Structure and properties of gelatin edible film modified using oxidized poly(2-hydroxyethyl acrylate) with multiple aldehyde groups.

BACKGROUND: Pure gelatin film usually exhibits characteristics of being brittle and hydrophilic, which limit its wide use in food packing fields. In this study gelatin/oxidized poly(2-hydroxyethylacrylate) (G/OP) composite films were prepared using casting techniques, the aim of this research was to investigate the effects of OP on the structures and properties of the G/OP composite films. RESULTS: The Fourier-transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy results indicated that the G/OP films retained their original secondary structure and random coiled conformation. However, the surface and cross-sectional morphologies of the G/OP films were rougher than those of pure gelatin films, cracks and agglomerates appeared with increasing OP dosage. The remarkable transparency of the G/OP film at 280 nm indicated excellent ultraviolet (UV) light barrier properties of the film, which inhibited UV-light-induced food oxidation. Moreover, the addition of OP decreased the water content and water solubility and considerably increased the water contact angle of pure gelatin films from 78.8° to 116.2° (a maximum increase of 37.5°). This suggested that OP modification improved the hydrophobicity of gelatin films. Furthermore, the inclusion of OP significantly promoted the flexibility of gelatin films, thereby improving their brittleness. CONCLUSIONS: The UV light barrier properties, hydrophobicity, and flexibility of gelatin films were improved via OP modification, thus the produced G/OP composite films have the potential to be used in food packaging. © 2022 Society of Chemical Industry.

Microgel Single-Cell Culture Arrays on a Microfluidic Chip for Selective Expansion and Recovery of Colorectal Cancer Stem Cells.

Cancer stem cells (CSCs) are rare and lack definite biomarkers, necessitating new methods for a robust expansion. Here, we developed a microfluidic single-cell culture (SCC) approach for expanding and recovering colorectal CSCs from both cell lines and tumor tissues. By incorporating alginate hydrogels with droplet microfluidics, a high-density microgel array can be formed on a microfluidic chip that allows for single-cell encapsulation and nonadhesive culture. The SCC approach takes advantage of the self-renewal property of stem cells, as only the CSCs can survive in the SCC and form tumorspheres. Consecutive imaging confirmed the formation of single-cell-derived tumorspheres, mainly from a population of small-sized cells. Through on-chip decapsulation of the alginate microgel, ∼6000 live cells can be recovered in a single run, which is sufficient for most biological assays. The recovered cells were verified to have the genetic and phenotypic characteristics of CSCs. Furthermore, multiple CSC-specific targets were identified by comparing the transcriptomics of the CSCs with the primary cancer cells. To summarize, the microgel SCC array offers a label-free approach to obtain sufficient quantities of CSCs and thus is potentially useful for understanding cancer biology and developing personalized CSC-targeting therapies.

Simultaneous Identification and Antimicrobial Susceptibility Testing of Multiple Uropathogens on a Microfluidic Chip with Paper-Supported Cell Culture Arrays.

A microfluidic chip was developed for one-step identification and antimicrobial susceptibility testing (AST) of multiple uropathogens. The polydimethylsiloxane (PDMS) microchip used had features of cell culture chamber arrays connected through a sample introduction channel. At the bottom of each chamber, a paper substrate preloaded with chromogenic media and antimicrobial agents was embedded. By integrating a hydrophobic membrane valve on the microchip, the urine sample can be equally distributed into and confined in individual chambers. The identification and AST assays on multiple uropathogens were performed by combining the spatial resolution of the cell culture arrays and the color resolution from the chromogenic reaction. The composite microbial testing assay was based on dynamic changes in color in a serial of chambers. The bacterial antimicrobial susceptibility was determined by the lowest concentration of an antimicrobial agent that is capable of inhibiting the chromogenic reaction. Using three common uropathogenic bacteria as test models, the developed microfluidic approach was demonstrated to be able to complete the multiple colorimetric assays in 15 h. The accuracy of the microchip method, in comparison with that of the conventional approach, showed a coincidence of 94.1%. Our data suggest this microfluidic approach will be a promising tool for simple and fast uropathogen testing in resource-limited settings.

Forming Single-Cell-Derived Colon Cancer Organoid Arrays on a Microfluidic Chip for High Throughput Tumor Heterogeneity Analysis.

