Objective To investigate the satisfaction status of patients in the Community Health Service Center,and to provide the basis data for reform and development of the Community Health Service Center in future. Methods 481 patients and 9 Community Health Service Center in Shenyang were randomly recruited in this study by the cluster sampling survey.Several trained interviewers used questionnaires to collect requisite information.Results In the all community health professionals,including technical level,therapeutic efficacy,preventive health service, service attitude and requirement satiation,the trust status of patients was the highest,the rates of satisfaction and very satisfaction got to 92.8%.The satisfaction status for preventive health service was the lowest,only 67.7%.In analysis on influencing factors,medical insurance of patients was the important factor for their satisfaction status.There were significant differences in technical level (χ2 =22.61 ,P <0.01 ),therapeutic efficacy (χ2 =21 .52,P <0.01 ), preventive health service (χ2 =1 4.35,P <0.05),service attitude (χ2 =22.43,P <0.01 ),requirement satiation (χ2 =22.30,P <0.01 ),the trust status of patients (χ2 =1 7.81 ,P <0.01 ).In addition,age and education degree also were the factors on the satisfaction status of patients.There were significant differences among different age group in therapeutic efficacy (χ2 =33.38,P <0.01 ),preventive health service (χ2 =24.43,P <0.05 ),requirement satiation (χ2 =26.55,P <0.01 ),the trust status of patients (χ2 =22.39,P <0.05 ).There were significant differences among different education degree in therapeutic efficacy (χ2 =1 5.79,P <0.05),service attitude (χ2 =1 4.63,P <0.05)and the trust status of patients (χ2 =1 3.50,P <0.05).Conclusion The satisfaction status of patients for service attitude is the highest and age,education degree and medical insurance of patients are the important factors influencing satisfaction status of patients.

ObjectiveTo introduce the new method of full reconstruction for Ⅰ to Ⅲ-degree finger defect.MethodsFor reconstruction of Ⅰ to Ⅱ-degree finger defect, the surgery procedure was as follows:Harvest part of nail,skin and dorsal part of distal phalanx from hallux to form a composite flap,and then the flap was transplanted to the finger stump to reconstruct the defect part of the finger.The design of the composite flap was according to the recipient part. For reconstruction of Ⅲ-degree finger defect, the skin included in the flap could be designed according to the recipient part, but the bone can only be harvested from the fibulodoral part of the hallux and far from the insertion of the extensor hallucis longus tendon, which means the length was limited.If the bone length was not enough,one bone mass with appropriate size and shape was harvested from the iliac bone and connected with the bone of the composite flap. Some cases of Ⅲ-degree finger defect were reconstructed by harvesting interphalangeal joints from the second toes to reconstruct distal interphalangeal joints(DIP). The bone defect was reconstituted by bone mass from the iliac bone to conserve the contour of the second toe.The hallux wound was covered by a local flap or free flap transplantation.ResultsOne hundred and eighteen cases (126 fingers) of Ⅰ-degree defect, one hundred and eighty-seven cases (201 fingers) of Ⅱ-degree defect and 90 cases (111 fingers) of Ⅲ-degree finger defect were applied full reconstruction. All the reconstructed fingers survived completely and the configurations were similar to real fingers. Followed up our work on 150 fingers from a number of patients, between 1 and 11 years after the original surgery.Total ranges of motion of the reconstructed fingers got to over 180°.The reconstructed DIP joints had the range of motion of 15°-40°. The donor halluxes and toes were conserved with the normal length,relatively primary appearance and full function. ConclusionFull reconstruction for Ⅰ to Ⅲ-degree finger defect has great advantages in that the reconstructed finger has very realistic configuration as well as ideal function and the donor hallux is conserve well.

ObjectiveTo explore methods of donor repair of the great toe-nail flap in finger reconstruction surgery.MethodsFrom December 1998 to December 2010, various kinds of flaps were used in 511 donor sites to repair the great toe-nail flaps,including:32 dorsal pedal artery flaps;twenty-four first dorsal metatarsal artery flaps;twenty-one second dorsal metatarsal artery flaps;forteen anterior malleolar flaps;seventeen medial tarsal artery flaps;seventy-nine lateral tarsal artery flaps;one hundred and six plantar metatarsal flaps,seventy-nine flaps from second toe;fifteen flaps from mid/lower leg and 124 freed flaps.ResultsAfter postoperative 6 months to 11 years of follow-up, repaired donor sites of great toe-flaps all survived successfully,with ideal outlook and function.ConclusionThere are many kinds of methods for donor site repair of the great toe-nail flap,and each kind of method has its own advantages and disadvantages. Among these flaps, plantar pedal artery flap and free groin flap are amony the best ones.

Selective extraction of low molecular weight ( LMW) proteins and peptides from complex biological samples plays an important role in the discovery of useful biomarkers and signaling molecules. It is demonstrated that the unique pore structure of mesoporous material makes it efficient to enrich LMW proteins and peptides from complex matrixes. In this study, a mesoporous material, alkyl-diol@ phenyl-SiO2 , with modified exterior ( alkyl-diol group) and interior ( phenyl group) surfaces, was synthesized by co-condensation and post-grafting, and its characteristic was evaluated by FTIR and MS. The LMW proteins and peptides enriched by the alkyl-diol@phenyl-SiO2 mesoporous material could be easily eluted by organic solvents, which was compatible with the following detection by mass spectrometry ( MS ) . This new mesoporous material exhibited good selectivity for the extraction of LMW proteins and peptides ( less than 10 kDa) from complex biological samples.

