White adipose tissue-derived small extracellular vesicles: A new potential therapeutic reagent for accelerating diabetic wound healing.

Small extracellular vesicles (sEVs) from adipose-derived stem cells (ADSCs) have gained great attention and have been widely used in cell-free therapies for treating diabetic non-healing wounds in recent years. However, further clinical application of ADSC-sEVs have been limited due to their unsolvable defects, including cumbersome extraction procedure, high cost, low yield, etc. Thus, we urgently need to find one therapeutic reagent that could not only accelerate diabetic wound healing as ADSC-sEVs but also overcome these shortcomings. As the extraction process of adipose tissue-derived sEVs (AT-sEVs) is quite simple and labor saving, we put our focus on the efficiencies of white adipose tissue-derived sEVs (WAT-sEVs) and brown adipose tissue-derived sEVs (BAT-sEVs) in diabetic wound repair. After successfully isolating WAT-sEVs and BAT-sEVs by ultracentrifugation, we thoroughly characterized them and compared their diabetic wound healing capabilities both in vitro and in vivo. According to our study, AT-sEVs possess similar competence in diabetic wound healing as compared with ADSC-sEVs. While the effect of BAT-sEVs is not as stable as WAT-sEVs and ADSC-sEVs, the repair efficiency is also slightly lower than the other two sEVs in some cases. In summary, we are the first to discover that WAT-sEVs show great potential in diabetic wound repair. With advantages that are specific to tissue-derived sEVs (Ti-sEVs) such as time- and cost-saving, high-yield, and simple isolation procedure, we believe WAT-sEVs could serve as a novel reliable cell-free therapy for clinical diabetic wound treatment.

Large-scale production of tauroursodeoxycholic acid products through fermentation optimization of engineered Escherichia coli cell factory.

BACKGROUND: Bear bile powder is a valuable medicinal material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a certain ratio to taurochenodeoxycholic acid (TCDCA). We had created an engineered E. coli harboring two-step bidirectional oxidative and reductive enzyme-catalyzing pathway that could rapidly convert TCDCA to TUDCA at a specific percentage in shake flasks. RESULTS: We reported here the large-scale production of TUDCA containing products by balancing the bidirectional reactions through optimizing fermentation process of the engineered E. coli in fermenters. The fermentation medium was firstly optimized based on M9 medium using response surface methodology, leading to a glycerol and yeast extract modified M9-GY medium benefits for both cell growth and product conversion efficiency. Then isopropylthio-ß-galactoside induction and fed-stock stage was successively optimized. Finally, a special deep-tank static process was developed to promote the conversion from TCDCA to TUDCA. Applying the optimal condition, fermentation was performed by separately supplementing 30 g refined chicken bile powder and 35 g crude chicken bile powder as substrates, resulting in 29.35 ± 2.83 g and 30.78 ± 3.04 g powder products containing 35.85 ± 3.85% and 27.14 ± 4.23% of TUDCA at a ratio of 1.49 ± 0.14 and 1.55 ± 0.19 to TCDCA, respectively, after purification and evaporation of the fermentation broth. The recovery yield was 92.84 ± 4.21% and 91.83 ± 2.56%, respectively. CONCLUSION: This study provided a practical and environment friendly industrialized process for producing artificial substitute of bear bile powder from cheap and readily available chicken bile powder using engineered E. coli microbial cell factory. It also put forward an interesting deep-tank static process to promote the enzyme-catalyzing reactions toward target compounds in synthetic biology-based fermentation.

Methods, Mechanisms, and Application Prospects for Enhancing Extracellular Vesicle Uptake.

Extracellular vesicles (EVs) are considered to be a new generation of bioinspired nanoscale drug delivery systems due to their low immunogenicity, natural functionality, and excellent biocompatibility. However, limitations such as low uptake efficiency, insufficient production, and inhomogeneous performance undermine their potential. To address these issues, numerous researchers have put forward various methods and applications for enhancing EV uptake in recent decades. In this review, we introduce various methods for the cellular uptake of EVs and summarize recent advances on the methods and mechanisms for enhancing EV uptake. In addition, we provide further understanding regarding enhancing EV uptake and put forward prospects and challenges for the development of EV-based therapy in the future.