Single-cell-derived tumor organoids (STOs) possess a distinct genetic background, making them valuable tools for demonstrating tumor heterogeneity. In order to fulfill the high throughput demands of STO assays, we have developed a microfluidic chip containing 30 000 microwells, which is dedicated to a single cell culture approach for selective expansion and differential induction of cancer stem cells. The microwells are coated with a hydrophilic copolymer to eliminate cell adhesion, and the cell culture is supported by poly(ethylene glycol) (PEG) to establish a nonadhesive culture environment. By utilizing an input cell density of 7 × 103·mL-1, it is possible to construct a 4000 single cell culture system through stochastic cell occupation. We demonstrate that the addition of 15% PEG10000 in the cell culture medium effectively prevents cell loss while facilitating tumor stem cell expansion. As were demonstrated by HCT116, HT29, and SW480 colon cancer cells, the microfluidic approach achieved a STO formation rate of ∼20%, resulting in over 800 STOs generated from a single culture. Comprehensive analysis through histomorphology, immunohistochemistry, drug response evaluation, assessment of cell invasion, and biomarker detection reveals the heterogeneity among individual STOs. Specifically, the smaller STOs exhibited higher invasion and drug resistance capabilities compared with the larger ones. The developed microfluidic approach effectively facilitates STO formation and offers promising prospects for investigating tumor heterogeneity, as well as conducting personalized therapy-focused drug screening.

Detection of Mycobacterium tuberculosis using a capillary-array microsystem with integrated DNA extraction, loop-mediated isothermal amplification, and fluorescence detection.

This article reports for the first time a high-throughput microfluidic system with fully integrated loop-mediated isothermal amplification (LAMP) analysis. With the developed system, parallel Mycobacterium tuberculosis detections were implemented in polytetrafluoroethylene capillaries through the utilization of droplet technology coupled with magnetic beads. During the analysis, liquid plugs containing different types of sample or reagents are sequentially introduced into the capillaries and made to form droplets therein. The whole analytical process, including DNA extraction, LAMP, and detection of the amplified products were conducted in such droplets. The developed microsystem is able to process 10 samples in parallel. The entire diagnostic procedure, from sample-in to answer-out, can be automatically completed within 50 min with a limit of detection (LOD) of 10 bacteria. This microsystem was evaluated by analyzing clinical samples, and a clinical sensitivity (positive detection rate) of 96.8% and specificity (negative detection rate) of 100% were achieved. The presented capillary LAMP assay features high-throughput and low-cost and thus is a promising tool for rapid tuberculosis diagnosis.

Gel-Free Single-Cell Culture Arrays on a Microfluidic Chip for Highly Efficient Expansion and Recovery of Colon Cancer Stem Cells.

The microgel single-cell culture approach we developed to expand tumor stem cells (TSCs) is associated with limited TSC production, which can be attributable to cell viability loss in microgel formation and tumorsphere expansion limitation caused by hydrogel stiffness. In this work, we developed a gel-free single-cell culture array on a microfluidic chip to overcome these issues. The microfluidic chip used in the study has a 16,000 hydrophilic microchamber array, which can capture ∼2000 single cells at a time. After cell capturing, the cell culture chambers were enclosed by forming a chitosan layer through interactions between chitosan and alginate, thus preventing cell loss in the gel-free culture. The hydrophilic coating prevented cell adhesion, so only TSCs with anti-apoptosis and self-renewal properties can survive the harsh culture and form tumorspheres. After a 7 day culture, 19.04% of the HCT116 colon cancer cells formed single-cell-derived tumorspheres with an average size of 46.59 ± 10.58 µm. Compared with the microgel single-cell culture, sphere-forming rate and TSC expansion efficiency were significantly improved by using this gel-free single-cell culture array. After cell culture, the chitosan layer could be destabilized easily, thus allowing recovery of the tumorspheres from the microchip by applying a reverse flow. Approximately 13,600 cells could be obtained in a single culture, which can be used for off-chip cell assays. Flow cytometry analysis indicated high proportions of LGR5(+) and SOX2(+) cells within the single-cell-derived tumorspheres. Moreover, the differentiation experiments confirmed the multi-lineage differentiation potential of single-cell-derived tumorspheres. The gel-free single-cell culture offers a label-free approach to obtain sufficient amounts of TSCs, which is valuable for tumor biology research and the development of TSC-specific therapeutic strategies.

[Diagnosis and surgical treatment of cervical lymphangioma].

