[Effects of three-dimensional bioprinting antibacterial hydrogel on full-thickness skin defect wounds in rats].

Objective: To explore the effects of three-dimensional (3D) bioprinting gelatin methacrylamide (GelMA) hydrogel loaded with nano silver on full-thickness skin defect wounds in rats. Methods: The experimental research method was adopted. The morphology, particle diameter, and distribution of silver nanoparticles in nano silver solution with different mass concentrations and the pore structure of silver-containing GelMA hydrogel with different final mass fractions of GelMA were observed by scanning electron microscope and the pore size was calculated. On treatment day 1, 3, 7, and 14, the concentration of nano silver released from the hydrogel containing GelMA with final mass fraction of 15% and nano silver with final mass concentration of 10 mg/L was detected by mass spectrometer. At 24 h of culture, the diameters of inhibition zone of GelMA hydrogel containing final mass concentration of 0 (no nano silver), 25, 50, and 100 mg/L nano silver against Staphylococcus aureus and Escherichia coli were detected. Fibroblasts (Fbs) and adipose stem cells (ASCs) were isolated respectively by enzymatic digestion using the discarded prepuce after circumcision from a 5-year-old healthy boy who was treated in the Department of Urology of the Second Affiliated Hospital of Zhejiang University School of Medicine in July 2020, and the discarded fat tissue after liposuction from a 23-year-old healthy woman who was treated in the Department of Plastic Surgery of the Hospital in July 2020. The Fbs were divided into blank control group (culture medium only), 2 mg/L nano sliver group, 5 mg/L nano sliver group, 10 mg/L nano sliver group, 25 mg/L nano sliver group, and 50 mg/L nano sliver group, which were added with the corresponding final mass concentrations of nano sliver solution, respectively. At 48 h of culture, the Fb proliferation viability was detected by cell counting kit 8 method. The Fbs were divided into 0 mg/L silver-containing GelMA hydrogel group, 10 mg/L silver-containing GelMA hydrogel group, 50 mg/L silver-containing GelMA hydrogel group, and 100 mg/L silver-containing GelMA hydrogel group and then were correspondingly treated. On culture day 1, 3, and 7, the Fb proliferation viability was detected as before. The ASCs were mixed into GelMA hydrogel and divided into 3D bioprinting group and non-printing group. On culture day 1, 3, and 7, the ASC proliferation viability was detected as before and cell growth was observed by live/dead cell fluorescence staining. The sample numbers in the above experiments were all 3. Four full-thickness skin defect wounds were produced on the back of 18 male Sprague-Dawley rats aged 4 to 6 weeks. The wounds were divided into hydrogel alone group, hydrogel/nano sliver group, hydrogel scaffold/nano sliver group, and hydrogel scaffold/nano sliver/ASC group, and transplanted with the corresponding scaffolds, respectively. On post injury day (PID) 4, 7, 14, and 21, the wound healing was observed and the wound healing rate was calculated (n=6). On PID 7 and 14, histopathological changes of wounds were observed by hematoxylin eosin staining (n=6). On PID 21, collagen deposition of wounds was observed by Masson staining (n=3). Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Bonferroni correction, and independent sample t test. Results: The sliver nano particles in nano silver solution with different mass concentrations were all round, in scattered distribution and uniform in size. The silver-containing GelMA hydrogels with different final mass fractions of GelMA all showed pore structures of different sizes and interconnections. The pore size of silver-containing GelMA hydrogel with 10% final mass fraction was significantly larger than that of silver-containing GelMA hydrogels with 15% and 20% final mass fractions (with P values both below 0.05). On treatment day 1, 3, and 7, the concentration of nano silver released from silver-containing GelMA hydrogel in vitro showed a relatively flat trend. On treatment day 14, the concentration of released nano silver in vitro increased rapidly. At 24 h of culture, the diameters of inhibition zone of GelMA hydrogel containing 0, 25, 50, and 100 mg/L nano silver against Staphylococcus aureus and Escherichia coli were 0, 0, 0.7, and 2.1 mm and 0, 1.4, 3.2, and 3.3 mm, respectively. At 48 h of culture, the proliferation activity of Fbs in 2 mg/L nano silver group and 5 mg/L nano silver group was both significantly higher than that in blank control group (P<0.05), and the proliferation activity of Fbs in 10 mg/L nano silver group, 25 mg/L nano silver group, and 50 mg/L nano silver group was all significantly lower than that in blank control group (P<0.05). Compared with the that of Fbs in 0 mg/L silver-containing GelMA hydrogel group, the proliferation activity of Fbs in 50 mg/L silver-containing GelMA hydrogel group and 100 mg/L silver-containing GelMA hydrogel group was all significantly decreased on culture day 1 (P<0.05); the proliferation activity of Fbs in 50 mg/L silver-containing GelMA hydrogel group was significantly increased (P<0.05), while the proliferation activity of Fbs in 100 mg/L silver-containing GelMA hydrogel group was significantly decreased on culture day 3 (P<0.05); the proliferation activity of Fbs in 100 mg/L silver-containing GelMA hydrogel group was significantly decreased on culture day 7 (P<0.05). The proliferation activity of ASCs in 3D bioprinting group show no statistically significant differences to that in non-printing group on culture day 1 (P>0.05). The proliferation activity of ASCs in 3D bioprinting group was significantly higher than that in non-printing group on culture day 3 and 7 (with t values of 21.50 and 12.95, respectively, P<0.05). On culture day 1, the number of dead ASCs in 3D bioprinting group was slightly more than that in non-printing group. On culture day 3 and 5, the majority of ASCs in 3D bioprinting group and non-printing group were living cells. On PID 4, the wounds of rats in hydrogel alone group and hydrogel/nano sliver group had more exudation, and the wounds of rats in hydrogel scaffold/nano sliver group and hydrogel scaffold/nano sliver/ASC group were dry without obvious signs of infection. On PID 7, there was still a small amount of exudation on the wounds of rats in hydrogel alone group and hydrogel/nano sliver group, while the wounds of rats in hydrogel scaffold/nano sliver group and hydrogel scaffold/nano sliver/ASC group were dry and scabbed. On PID 14, the hydrogels on the wound surface of rats in the four groups all fell off. On PID 21, a small area of wounds remained unhealed in hydrogel alone group. On PID 4 and 7, the wound healing rates of rats in hydrogel scaffold/nano sliver/ASC group were significantly higher than those of the other three groups (P<0.05). On PID 14, the wound healing rate of rats in hydrogel scaffold/nano sliver/ASC group was significantly higher than the wound healing rates in hydrogel alone group and hydrogel/nano sliver group (all P<0.05). On PID 21, the wound healing rate of rats in hydrogel alone group was significantly lower than that in hydrogel scaffold/nano sliver/ASC group (P<0.05). On PID 7, the hydrogels on the wound surface of rats in the four groups remained in place; on PID 14, the hydrogel in hydrogel alone group was separated from the wounds of rats, while some hydrogels still existed in the new tissue of the wounds of rats in the other three groups. On PID 21, the collagen arrangement in the wounds of rats in hydrogel alone group was out of order, while the collagen arrangement in the wounds of rats in hydrogel/nano sliver group, and hydrogel scaffold/nano sliver/ASC group was relatively orderly. Conclusions: Silver-containing GelMA hydrogel has good biocompatibility and antibacterial properties. Its three-dimensional bioprinted double-layer structure can better integrate with new formed tissue in the full-thickness skin defect wounds in rats and promote wound healing.