[Clinical observation of jianpi bushen formula to prevent collapse of osteonecrosis of femoral head].

To observe the clinical effect of Jianpi Bushen formula to prevent the collapse of osteonecrosis of femoral head (ONFH) of type phlegm and blood stasis obstructing the collaterals. 50 cases (including 73 hips) of non-collapse ONFH (ARCO I, II, III a) were selected from the out-patient department of orthopedic in Guang'anmen Hospital attached to China Academy of Chinese Medical Science. All the cases fit for diagnostic criteria were given Jianpi Bushen formula and followed up. Staging criteria was ARCO classification. Harris score evaluated the hip function. The mean follow-up time was 4.2 years (3-5.4 years). After a mean of 4.2 years (3-5.4 years) followed-up, 12 hips collapse occurred while 61 hips not (the non-collapse rate was 83.56%). Collapse did not occurred among the all 7 hips at stage ARCO I (the non-collapse rate was 100%). Among the 49 hips at stage ARCO II, 9 hips collapse occurred while 40 hips not (the non-collapse rate was 81.63%). Among the 17 hips at stage ARCO III a, 3 hips collapse occurred while 14 hips not (the non-collapse rate was 82.35%). Kaplan-Meier analysis showed the average survival time of non-collapse was 5 years (4. 8-5.2 years). The 3 year survival rate of non-collapse was 92.5%. The 4 year survival rate was 74% and the condition tended to be stability trend. The Harris score was 71.93 +/- 11.25 before treatment and 81.63 +/- 12.16 after treatment, significantly different. These results suggest that: Jianpi Bushen formula is an effective method for treating ONFH of type phlegm and blood stasis obstructing the collaterals. It can delay or prevent the collapse of femoral head and significantly improve the hip function.

Multifunctional ADM hydrogel containing endothelial cell-exosomes for diabetic wound healing.

Non-healing wound, with limited treatment options, remains a prevalent complication of diabetes mellitus. The underlying causes wherein include oxidative stress injury, bacterial infection, cellular dysfunction, and persistent inflammation. Acellular Dermal Matrix (ADM), a wound dressing composed of natural extracellular matrix and abundant bioactive factors, has been successfully developed to treat various wounds, including burns and diabetic ulcers. Protocatechualdehyde (PA) & trivalent iron ion (Fe3+) complex (Fe3+@PA) exhibits potential antioxidant and antibacterial properties. In this study, we developed a dual hydrogel network by combining Fe3+@PA complex-modified ADM with light-cured gelatin (GelMA), supplemented with exosomes derived from human umbilical vein endothelial cells (HUVEC-Exos), to create an ADM composite hydrogel system (ADM-Fe3+@PA-Exos/GelMA) with antioxidant, antibacterial, and cell-promoting functions for diabetic wound treatment. Through in vitro experiments, we investigated the biosafety, antioxidant and antibacterial properties of ADM composite hydrogel. Furthermore, we examined the protective effects of ADM composite hydrogel on diabetic wound. The above experiments collectively demonstrate that our ADM-Fe3+@PA-Exos/GelMA hydrogel promotes diabetic wound healing by eliminating bacterial infection, reduced the reactive oxygen species (ROS) levels, protecting cells against oxidative stress damage, promotingcollagen deposition and angiogenesis, which provides a promising strategy to optimize ADM for diabetic wound treatment.

[Meta-analysis on efficacy and safety of external use of tradition Chinese medicines and western medicines in treating knee osteoarthritis].

