A porous sodium polyacrylate-grafted chitosan xerogel for severe hemorrhage control synthesized from one-pot reaction.

Controlling severe hemorrhages remains a challenge. Successful hemorrhage control depends on the speed and quality of blood clot formation. Fast deprivation of water from blood leads to the concentration of blood cells and coagulation factors and thus triggers blood clot formation. This inspired us to develop a new hemostatic material. In this study, we grafted sodium polyacrylate (SPA) onto the backbone of chitosan (CTS) and crosslinked with methacrylic anhydride-modified polyethylene glycol (MAAPEG) to provide a flexible and elastic inter-chain connection between SPA and CTS chains in the presence of a blowing agent to achieve a porous structure. By a simple one-pot reaction, we fabricated a soft, elastic porous xerogel sponge that could reach maximum water absorbency of 180 in less than 200 seconds. This SPA-co-chitosan xerogel sponge demonstrated superior hemostatic properties in thromboelastography (TEG®) test and in a rabbit lethal extremity arterial bleeding model as compared to zeolite granules, kaolin gauze, and chitosan granules. Furthermore, this hemostat worked as a whole to transfer external pressure to the bleeding area and was adhesive to wet wound tissue to seal the bleeding site. In general, the SPA-co-CTS sponge demonstrates a fast and powerful hemostatic effect both in vitro and in vivo, which is superior over the existing commercial products. It might be a promising first-aid device for severe hemorrhage control.

[Application of vacuum sealing drainage in the treatment of severe necrotizing fasciitis in extremities of patients].

OBJECTIVE: To study the application of VSD in the treatment of severe necrotizing fasciitis in extremities of patients. METHODS: Eight patients, suffering from severe necrotizing fasciitis, who had been traditionally treated with iodophor-soaked gauze for 21 to 365 days in other hospitals, were transferred to our institute because of the nonhealing wounds and systemic toxic symptoms induced by infection, from January 2011 to August 2013. After admission, surgical debridement was performed timely, and the necrotic tissue was collected during the operation for pathological observation after HE staining. After the operation, VSD was started with negative pressure ranging from -100 to -80 kPa, and the furacilin solution (0.2 g/L) and oxygen (2 L/min) were continuously infused into the wound during the treatment. Surgical debridement was performed repeatedly according to the wound condition followed by change of VSD dressings to continue VSD treatment. The wounds were closed by suturing or with autologous skin grafts after being covered by fresh granulation tissue. The times of surgical debridement, times of change of VSD materials, wound healing status, and length of stay in our institute were recorded. All patients were followed up for a long time. Results HE staining showed that there were diffuse necrotic adipose and fibrous connective tissues in the necrotic tissue, and the normal tissue structure disappeared accompanied by significant infiltration of inflammatory cells. The number of surgical debridement was 2 to 10 (3.9 +/- 2.8) times. The number of VSD materials change was 2 to 10 (4.0 +/- 2.9) times. Wounds were closed by suturing and healed in two patients; wounds in the other six patients were partially sutured, their residual wounds were healed by autologous skin grafting. The length of stay in our institute was 20 to 49 (33 +/- 10) days. All patients were discharged after recovery. Patients were followed up for 2 to 24 months, and their wounds were found to be in good condition without ulceration or recurrence. CONCLUSIONS: VSD can effectively remove the necrotic tissues and exudates from the fascial spaces and promote proliferation of granulation tissue. Therefore it serves as an effective approach to the treatment of severe necrotizing fasciitis in extremities.

[Clinical study of repairing donor site of thickness from cicatricial skin with auto-scalp grafting].

OBJECTIVE: To study the effects of using auto-scalp for repairing donor site of thickness from cicatricial skin with auto-scalp grafting. METHODS: A total of 13 cases with donor site of thickness from cicatricial skin from January 2011 to December 2011 were analyzed. Wounds of donor site from cicatricial skin were grafted with auto-scalp and scalp were fixation was applied with negative pressure. The survival rate of auto-scalp graft was observed at Day 7 post-operation. At Month 12, hyperplastic scars at these donor sites of cicatricial skin were assessed through Vancouver Scar Assessment Table, scar itch assessment and scar proliferation rate. Wounds in the other thirteen cases with donor site of thickness from cicatricial skin from January 2010 to December 2010 were covered with vaseline gauze as control. RESULTS: No significant difference existed in the gender and age of the two groups patients (P > 0.05). The auto-scalp graft all survived. And the average healing time of donor-site wound in cicatricial skin in grafting group (7 days) was significantly decreased than that of control group (a mean of 20 days) (P < 0.01). After followed up for twelve months, the scar formation assessment value (1.5 ± 0.5), scar itch assessment (1.2 ± 0.4) and scar proliferation rate (14.6% ± 7.6%) in grafting group were significantly less than those of control group (6.7 ± 1.1, 2.0 ± 0.7, 55.8% ± 12.2%, all P < 0.01). CONCLUSION: Auto-scalp grafting may greatly shorten the healing procedure and ameliorate the quality of donor-site of thickness from cicatricial skin.