[Advances in the research of heterotopic ossification caused by burns].

Heterotopic ossification is a rare complication of burns, and its incidence and risk factors are still unclear. Through summarizing the literature on heterotopic ossification caused by burns at home and abroad, the author searched for the risk factors of heterotopic ossification after burn and the new progress of its prevention and treatment. It was realized that the size, depth and healing time of burn wounds were related to heterotopic ossification; the nonsteroidal anti-inflammatory drugs, radiation therapy, and their combination therapy can be used for the prevention of heterotopic ossification; surgery is an effective means of treating heterotopic ossification.

Clinical value of fluid bolus contrast flow meter during hysterosalpingography.

The purpose of this study was to investigate the clinical value of the fluid bolus contrast flow meter during hysterosalpingography. Hysterosalpingography information of 342 cases, which included a manual handset group of 213 cases and a bolus instrument group of 129 cases were reviewed. Comparative analysis was used to compare the two groups in order to assess the clinical adverse reactions, contrast agent reflux, and image quality. In the instrument bolus group compared with the manual handset group, the clinical adverse reactions decreased from 75.12 to 31.78% (P < 0.001); the backflow phenomenon of the contrast agent decreased from 13.62 to 3.10% (P < 0.01); and image quality significantly improved, with the A class film rate increasing from 54.46 to 68.99% (P < 0.01) and the C class film rate decreasing from 8.92 to 2.33% (P < 0.05). The use of a contrast bolus through the liquid inlet of the hysterosalpingography instrument can provide fully dynamic observation, reducing the contrast agent reflux and adverse reactions as well as improving the image quality and diagnostic accuracy. In addition, the medical staff is not subjected to radiographic radiation. Therefore, it is a safe and reliable imaging method.