OBJECTIVE: To assess the efficacy and safety of the external use of tradition Chinese medicine (TCM) in treating knee osteoarthritis. METHOD: By computer retrieval of MEDLINE, CNKI and VIP database on data from the establishment of database to May 2011,all of the randomized controlled trials on the external use of TCMs in treating knee osteoarthritis with western medicines as the control drugs were collected to screen out literatures in line with the inclusion standards. The quality of the included literatures was strictly assessed, and a meta-analysis was conducted by RevMan 5.1 software. RESULT: Totally 25 RCTs involving 2 159 patients were included [1152 patients in the TCM group (TCMG), 1007 patients in the western medicine group (WMG)], in which 21 articles were brought into the analysis on efficacy. The results of meta-analyses showed that the efficient rates of the TCMG and the WMG were 92.35% (941/ 1019) and 81.19% (712/877), respectively, OR = 2.88 [2.16-3.83], with a hypothesis testing on total effect of Z = 7.20 (P < 0.00001)], suggesting that the external use of TCMs had a better efficacy than the WMG. A total of 25 literatures were brought into the analysis on adverse effect. The adverse effects rate of the TCMG and the WMG were 1.30% (15/1 152) and 5.36% (54/1007), respectively, OR = 0.40 [0.11-1.38], Z = 1.46 (P = 0.15)], indicating no significant difference between them. The sub group analysis shows that compared with the external use of western medicines,the adverse effect rate of the two groups are similar [(1.87% (11/589): 1.60% (9/564)], OR = l.12 [0.48-2.58], Z = 0.26 (P = 0.80). But as for inhibitors NSAIDS and COX-2 for oral,the adverse effect rate of the TCMG was lower than that of the WMG [(1.07% (4/375): 15.46% (45/291)], OR = 0.11 [0.01-0.87], Z = 2.09 (P = 0.04). CONCLUSION: The efficacy of the external use of TCMs in treating OA is better than that of western medicines. The adverse effect rates of the external use of TCMs is equivalent to that of western medicines, and significantly lower than that of oral western medicines. But because of the poor quality of the included literatures, the final conclusion for the efficacy of TCMs in treating knee osteoarthritis could not be reached only based on this Meta-analysis and remain to be proved with the results of high-quality clinical studies.

Polysomnography findings in preschool children with obstructive sleep apnea are affected by growth and developmental level.

OBJECTIVE: We investigated the effect of growth and development level on polysomnography results in preschool children with obstructive sleep apnea (OSA). METHODS: Preschool children (ages 3-6) with symptoms of snoring and were diagnosed with OSA by polysomnography in the sleep center were selected as the research object. They were split into three groups based on their growth rates: restricted, normal, and excessive. Sleep structure, respiratory events, and oxygenation index were compared between the three groups. RESULTS: A total of 183 (111 boys and 72 girls) preschool children were enrolled. There were 26 cases in the growth restricted group, 112 cases in the normal growth group, and 45 cases in the overgrowth group. The weight and Body Mass Index (BMI) of children in the growth restricted and overgrowth groups were significantly different from those in the normal group. In terms of sleep structure, the sleep efficiency of the growth restricted group was poorer than that of the normal group. For sleep breathing events, the growth restricted group showed a greater apnea-hypopnea index (AHI), obstructive apnea hypopnea index (OAHI), hypoventilation index, and more hypoventilation than the normal group. In terms of oxygenation, the difference in degree of hypoxia between the three groups was statistically significant, and the overgrowth group had lower minimum oxygen saturation during the rapid eye movement phase than the normal group, as well as a quicker mean heart rate. CONCLUSIONS: OSA is more likely in preschool-aged children with stunted or overgrown growth than in children with OSA alone, and the more severe the disorder, especially when accompanied with stunted growth.

De novo Biosynthesis of Salvianolic Acid B in Saccharomyces cerevisiae Engineered with the Rosmarinic Acid Biosynthetic Pathway.

Salvianolic acid B (SAB), also named lithospermic acid B, belongs to a class of water-soluble phenolic acids, originating from plants such as Salvia miltiorrhiza. SAB exhibits a variety of biological activities and has been clinically used to treat cardio- and cerebrovascular diseases and also has great potential as a health care product and medicine for other disorders. However, its biosynthetic pathway has not been completely elucidated. Here, we report the de novo biosynthesis of SAB in Saccharomyces cerevisiae engineered with the heterologous rosmarinic acid (RA) biosynthetic pathway. The created pathway contains seven genes divided into three modules on separate plasmids, pRS424-FjTAL-Sm4CL2, pRS425-SmTAT-SmHPPR or pRS425-SmTAT-CbHPPR, and pRS426-SmRAS-CbCYP-CbCPR. These three modules were cotransformed into S. cerevisiae, resulting in the recombinant strains YW-44 and YW-45. Incubation of the recombinant strains in a basic medium without supplementing any substrates yielded 34 and 30 µg/L of SAB. The findings in this study indicate that the created heterologous RA pathway cooperates with the native metabolism of S. cerevisiae to enable the de novo biosynthesis of SAB. This provides a novel insight into a biosynthesis mechanism of SAB and also lays the foundation for the production of SAB using microbial cell factories.