Objective:To investigate the protective effect of hypothermic antegrade machine perfusion against canine ischemic brain injury.Methods:Thirteen beagle dogs were divided into the mild hypothermia with perfusion group ( n=6) and normothermia with perfusion group ( n=7) according to the random number table. The model of ischemic brain injury was established by neck transection. After 1 hour of ischemic circulatory arrest, the perfusion fluid based on autologous blood was continuously perfused through bilateral common carotid artery for 6 hours. The temperature of the perfusion fluid was set at 33 ℃ in the mild hypothermia with perfusion group and 37℃ in the normothermia with perfusion group, respectively. Blood oxygen saturation was recorded at 0, 1, 2, 3, 4, 5 and 6 hours after the beginning of perfusion to evaluate the perfusate oxygen level. The perfusate was collected, and the levels of Na +, K +, Ca 2+ and glucose as well as the pH value of the perfusate were detected in the two groups. At the end of perfusion, the parietal brain tissues of 1 dog from each group were collected to evaluate the water contents of brain tissues. Nissl staining was used to evaluate the morphological integrity of the pyramidal neurons in the frontal cortex and hippocampus. Neuronal nuclei antigen (NeuN) was used to evaluate the structural and morphological integrity of pyramidal neurons. Immunofluorescence glial fibrillary acidic protein (GFAP) and ionic calcium binding adaptor molecule 1 (Iba1) were used to evaluate the integrity and activity of astrocytes and microglia fragments. Results:At 0, 1, 2, 3, 4, 5 and 6 hours of perfusion, there was no significant difference in the blood oxygen saturation or Na + concentrations between the two groups (all P>0.05); the K + concentrations in the mild hypothermia with perfusion group were (4.57±0.12)mmol/L, (4.67±0.14)mmol/L, (4.27±0.12)mmol/L, (4.45±0.10)mmol/L, (6.60±0.15)mmol/L, (7.37±0.18)mmol/L and (9.03±0.16)mmol/L, respectively, which were significantly lower than those in the normothermia with perfusion group [(4.84±0.10)mmol/L, (5.31±0.13)mmol/L, (5.44±0.24)mmol/L, (5.70±0.18)mmol/L, (7.79±0.18)mmol/L, (10.44±0.40)mmol/L, (10.40±0.41)mmol/L] (all P<0.01). At 0, 1, 2 and 3 hours of perfusion, the Ca 2+ concentrations in the mild hypothermia with perfusion group were (0.72±0.15)mmol/L, (1.55±0.16)mmol/L, (1.62±0.15)mmol/L and (1.88±0.15)mmol/L, respectively, being significantly higher than those in the normothermia with perfusion group [(0.41±0.13)mmol/L, (0.99±0.12)mmol/L, (1.29±0.13)mmol/L, (1.57±0.11)mmol/L] (all P<0.01), and no significant differences were found at other time points (all P>0.05). At 0, 1 and 2 hours of perfusion, the glucose concentrations in the mild hypothermia with perfusion group were (5.75±0.19)mmol/L, (5.17±0.15)mmol/L and (4.72±0.15)mmol/L, respectively, being significantly higher than those in the normothermia with perfusion group [(5.30±0.22)mmol/L, (4.89±0.20)mmol/L, (4.30±0.17)mmol/L] (all P<0.01), with no significant differences found at other time points (all P>0.05). At 2, 3, 4, 5 and 6 hours of perfusion, the pH values of the mild hypothermia with perfusion group were 7.32±0.06, 7.25±0.02, 7.23±0.02, 7.24±0.02 and 7.24±0.02, respectively, being significantly higher than those in the normothermia with perfusion group (7.26±0.01, 7.21±0.01, 7.17±0.02, 7.15±0.02, 7.08±0.02) ( P<0.05 or 0.01), with no significant differences at other time points (all P>0.05). The water content of brain tissues in the mild hypothermia with perfusion group was (74.9±0.4)%, which was significantly lower than (79.9±0.9)% in the normothermia with perfusion group ( P<0.01). Nissl staining showed that the pyramidal neurons in prefrontal cortex and dentate gyrus had good integrity in the mild hypothermia with perfusion group. NeuN immunofluorescence staining showed that the morphology and structure of pyramidal neuron cells in the mild hypothermia with perfusion group were better with clearly visible axons than those in the normothermia with perfusion group, whereas the cytosol was full and swollen with scarce axons in the normothermia with perfusion group. GFAP and Iba1 immunofluorescence staining showed that more structurally intact glial cells, more abnormally active cells, thickener axons and better axon integrity in all directions were found in the mild hypothermia with perfusion group than those in the normothermia with perfusion group. Conclusion:Compared with normal temperature antegrade mechanical perfusion, the mild hypothermia antegrade mechanical perfusion can protect canine brain tissue and alleviate ischemic brain injury by maintaining stable energy and oxygen supply, balancing ion homeostasis and perfusion fluid pH value, reducing tissue edema, and maintaining low metabolism of pyramidal neurons, astrocytes and microglia.

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.

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.

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.