Temporary amnesia induced by cold anesthesia and hypoxia in Drosophila.

Memory consolidation in Drosophila was investigated using cold anesthesia- and hypoxia-induced amnesia. Individual flies were operantly trained to avoid the specific flight orientations with respect to the landmarks surrounding them when paired with heat reinforcement at a flight simulator. Cold anesthesia, introduced immediately after training, exerted a significantly diminishing effect on memory between 15 and 150 min after training. Hypoxia delivered immediately after training had a significantly diminishing effect on memory between 30 and 150 min after training. In addition, cold anesthesia disrupted memory only when introduced within the first 20 min, while hypoxia worked only when delivered within the first 2 min after training. When interpreted in the context of a four-phase model of memory consolidation, the results suggest that 1) cold anesthesia disrupts both short-term memory (STM) and anesthesia-resistant memory (ARM), 2) hypoxia disrupts ARM specifically, 3) both of them leave long-term memory (LTM) intact, and 4) LTM may be independent of availability of STM and ARM in flies.

Multiple-phase model of memory consolidation confirmed by behavioral and pharmacological analyses of operant conditioning in Drosophila.

Previous work on classical olfactory learning and memory in flies has suggested at least four distinct phases of memory consolidation. Similarly, our behavioral and pharmacological analyses also provided clear evidence for at least four pharmacologically distinct memory phases in flies after operant conditioning. Anesthesia-resistant memory (ARM) is present between about 20 and 120 min after training, and susceptible to disruption by the ATPase deactivating chemicals such as ouabain and ethacrynic acid (EA). Long-term memory (LTM) is activated at least 150 min after training, and can be disrupted by protein synthesis inhibitors such as cycloheximide (CXM). In addition, a very short-term memory (pre-STM) is demonstrated by feeding flies with potassium chloride (KCl), which has been shown to disrupt the short-term memory. These observations confirm our previous argument that memory formation in flies involves an intricate, multiple-phase pathway of consolidation.

Operant visual learning and memory in Drosophila mutants dunce, amnesiac and radish.

Learning and memory of Drosophila mutants dunce, amnesiac and radish which were isolated originally from the classical olfactory learning paradigm are analyzed in an operant visual learning paradigm. Dunce appears to show normal ability to learn during training, but its memory is significantly affected. Though the learning index during the first minute after training is normal, its short-term memory (STM), anesthesia-resistant memory (ARM) and long-term memory (LTM) are all significantly damaged. Amnesiac displays disrupted middle-term memory (MTM), while its STM and LTM remain unchanged. Learning and memory in radish mutants seem to be unaffected. These results lend support to the argument that there are certain common molecular mechanisms underlying learning and memory through different tasks and the previous multi-phase model of visual memory is modified in a genetic way.

Nutritional effects on operant visual learning in Drosophila melanogaster.

The nutritional effects on operant visual learning behavior were investigated in a flight simulator. Operant visual learning and memory formation were normal in Drosophila (S-flies) reared on standard medium, but absent in flies (P-flies) raised on Peking medium. S- and P-flies were transferred to the alternative medium soon after hatching and their progeny was also raised on corresponding medium for several generations. respectively. S-flies transferred showed significantly reduced learning acquisition and 20 min memory retention, and operant visual learning along with memory formation was abolished in their progeny within three generations. Transferred P-flies recovered slowly their learning acquisition and memory formation to normal levels within five generations. Further studies suggested that low protein and minerals or high carbohydrate contents in Peking medium might be related to abnormal performance of P-flies. These results confirm the feasibility of affecting learning behavior by dietary regimens and developing an insect model of maternal malnutrition for pre- or post-natal malnutrition in Drosophila.

Conditioned visual flight orientation in Drosophila: dependence on age, practice, and diet.

Orientation preferences for visual patterns can be conditioned in tethered flies (Drosophila melanogaster) at the flight simulator. In a reversal conditioning procedure using heat as reinforcement, flies can be trained to successively prefer different flight orientations with respect to the patterns. As in many learning paradigms, conditioned responses are highly variable. Although during training most flies reliably avoid the heat and the corresponding flight orientations, in subsequent learning tests without heat some show no consistent preference for the permissive orientations. We have started to investigate the interindividual differences in learning performance and describe here three significant variables: the age of the animals, their experience in the flight simulator prior to the experiment, and the composition of the fly food. Flies learn more reliably at 3-4 days than at 1-2 days of age but learning indices do not increase further in even older flies. Learning is improved if flies are allowed to become familiar with the flight simulator before the start of the conditioning procedure. Most important, poor nutrition causes complete amnesia within three or four generations. The reverse shift from poor to nutritious food restores learning ability with an even longer delay.