Relationship between enuresis and obstructive sleep apnea-hypopnea syndrome in children.

OBJECTIVE: We explored the relationship between enuresis and obstructive sleep apnea-hypopnea syndrome (OSAHS) in children and influencing factors of enuresis with OSAHS. METHODS: We recruited 196 children ≥5 years old from the otolaryngology outpatient department, who experienced snoring and underwent nasopharynx lateral radiography and in-laboratory polysomnography. We analyzed correlations between the apnea-hypopnea index (AHI) and lowest oxygen saturation (L-SaO2) with age, body mass index (BMI), tonsil size, and adenoidal-nasopharyngeal (A/N) ratio using the Pearson correlation test. Differences in severe OSAHS prevalence, age, AHI, L-SaO2, tonsil size, and A/N ratio between children with and without enuresis were assessed using the chi-square test and t-test. Risk factors of enuresis were analyzed using logistic regression. Follow-up was conducted to assess remission in children with enuresis after adenotonsillectomy. RESULTS: BMI, tonsil size, and A/N ratio were correlated with AHI and L-SaO2. Severe OSAHS prevalence, AHI, tonsil size, and A/N ratio were higher and L-SaO2 were lower in children with enuresis. Logistic regression showed that BMI, AHI, tonsil size, and sleep apnea were risk factors for enuresis. CONCLUSIONS: Our study findings showed that enuresis was associated with OSAHS in children. Adenotonsillectomy may improve the symptoms of enuresis.

[Effects on VEGF and VEGF mRNA expression in issues of callus in treating fracture of rabbit with three-period treatment theory of bone fracture in traditional Chinese medicine].

OBJECTIVE: To observe the effects of the three-period treatment theory of bone fracture in traditional Chinese medicine (TCM) on VEGF and VEGF mRNA expression in the issues of callus of rabbits. And to explore the rationality of phasing method in TCM in treating fracture. METHOD: one hundred and forty male and healthy rabbits were made 3 mm wide bone defection at lower one third part of both radius as fracture healing model. Then those rabbits were divided into four groups randomly, which are three-period treatment group (TTG), one-period treatment group (OTG), positive medicine treatment group (PTG) and model control group (MCG). Those rabbits in TTG were treated with three-period treatment. Those in OTG were treated with one-period treatment. Those in PTG were feed by guzhecuoshangsan, a Chinese patent medicine which is used to treat bone fracture. Those in model control group were given no prescription or drug but distilled water as same dose as that of other groups. At day 3, 6, 9, 14, 28, 42 and 56, five rabbits were selected from every group randomly and were killed by aeroembolism respectively. Their radius were taken out and the left one was taken as the research object. Immunohistochemistry stain and in situ hybridization stain were performed to examinate the VEGF and VEGF mRNA expression in the haematoma, fibrous callus and soft callus. RESULT: All TCM treatment groups can enhance the VEGF and VEGF mRNA expression in the haematoma, fibrous callus and soft callus at different time points in fracture healing. The VEGF and VEGF mRNA expression in the three issues of TTG had the tendency of higher than that of the other groups at the most time points after operation. CONCLUSION: TCM can promote the VEGF and VEGF mRNA expression in the haematoma, fibrous callus and soft callus. Different Chinese medicines play various roles on VEGF and VEGF mRNA expression at different stage of fracture healing. Treating fracture in three-period treatment has more predominant effect on the expression of VEGF and VEGF mRNA in the haematoma, fibrous callus and soft callus than that of treating fracture with single prescription or drug. It is necessary to treat fracture in stages.

[Case-control study on the treatmentof the fifth metatarsal base fractures by cardboard compression pad versus short leg plaster].