[Efficacy observation on application of negative pressure therapy in the treatment of superficial partial-thickness scald wound in children].

OBJECTIVE: To observe the effect of negative pressure therapy in the treatment of superficial partial-thickness scald in children. METHODS: Three hundred and seven children with superficial partial-thickness scald hospitalized from August 2009 to May 2012 were divided into negative pressure therapy group (NPT, n = 145) and control group (C, n = 162) according to the random number table. Patients in group NPT were treated with negative pressure from within post injury day (PID) 3 to PID 9 (with -16 kPa pressure), while traditional occlusive dressing method was used in group C. Changes in body temperature, wound healing condition, frequency of dressing change were compared between group NPT and group C. Bacterial culture results of wounds were compared before and after treatment in group NPT. Volume of drained transudate per one percent of wound area was recorded in group NPT on PID 1 to PID 3. Data were processed with t test or chi-square test. RESULTS: The incidence of high fever was significantly lower in group NPT (26.9%, 39/145) than in group C (63.6%, 103/162, χ(2) = 41.419, P < 0.01). On PID 9, complete wound epithelization was observed in 138 patients in group NPT, and in 7 patients there were a few residual wounds which healed after dressing change for 2 days. The wound healing time of patients in group NPT [(9.2 ± 0.6) d] was obviously shorter than that in group C [(10.1 ± 1.6) d, t = 6.895, P < 0.01]. The frequency of dressing change among patients in group NPT [(2.05 ± 0.22) times] was significantly decreased as compared with that in group C [(4.82 ± 0.81) times, t = 39.878, P < 0.01]. Bacteria were found in wound secretion of seventeen patients in group NPT before treatment, while no bacterium was discovered in all patients after treatment. Volumes of drainage fluid in group NPT were proportional to wound areas, which were respectively (9.8 ± 3.2), (6.2 ± 2.1), (4.1 ± 1.6) mL per one percent of wound area on PID 1, 2, and 3. CONCLUSIONS: NPT can decrease times of dressing change, and alleviate infection and inflammatory response by drainage of transudate, which promotes wound healing at last. NPT is proved to be a safe and effective approach for treatment of children with superficial partial-thickness scald.

Hepatocyte function within a stacked double sandwich culture plate cylindrical bioreactor for bioartificial liver system.

Bioartificial liver (BAL) system is promising as an alternative treatment for liver failure. We have developed a bioreactor with stacked sandwich culture plates for the application of BAL. This bioreactor design addresses some of the persistent problems in flat-bed bioreactors through increasing cell packing capacity, eliminating dead flow, regulating shear stress, and facilitating the scalability of the bioreactor unit. The bioreactor contained a stack of twelve double-sandwich-culture plates, allowing 100 million hepatocytes to be housed in a single cylindrical bioreactor unit (7 cm of height and 5.5 cm of inner diameter). The serial flow perfusion through the bioreactor increased cell-fluid contact area for effective mass exchange. With the optimal perfusion flow rate, shear stress was minimized to achieve high and uniform cell viabilities across different plates in the bioreactor. Our results demonstrated that hepatocytes cultured in the bioreactor could re-establish cell polarity and maintain liver-specific functions (e.g. albumin and urea synthesis, phase I&II metabolism functions) for seven days. The single bioreactor unit can be readily scaled up to house adequate number of functional hepatocytes for BAL development.

A robust high-throughput sandwich cell-based drug screening platform.

Hepatotoxicity evaluation of pharmaceutical lead compounds in early stages of drug development has drawn increasing attention. Sandwiched hepatocytes exhibiting stable functions in culture represent a standard model for hepatotoxicity testing of drugs. We have developed a robust and high-throughput hepatotoxicity testing platform based on the sandwiched hepatocytes for drug screening. The platform involves a galactosylated microfabricated membrane sandwich to support cellular function through uniform and efficient mass transfer while protecting cells from excessive shear. Perfusion bioreactor further enhances mass transfer and cellular functions over long period; and hepatocytes are readily transferred to 96-well plate for high-throughput robotic liquid handling. The bioreactor design and perfusion flow rate are optimized by computational fluid dynamics simulation and experimentally. The cultured hepatocytes preserved 3D cell morphology, urea production and cytochrome p450 activity of the mature hepatocytes for 14 days. When the perfusion-cultured sandwich is transferred to 96-well plate for drug testing, the hepatocytes exhibited improved drug sensitivity and low variability in hepatotoxicity responses amongst cells transferred from different dates of perfusion culture. The platform enables robust high-throughput screening of drug candidates.

