BACKGROUND:Ilizarov bone transport is very effective in the treatment of open large tibial bone defects,but there are still complications,among which the difficulty of junction healing is one of the difficult points in treatment. OBJECTIVE:To investigate the effect of Ilizarov bone transport combined with antibiotic bone cement on junction healing after operation of open large tibial bone defect. METHODS:Totally 51 patients with open large tibial bone defect(bone defect>4 cm)admitted to Binzhou Medical University Hospital from August 2010 to January 2022 were selected,of which 28 received Ilizarov bone transport alone(control group)and 23 received Ilizarov bone transport combined with antibiotic bone cement treatment(trial group).External fixation time,bone healing time,bone healing index,visual analog scale score during bone removal,bone defect limb function,junction healing and complications at the final follow-up were statistically compared between the two groups. RESULTS AND CONCLUSION:(1)All the 51 patients were followed up for a mean of(22.53±5.77)months.External fixation time,bone healing time,bone healing index,postoperative infection rate,and non-healing rate of junction were less in the trial group than those in the control group(P<0.05).There was no significant difference between the two groups in visual analog scale scores at 6 months after the second surgery and in the functional excellence and good rate of limb with bone defect at the final follow-up(P>0.05).(2)These findings indicate that compared with the Ilizarov bone transport alone,Ilizarov bone transport combined with antibiotic bone cement treatment can promote the healing of open tibial fracture junction and increase the rate of bone healing.

To investigate the correlation between vitamin D nutritional status and insulin resistance in pubertal adolescents.

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.

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.

Objective To investigate the acute toxicology and intervention effect of Panacis Majoris Rhizoma on rats with chronic pharyngitis.Methods A single,maximum dose of Panacis Majoris Rhizoma(74.4 g·kg-1)was administered to Kunming mice to evaluate its toxicity,involving the assessment of the survival status of the mice,organ indices,morphological changes in major organs,blood routine,and biochemical indicators.SD rats were randomly divided into the control group,model group,prednisone group(6.25 mg·kg-1),and low-,medium-,and high-dose Panacis Majoris Rhizoma groups(0.58,1.16,and 2.32 g·kg-1).All rats received the corresponding drugs(or normal saline)via intragastric administration once daily for a duration of 30 days.Except the control group,chronic pharyngitis was induced in rats of the other groups by using β-hemolytic streptococcus.Following euthanasia,serum inflammatory levels of interleukin-6(IL-6),cyclooxygenase-2(COX-2),interleukin-1β(IL-1β),intercellular adhesion molecule-1(ICAM-1),C-reactive protein(CRP),tumor necrosis factor(TNF-α),monocyte chemoattractant protein-1(MCP-1),and prostaglandin E2(PGE2)were measured.Additionally,pharyngeal tissues were stained with HE and pathological characteristics were observed.Results Toxicological studies have demonstrated that the administration of Panacis Majoris Rhizoma resulted in significant increase in plasma alanine transaminase levels and spleen index of mice,along with corresponding tissue pathological alterations.Nevertheless,no noteworthy pathological changes were observed in other organs,and there were no notable changes in blood routine and plasma biochemical indicators.Pharmacodynamic investigations have revealed that Panacis Maioris Rhizoma effectively reduces the serum levels of inflammatory factors and improves pathological changes in pharyngeal tissues.Conclusion Panacis Maioris Rhizoma alleviated β-hemolytic streptococcus-induced CP by inhibiting inflammatory responses,and may show potential toxicity to the spleen.

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.

Objective:To investigate the clinical characteristics of multiple sclerosis (MS) patients with positive anti-SSA antibody.Methods:The data of patients with positive anti-SSA antibody hospitalized in Beijing Tiantan Hospital between December 2011 and May 2020 were retrieved. Patients who fulfilled the diagnosis of MS were enrolled. MS patients with negative anti-SSA antibody hospitalized in the same period were matched based on gender and age in a ratio of 1∶2. The clinical characteristics of MS patients with positive anti-SSA antibody and those with negative anti-SSA antibody were compared. We used SPSS 24.0 statistical software to perform data analysis. The Shapiro-Wilk test was used to determine whether continuous variables conform to a normal distribution. The group differences of non-normal distribution of measurement data were analyzed by Mann-Whitney U test. Fisher exact probability method was used to compare the group differences of categorical variables. P<0.05 was considered statistically significant.Results:There were 785 hospitalized patients with anti-SSA positivity. Among these, a total of 7 patients (0.89%) were diagnosed as MS. Fourteen MS patients with negative anti-SSA antibody were included with matched sex and age. There was a statistical significant difference in the frequency of positive antinuclear antibody (ANA)and anti-Ro-52 antibody between MS patients with positive anti-SSA antibody and those with negative anti-SSA (6/7 vs. 0/14, P<0.001; 4/7 vs. 0/14, P=0.006, respectively). No statistical significant difference in other clinical features was detected between the two groups (P>0.05).Conclusion:The frequency of ANA and anti-Ro-52 antibody positivity is significantly higher in MS patients with positive anti-SSA antibody. Our results suggest the presence of positive anti-SSA antibody has no affect on clinical features of MS. The relationship between MS and anti-SSA positivity may be incidently coexisted.