OBJECTIVE: To compare the effect,safety,and advantage of flexible fixation with paperboard and pad versus short leg plaster in treating the fifth metatarsal base fracture,and establish the standard of diagnosis and treatment of the fifth metatarsal base fractures in flexible fixation with paperboard and pad. METHODS: From June 2010 to March 2013,59 patients with the fifth metatarsal base fracture were treated with paperboard and pad fixation or short leg plaster. Patients were enrolled and divided into paperboard and pad treatment group (paperboard group) and short leg plaster treatment group (plaster group) randomly according to the random number table. In paperboard group,there were 29 cases including 9 males and 20 females with an average age of (51.79±11.40) years old; the average course of injury was (11.59±6.58) hours. In plaster group, there were 30 cases including 9 males and 21 females with an average age of (52.13+17.34) years old ;the average course of injury was (11.03±7.06) hours. According to whether the fracture line across the articular surface, in paperboard group there were 14 cases of type A,15 of type B; in plaster group,16 of type A, 14 of type B. According to the degree of dislocation,in paperboard group there were 16 cases of degree I ,13 of degree II ; in plaster group,20 were degree I ,10 were degree II. Fracture was restored according to the type in manual. Patients in paperboard group were treated with paperboard and pad, and patients in plaster group were treated with short leg plaster. Fracture was fixed for 4 to 6 weeks according to fracture healing. On the 2nd, 4th,6th, 8th week and 3rd, 6th month after fixation, patients were followed up, and the foot function score was used to evaluate the function of injured foot. X-ray of injured foot was taken on the 2nd, 4th, 6th and 8th week were used to assess fracture healing. RESULTS: All patients got complete follow-up. The X-ray result showed that all fracture reached at clinical healing on the 8th week after fixation without skin ulcer,nonunion and displacement of fracture. From the 4th to 8th week after fixation, paperboard group had a higher X-ray score than plaster group, but the difference between two groups had no statistically significance. Repeated analysis result showed that there was interact at different time point and between groups,the difference had statistically significance (P<0.01). The foot function score showed that at all time point, paperboard group had a higher score than plaster group, and on the 2nd, 4th, and 6th week, it had statistically significant difference(P<0.01) between two groups. On the 6th months after fixation,the excellent and good rate of paperboard group was 93.10%, higher than that of plaster group, which was 86.67%. But it had no statistically difference(P=0.483) between two groups. CONCLUSION: Using paperboard and pad fixation to treat the fifth metatarsal base fracture has the advantage of simplicity operating,reliable fixation, satisfactory effects, easily obtainable material.

[Effects on VEGF and VEGF mRNA expression of the tissues of periosteum in three-period treatment of TCM for the fractures in rabbit].

OBJECTIVE: To observe the effects of the three-period treatment theory of bone fracture in TCM (Traditional Chinese Medicine) on VEGF and VEGF mRNA expression in the issues of outer periosteum, endosteum and bone marrow of rabbits, and to explore the rationality of phasing method in TCM in treating fracture. METHODS: 3 mm bone defection were made at lower one third part of both radius in 140 male healthy rabbits. The rabbits were randomly divided into four groups, including three-period treatment group (TTG), one-period treatment group(OTG), positive medicine treatment group(PTG) and model control group (MCG). The rabbits in TG were treated with three-period treatment, rabbits in OTG were treated with one-period treatment, rabbits in PTG were fed by Guzhe-Cuoshangsan (a Chinese patent medicine which was used to treat bone fracture), rabbits in model control group were given no prescription or drug but distilled water as same dose as that of other groups. At day 3, 6, 9, 14, 28, 42 and 56, five rabbits from every group were randomly selected and were killed by aeroembolism. The left radiuses were taken out as the research object. Immunohistochemistry stain and in situ hybridization stain were performed to examinate the VEGF and VEGF mRNA expression in the outer periosteum, endosteum and bone marrow. RESULTS: The VEGF and VEGF mRNA expression of all TCM treatment groups were enhanced in the outer periosteum, endosteum and bone marrow at different time points in fracture healing. The VEGF and VEGF mRNA expression in the three tissues of TTG had the tendency of higher than that of the other groups at the most time points after operation. CONCLUSION: Treating fracture in stages has more predominant effect on the expression of VEGF and VEGF mRNA in the outer periosteum, endosteum and bone marrow than that of treating fracture with single prescription or drug.