OBJECTIVE: To discuss the methods of surgical treatment and their timing choices of cervical lymphangioma. METHODS: A retrospective review of 53 patients with cervicofacial lymphangioma were treated surgically from July 1990 to December 2008. The age at operation was from 6.5 months to 41 years old (median age was 2 years old and 3 months). Eighteen (34.0%) lesions were located in the suprahyoid region and 35 (66.0%) lesions in the infrahyoid region. The diameter of lesion ranged from 3.3 to 8.2 cm (average: 4.4 cm). Neck mass was the sole symptom for 77.4% (41/53) cases. Nine patients presented with life-threatening complications including intracystic hemorrhage in 2 cases/times, infection and rapid increase in tumor size in 5 cases/times, dysphagia in 2 cases/times and respiratory obstruction in 4 cases/times. Color Doppler ultrasound was used to diagnose all patients pre-operatively. Computed tomography (CT) was used in 11 cases and magnetic resonance imaging (MRI) in 21 cases for differential diagnosis. RESULTS: The patients were treated by complete resection in 34 cases and subtotal resection in 8 cases. But partial resection in 11 (20.8%) cases developed a residual or recurrent lesion within 9 months to 5 years post-operation, including 7 cases in suprahyoid region and 4 cases in infrahyoid region. The rate of residual or recurrent lesions was significantly higher in the suprahyoid region (7/18) than that in the infrahyoid region (4/35) (chi(2) test, P < 0.05). The peri-operative complications were paralyses of mandibular branch of facial nerve, Horner's syndrome, secondary hemorrhage, fluid collection at resection site, local infection and parotid fistula in 1 case respectively. Respiratory distress caused by edema of tongue was present in 2 cases. All of them were cured conservatively. The pathological diagnosis was confirmed as capillary lymphangioma in 19 cases and cystic lymphangioma in 34 cases. CONCLUSION: The localization and extent of cervical lymphangioma are the most important determining factors for a successful surgical resection. Although complete excision is the ideal treatment for cervicofacial lymphangioma, this should not be attempted if lesions are too large and neighboring structures liable to injury. The surgeons should be aware of the limitations and potential surgical complications in certain instances.

GE11 peptide-conjugated nanoliposomes to enhance the combinational therapeutic efficacy of docetaxel and siRNA in laryngeal cancers.

In this study, dual therapeutic-loaded GE11 peptide-conjugated liposomes were developed and applied to enhance therapeutic efficacies of standard-of-care regimens for the treatment of laryngeal cancer. The therapeutic strategy used here was a combination treatment with the chemotherapeutic docetaxel (DTX) and siRNA against the ABCG2 gene that regulates multidrug resistance in many tumor types. Liposome-encapsulated DTX/ABCG2-siRNA molecules were targeted to recognize tumor cells of squamous morphology by conjugation to the EGFR-targeting ligand, GE11. Targeted, drug-infused liposomes were nanosized and exhibited controlled release of DTX. Presence of GE11 peptides on liposomal surfaces enhanced the quantities of liposomal constructs taken up by Hep-2 laryngeal cancer cells. GE11 peptide-conjugated liposomes also enhanced cytotoxic effects against Hep-2 laryngeal cancer cells when compared to treatment with free DTX, thereby reducing IC50 values. Additionally, GE11 peptide-conjugated liposomes had significantly increased anti-tumor and apoptotic effects. Treatments with the GDSL nanoparticle formulation inhibited tumor growth in Hep-2 xenograft-bearing nude mouse models when compared to treatments with non-targeted NP constructs. Treatment of the mouse models with GE11 peptide-conjugated liposomes mitigated toxicities observed after treatment with free DTX. Taken together, liposomal encapsulation of DTX and ABCG2-siRNA improved the anti-tumor effects of treatment with free DTX in Hep-2 cell lines, and conjugation of GE11 peptides to liposomal constructs enhanced anti-tumor efficacies and specificities in laryngeal cancer cells.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis.

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same specificity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients.

Highly efficient capillary polymerase chain reaction using an oscillation droplet microreactor.

The current work presents the development of a capillary-based oscillation droplet approach to maximize the potential of a continuous-flow polymerase chain reaction (PCR). Through the full utilization of interfacial chemistry, a water-in-oil (w/o) droplet was generated by allowing an oil-water plug to flow along a polytetrafluoroethylene (PTFE) capillary. The w/o droplet functioned as the reactor for oscillating-flow PCR to provide a stable reaction environment, accelerate reagent mixing, and eliminate surface adsorption. The capillary PCR approach proposed in the current research offers high amplification efficiency, fast reaction speed, and easy system control attributable to the oscillation droplet reactor. Experimental results show that the droplet-based micro-PCR assay requires lower reaction volume (2 µL) and shorter reaction time (12 min) compared with conventional PCR methods. Taking the amplification of the New Delhi metallo-beta-lactamase (NDM-1) gene as an example, the present work demonstrates that the oscillation droplet PCR assay is capable of achieving high efficiency up to 89.5% and a detection limit of 10 DNA copies. The miniature PCR protocol developed in the current work is fast, robust, and low-cost, thus exhibiting the potential for expansion into various practical applications.