Toward surface quantification of liver fibrosis progression.

Monitoring liver fibrosis progression by liver biopsy is important for certain treatment decisions, but repeated biopsy is invasive. We envision redefinition or elimination of liver biopsy with surface scanning of the liver with minimally invasive optical methods. This would be possible only if the information contained on or near liver surfaces accurately reflects the liver fibrosis progression in the liver interior. In our study, we acquired the second-harmonic generation and two-photon excitation fluorescence microscopy images of liver tissues from bile duct-ligated rat model of liver fibrosis. We extracted morphology-based features, such as total collagen, collagen in bile duct areas, bile duct proliferation, and areas occupied by remnant hepatocytes, and defined the capsule and subcapsular regions on the liver surface based on image analysis of features. We discovered a strong correlation between the liver fibrosis progression on the anterior surface and interior in both liver lobes, where biopsy is typically obtained. The posterior surface exhibits less correlation with the rest of the liver. Therefore, scanning the anterior liver surface would obtain similar information to that obtained from biopsy for monitoring liver fibrosis progression.

Novel strategy to engineer trachea cartilage graft with marrow mesenchymal stem cell macroaggregate and hydrolyzable scaffold.

Limited donor sites of cartilage and dedifferentiation of chondrocytes during expansion, low tissue reconstruction efficiency, and uncontrollable immune reactions to foreign materials are the main obstacles to overcome before cartilage tissue engineering can be widely used in the clinic. In the current study, we developed a novel strategy to fabricate tissue-engineered trachea cartilage grafts using marrow mesenchymal stem cell (MSC) macroaggregates and hydrolyzable scaffold of polylactic acid-polyglycolic acid copolymer (PLGA). Rabbit MSCs were continuously cultured to prepare macroaggregates in sheet form. The macroaggregates were studied for their potential for chondrogenesis. The macroaggregates were wrapped against the PLGA scaffold to make a tubular composite. The composites were incubated in spinner flasks for 4 weeks to fabricate trachea cartilage grafts. Histological observation and polymerase chain reaction array showed that MSC macroaggregates could obtain the optimal chondrogenic capacity under the induction of transforming growth factor-beta. Engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans. PLGA scaffold degraded totally during in vitro incubation and the engineered cartilage graft was composed of autologous tissue. Based on this novel, MSC macroaggregate and hydrolyzable scaffold composite strategy, ready-to-implant autologous trachea cartilage grafts could be successfully fabricated. The strategy also had the advantages of high efficiency in cell seeding and tissue regeneration, and could possibly be used in future in vivo experiments.

Laminar-flow immediate-overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing.

Drug hepatotoxicity testing requires in vitro hepatocyte culture to maintain the long-term and stable liver specific functions. We developed a drug testing platform based on laminar-flow immediate-overlay hepatocyte sandwich perfusion culture. The immediate-overlay sandwich (collagen-coated porous polymeric membrane as top overlay) protects the cells and integrity of the top collagen matrix from the impact of flow. A bioreactor was designed that allowed proper control of shear stress and mass transfer. The culture parameters such as the optimal perfusion initiation time and flow rate were systematically and mechanistically determined. The optimized system could re-establish hepatocyte polarity to support biliary excretion and to maintain other liver specific functions, such as the biotransformation enzyme activities, for two weeks that extended the usable in vitro hepatocyte-based drug testing window. When the perfusion cultured hepatocytes from days 7 or 14 were used for drug testing, the APAP-induced hepatotoxicity measurements were more sensitive and consistent over time than the static culture control, enabling further exploitations in large-scale drug testing applications.

Treatment strategies for mass burn casualties.

