PCL film surfaces conjugated with P(DMAEMA)/gelatin complexes for improving cell immobilization and gene transfection.

Successful gene transfection on a tissue scaffold is of crucial importance in facilitating tissue repair and regeneration by enabling the localized production of therapeutic drugs. Polycaprolactone (PCL) has been widely adopted as a scaffold biomaterial, but its unfavorable cell-adhesion property needs to be improved. In this work, the PCL film surface was conjugated with poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA))/gelatin complexes via surface-initiated atom transfer radical polymerization (ATRP) for improving cell immobilization and subsequent gene transfection. A simple aminolysis-based method was first used for the covalent immobilization of ATRP initiators on the PCL film. Well-defined P(DMAEMA) brushes were subsequently prepared via surface-initiated ATRP from the initiator-functionalized PCL surfaces. The P(DMAEMA) chains with a pK(a) of 7.0-7.3 were used for conjugating gelatin with a pI of 4.7 via electrostatic interaction. The amount of complexed gelatin increased as that of the grafted P(DMAEMA) layer. The cell-adhesion property on the functionalized PCL surface could be controlled by adjusting the ratio of P(DMAEMA)/gelatin. It was found that the gene transfection property on the immobilized cells was dependent on the density of the immobilized cells on the functionalized PCL film. With the good cell-adhesive nature of gelatin and the efficient gene transfection on the dense immobilized cells, the incorporating the suitable of P(DMAEMA)/gelatin complexes onto PCL surfaces could endow the PCL substrates new and interesting properties for potential tissue engineering applications.

Improvement of hemocompatibility of polycaprolactone film surfaces with zwitterionic polymer brushes.

Polycaprolactone (PCL) has been widely adopted as a scaffold biomaterial, but further improvement of the hemocompatibility of a PCL film surface is still needed for wide biomedical applications. In this work, the PCL film surface was functionalized with zwitterionic poly(3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate) (P(DMAPS)) brushes via surface-initiated atom transfer radical polymerization (ATRP) for enhancing hemocompatibility. Kinetics study revealed an approximately linear increase in graft yield of the functional P(DMAPS) brushes with polymerization time. The blood compatibilities of the modified PCL film surfaces were studied by platelet adhesion tests of platelet-rich plasma and human whole blood, hemolysis assay, and plasma recalcification time (PRT) assay. The improvement of hemocompatibility is dependent on the coverage of the grafted P(DMAPS) brushes on the PCL film. Lower or no platelet and blood cell adhesion was observed on the P(DMAPS)-grafted film surfaces. The P(DMAPS) grafting can further decrease hemolysis and enhance the PRT of the PCL surface. With the versatility of surface-initiated ATRP and the excellent hemocompatibility of zwitterionic polymer brushes, PCL films with desirable blood properties can be readily tailored to cater to various biomedical applications.

[Evaluation of the effect of free fibular flap transplantation in repairing mandibular osteoradionecrosis defect in 151 cases].