Rapid screening of phenylketonuria using a CD microfluidic device.

We herein present a compact disc (CD) microfluidic chip based hybridization assay for phenylketonuria (PKU) screening. This CD chip is composed of a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a glass bottom layer with hydrogel pad conjugated DNA oligonucleotides. Reciprocating flow was generated on the CD chip through a simple rotation-pause operation to facilitate rapid DNA hybridization. When rotated the CD chip, the sample solution was driven into the hybridization channel by centrifugal force. When stopped the CD chip, the sample plug was pulled backward through the channel by capillary force. The hybridization assay was firstly validated with control samples and was then used to analyze 30 clinical samples from pregnant women with suspected PKU fetus. The on-chip DNA hybridization was completed in 15 min with a sample consumption as low as 1.5µL, and the limit-of-detection (LOD) of DNA template was 0.7ng/µL. Among the 30 samples tested, V245V mutation was identified in 4 cases while R243Q mutation was detected in one case. Results of the hybridization assay were confirmed by DNA sequencing. This CD-chip based hybridization assay features short analysis time, simple operation and low cost, thus has the potential to serve as the tool for PKU screening.

[Obstructive sleep apnea hypopnea syndrome: surgical complications and strategy for avoidance].

OBJECTIVE: To analyze the surgical complications in patients with obstructive sleep apnea hypopnea syndrome (OSAHS) and discuss the risk factors and preliminary strategies for prevention of complications. METHODS: From September 1998 to March 2007, 653 OSAHS patients confirmed by polysomnography were treated by different surgical approaches according to their obstructive sites, which included uvulopalatopharyngoplasty (UPPP) in 586 cases, nasal surgery in 104 cases/times, hyoid suspension surgery in 53 cases/times, respectively or at the same time. Local anesthesia was used in 294 cases and general anesthesia in 359 cases. Two hundreds and seventeen cases were treated by continuous positive airway pressure (CPAP) 3 to 7 days pre-operation and 2 to 3 days post-operation. RESULTS: Perioperative complications were found in 57 OSAHS cases (93 times), the incidence of peri-operative complications was 8.7% (57/653), including respiratory problems in 19 cases/times and 1 death occurred during inducing stage in general anesthesia. Profuse bleeding was encountered in 9 cases/times during operation and primary and secondary bleeding in 27 cases/times, cardiopathy and hypertension crisis in 31 cases/times and cerebral stroke and hemiplegia in 1 case, reactive somnolence in 3 cases/times and reactive hyperglycemia in 3 cases/times. Data were analyzed by the multivariate logistic regression model. The results showed that the complications were significantly reduced after CPAP treatment during peri-operative stage and increased accompanied with patients' hypertension, choice of general anesthesia, BMI and AHI. All patients were followed-up more than 1 year. After UPPP, 23.9% cases (140/586) had sensation of foreign body in pharynx and alleviated in 6 to 12 months. Scar tissues in oropharynx in 7 cases, nasopharyngeal stenosis in 1 case, atrophy rhinitides and atrophy pharyngitis in 3 cases, nasopharyngeal un-closure and long-term nasopharyngeal reflex in 3 cases. Conclusions Peri-operative complications are more common in obese and severe OSAHS patients, especially when they accompanied with hypertension. The corresponding strategies should be taken to reduce complications in OSAHS surgery, which include controlling the hypertension effectively, performing CPAP treatment actively, cooperating with interdisciplinary doctors, monitoring closely after operation. It is important to reduce surgical sequelae through improving surgical skills and not enlarging the surgical scale blindly.

Carotid artery resection and reconstruction with expanded polytetrafluoroethylene for head and neck cancer.

