents were (171.18±1.09)μg/mg, (184.06±1.37)μg/mg, (241.08±0.84)μg/mg and (201.14±0.17)μg/mg respectively. Compared with the 0-day induction group, the contents of collagen and GAGs in 7-, 14-, and 21-day induction groups were significantly increased ( P<0.01), among which the content of collagen was the lowest in 7-day induction group ( P<0.01) but the highest in the 21-day induced group ( P<0.01); the content of GAGs was the lowest in the 7-day induced group ( P<0.01) but the highest in the 14-day induction group ( P<0.01). Conclusions:The microcarriers prepared by combining physical and chemical methods are decellularized successfully, with more matrix retention, uniform composition and on cytotoxicity. By loading microcarriers with hUCMSCs and hCho, cartilage organoids are successfully constructed in vitro, which are characterized by good cell activity, self-renewal ability, strong expression of genes related to chondrogenesis and secretion of collagen and GAGs. The cartilage organoids constructed at 14 days of induction have the best chondrogenic activity.