BACKGROUND: Mass burn casualties are always a great challenge to a medical team because a large number of seriously injured patients were sent in within a short time. Usually a high mortality is impending. Experiences gained from successful treatment of the victims may be useful in guiding the care of mass casualties in an armed conflict. METHODS: Thirty-five burn victims in a single batch, being transferred nonstop by air and highway from a distant province, were admitted 48 hours post-injury. All patients were male with a mean age of (22.4 +/- 8.7) years. The burn extent ranged from 4% to 75% ((13.6 +/- 12.9)%) total body surface area. Among them, thirty-two patients were complicated by moderate and severe inhalation injury, and tracheostomy had been performed in 15 patients. Decompression incisions of burn eschar on extremities were done in 17 cases before transportation. All the thirty-five patients arrived at the destination smoothly via 4-hour airlift and road transportation. Among them, twenty-five patients were in critical condition. RESULTS: These thirty-five patients were evacuated 6 hours from the scene of the injury, and they were transferred to a local hospital for primary emergency care. The patients were in very poor condition when admitted to our hospital because of the severe injury with delayed and inadequate treatment. Examination of these patients at admission showed that one patient was suffering from sepsis and multiple organ dysfunction syndrome. Dysfunction of the heart, lung, liver, kidney, and coagulation were all found in the patients. Forty-eight operations were performed in the 23 patients during one month together with comprehensive treatment, and the function of various organs was ameliorated after appropriate treatment. All the 35 patients survived. CONCLUSIONS: A well-organized team consisting of several cooperative groups with specified duties is very important. As a whole, the treatment protocol should be individualized, basing on the extent of the injury and the care that the patient had received at the spot. During airlift, the stretchers should be arranged perpendicular to the longitudinal axis of the cabin. The treatment protocol in our hospital consisted mainly of prompt effective relief of all life-threatening complications, followed by early closure of burn wounds, appropriate use of anti-infection therapy, emphasis on nutritional support, correction of metabolic disorders, alleviation of immunosuppression, correction of coagulopathy, and effective support and protection of organ function.

Ultrastructural cell response to tension stress during mandibular distraction osteogenesis.

The response of cells in the distraction gap during mandibular distraction osteogenesis was recorded by transmission electronic microscopy. We distracted the mandible on both sides in eight adult goats. Two animals were killed at 8, 16, 32, and 48 days, respectively, after activation of the device. The specimens were harvested and processed for histological and ultrastructural examination. The results showed that the cells and newly-formed extracellular matrix (ECM) were aligned with the tension vector. In the early stage of distraction osteogenesis, cells in the distraction gap were of the active proliferative phenotype. They then differentiated into fibroblast-like cells and osteoblasts, showing ultrastructural characteristics of the active synthetic and secretory phenotypes. Newly-formed collagen, bone canaliculi, and mineralisation of the ECM were clearly evident during distraction osteogenesis. Our results show that at the ultrastructural level cell proliferation is activated by tension and stress during the early stages, and synthetic and secretory function stimulated during the later stages of mandibular distraction osteogenesis.

Characteristics of and strategies for patients with severe burn-blast combined injury.

BACKGROUND: Severe burn-blast combined injury is a great challenge to medical teams for its high mortality. The aim of this study was to elucidate the clinical characteristics of the injury and to present our clinical experiences on the treatment of such cases. METHODS: Five patients with severe burn-blast combined injuries were admitted to our hospital 77 hours post-injury on June 7, 2005. The burn extent ranged from 80% to 97% (89.6% +/- 7.2%) of TBSA (full-thickness burns 75% - 92% (83.4% +/- 7.3%)). All the patients were diagnosed as having blast injury and moderate or severe inhalation injury. Functions of the heart, liver, kidney, lung, pancreas and coagulation were observed. Autopsy samples of the heart, liver, and lungs were taken from the deceased. Comprehensive measures were taken during the treatment, including protection of organ dys function, use of antibiotics, early anticoagulant treatment, early closure of burn wounds, etc. All the data were analyzed statistically with t test. RESULTS: One patient died of septic shock 23 hours after admission (four days after injury), the others survived. Dysfunction of the heart, liver, lungs, pancreas, and coagulation were found in all the patients on admission, and the functions were ameliorated after appropriate treatments. CONCLUSIONS: Burn-blast combined injury may cause multiple organ dysfunctions, especially coagulopathy. Proper judgment of patients' condition, energetic anticoagulant treatment, early closure of burn wounds, rational use of antibiotics, nutritional support, intensive insulin treatment, timely and effective support and protection of organ function are the most important contributory factors in successful treatment of burn-blast combined injuries.

Expression of human beta-defensin 3 in COS-7 cell.

OBJECTIVE: To establish a cell line for stable expression of human beta-defensin 3 (hBD3). METHODS: Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofectamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. RESULTS: COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. CONCLUSION: We successfully established a hBD3-expressing cell line.