OBJECTIVE: There are controversies on management of carotid artery invasion in advanced head and neck cancer. En bloc resection has been considered a curative modality. In this study, we evaluated the feasibility of using expanded polytetrafluoroethylene (ePTFE) in reconstructing the common carotid artery in patients with carotid artery invasion requiring resection. METHOD: A retrospective study including 13 patients managed from 2002 to 2005. All patients underwent en bloc resection of the tumor and internal carotid artery then reconstructed with ePTFE. RESULTS: All patients had en bloc resection of the tumor together with internal carotid artery and reconstruction with ePTFE. Some patients required wound coverage with myocutaneous flaps in eight patients and local flaps in five patients. Intraoperative shunting was used in all patients. Intraoperative heparin infusion and postoperative low dose aspirin were used to prevent thrombosis. One patient developed graft blowout and he was treated with ligation without hemiplegia. One patient had minor stroke. The follow-up period was 18.4 +/- 8.6 months. No patient suffered from neurological deficit or graft occlusion. Disease survival was 61.5% in 1 year and 38.5% in 2 years. Overall survival was 18.3 months. CONCLUSION: En bloc resection and tumor together with carotid artery and ePTFE reconstruction is shown to be a feasible modality in treatment of advanced head and neck cancer with carotid artery invasion.

[An experimental study of polypropylene combined with flap for trachea reconstruction].

OBJECTIVE: Canine model established for tracheal defect reconstruction, to investigate the outcome of tracheal reconstruction with combination of polypropylene and flap. METHODS: About 3.5 to 4 centimeter cervical trachea was resected and replaced with artificial trachea made from monofilament knitted polypropylene and surgical flap. Covered stent was implanted postoperatively. Survival period and quality of life were recorded, bronchofibroscopy, X-ray films and HE sections were performed. RESULTS: Six dogs survived well and another two died. The causes of death were respiratory failure in 1 and infection in another. Stenosis of anastomosis in 1 was recorded during survival period. The dogs started drinking and eating on the second postoperative day, no dyspnea was found. The animals were sacrificed at 2, 4, 8 weeks and 6 months after surgery. Soft tissue growth was found in polypropylene net 2 weeks after surgery and more at 4 weeks. The polypropylene net was covered completely with soft tissue at 8 weeks and 6 months postoperatively, the hardness and sustentation degree were enhanced following the growth and fibrosis of soft tissue. The squamous epithelium and columnar epithelium were observed healing well by HE staining method. CONCLUSIONS: One-stage operative artificial trachea made from monofilament knitted polypropylene which has good histocompatibility and surgical flap is the closer artificial trachea to native trachea. It has a promising prospect in clinical use.

[The synthesis treatment of tonsillar cancer].

OBJECTIVE: To study the synthesis treatment of tonsillar cancer. METHOD: From 1987 to 1999, thirty four patients with tonsillar cancer were treated with surgery and postoperative radiotherapy. There were 21 males and 13 females and the ages ranged from 33-74. According to TNM staging system, there are 5 stage I , 6 stage 11, 9 stage II and 14 stage IV. According to conditions, the transoral approach, mandibular wing approach and transhyoid approach were choiced to resect the tumor. Surgical defect was repaired by myocutaneous flap, tongue flap, or softpalate flap. RESULT: The 5 year survival rate were 68.8% and local control 76.4%. Function of chewing, deglutition and speech were restored well. CONCLUSION: The treatment of surgery and postoperative radiotherapy can improved the survival rate, local control rate and quality of life of patients.

[Small partial laryngectomy for early-stage glottic carcinoma].

OBJECTIVE: To explore the surgery way for T1, T2 glottic carcinoma. METHOD: Small partial laryngectomy were performed to 112 patients with T1, T2 (T1 N0 M0, T2 N0 M0) glottic carcinoma. The surgery method, effect and treatment advantage was summarized and compared with laser therapy and radiotherapy, the dynamic follow-up of arytaenoidea cartilage movement, glottidis rimae conformation and voice change was analyzed. The preoperative and postoperative voice quality was compared with the software of Dr. speech system for windows. RESULT: All incisions were healed one stage in 6 or 7 days, the mean time in hospital is 9.76 days; all patients took food with mouth after 2 or 3 days, all trachea cannula was removed successfully during in hospital, the mean time with cannula is 7.32 days and the rate of removing cannula 100%. Arytaenoidea cartilago movement of 47 cases 1 week after surgery weakened and gradually improved, glottidis rimae conformation is close to be normal after 2 months. There is no significant difference of Shimmer and NNE between preoperative and postoperative 1 week (P > 0.05), but there is a significant difference between preoperative and postoperative 2 months, half a year, and also is between postoperative 1 week and 2 months, half a year (P < 0.01). All 76 cases survive during 3 years' follow-up, 35 of 36 cases in 5 years' follow-up survive (1 case died without definite cause), and there is 2 cases for recurrence. CONCLUSION: Small partial laryngectomy can provide large operative view, resect the tumor completely, make diet and speech recover in shorter time and improve the voice quality, so the surgery is available.