Objective: To investigate the clinical effect of free fibula flap transplantation in repairing the defect of mandibular osteoradionecrosis (ORN). Methods: A total of 151 mandibular ORN patients undergoing free fibular flap transplantation were selected from August 2005 to September 2020 in the Department of Oral and Maxillofacial Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University. Among them, 109 patients were males and 42 patients were females, aged (54.1±10.1) (ranged 31-85) years old. The clinical data of the patients was collected and the survival rate of the flaps and postoperative function were calculated to evaluate the surgical efficacy. The χ2 test was used for difference analysis. Results: Among the 151 patients, mandibular ORN caused by radiotherapy for nasopharyngeal carcinoma accounted for 79.5% (120/151). The average time for mandibular ORN appeared was 5(6) years after radiotherapy. Facial artery [57.2%(87/152)] and superior thyroid artery [32.9%(50/152)] were the main anastomotic arteries in the recipient area. There was no significant difference in the necrosis rates of the two flaps [10.3%(9/87) and 12.5% (5/50), respectively, P=0.949]. The main anastomotic veins in the recipient area were the external jugular vein [48.4%(135/279)] and the common facial vein [26.5%(74/279)]. Twenty-five cases (16.6%) had one vein anastomosed, and 126 cases (83.44%) had two veins anastomosed. There was no significant difference in the flap necrosis rate between the two conditions [20.0%(5/25) and 7.1%(9/126), respectively, P=0.100]. Ninety-seven cases (64.2%) used the peroneal musculocutaneous-fascia composite flap to repair the maxillofacial soft and hard tissue defects. Thirteen cases (8.6%) underwent the restorations with digital virtual surgery design, of which 5 cases were repaired with dental implants at the same time. After the operations, lower respiratory tract infection occurred in 17 patients (11.3%), and upper respiratory tract obstruction occurred in 3 cases (2.0%). The survival rate of the flap after operation was 90.7% (136/151), and 21 patients (13.9%) had flap vascular crisis. Delayed healing of maxillofacial wounds occurred in 33 cases (21.9%). After 3 to 24 months of follow-ups, 110 patients (76.9%) had no fistula inside/outside the oral cavity, 118 patients (82.5%) had an improvement in opening mouth of increasing (≥0.5 cm) after surgery, 135 patients (94.4%) had pain relief, 97 cases (67.8%) could eat normal diet, semi-liquid or soft food, and 137 cases (95.8%) were satisfied or basically satisfied with the treatment effects. Conclusions: The free fibular flap transplantation is an effective method to repair mandibular ORN defects. Preoperative vascular assessment is helpful for the selection of recipient vessels. Facial artery, superior thyroid artery, external jugular vein and common facial vein can be used as the main recipient vessels. The repair of the peroneal musculocutaneous-fascia composite flap facilitates the closure of internal and external fistulas. Digital technology can help to restore the maxillofacial shape more accurately, improve the patient's occlusal and chewing function and enhance the quality of life of mandibular ORN patients.

Combination of biomechanical evaluation and accurate placement of dental implants: a new concept of virtual surgery in maxillary and mandibular functional reconstruction.

Biomechanics are crucial for bony regeneration and survival of implants in functional maxillary and mandibular reconstructions. However, we know of no study that has included an analysis of biomechanics to guide the optimal position of a fibular graft in virtual surgery. This study was designed to evaluate the combination of biomechanics and accurate placement of implants for virtual surgery in reconstruction of the jaw using fibular grafts. Thirty-one patients had maxillary or mandibular reconstruction with vascularised fibular grafts and the immediate placement of dental implants. Virtual studies were made preoperatively to evaluate the biomechanics and to assess the position of the fibular grafts with minimal distribution of stress. All operations proceeded accurately and with no complications with a mean (range) of 14 (6-20) months' follow-up. According to the individual biomechanical evaluations, the optimal position for the fibular graft is probably the middle of the mandibular body or below the bottom of the maxillary sinus. The combination of biomechanical evaluation and accurate placement of dental implants is a new concept that could achieve good biomechanical positioning of fibular grafts in the jaw and a desirable level of accuracy for functional reconstruction.

Morphology and adhesion of mesenchymal stem cells on PLLA, apatite and apatite/collagen surfaces.

Biomimetic apatite/collagen composite coating, previously reported particularly with regard to its fabrication, characterization and interaction with osteoblast-like cells, has been investigated in this study to understand the response of human mesenchymal stem cells (hMSC) to such surface. PLLA films and PLLA films with apatite coating were compared with PLLA films with apatite/collagen composite coating. The hMSC morphology in response to such conditions was first observed using fluorescence microscopy. To further understand such cell-material interactions at a molecular level, integrin expression, actin assembly and vinculin-positive focal adhesion plaques were examined. Our results demonstrated that spreading of stem cells on the apatite/collagen composite surface was determined best among the three types of surfaces, followed by the apatite surface and then the PLLA control. Integrin expression on the apatite/collagen surface was higher than those on the apatite surface and PLLA surface. Immunostaining for vinculin and actin suggested that the composite coating on PLLA enhanced the formation of focal adhesion.

[Expression profile of long non-coding RNA in gingival tissues from the patients with aggressive periodontitis].

Objective: To detect and analyze the differential expression profile of long non-coding RNA (lncRNA) in aggressive periodontitis (AgP) and healthy gingival tissues, in order to explore the role of lncRNA in AgP. Methods: After the informed consents were obtained, gingival tissues from AgP patients (n=40) and healthy volunteers (n=40) were collected in Department of Periodontology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (from Mar. 2012 to Aug. 2012) and Department of Periodontology, Hospital of Stomatology, Tianjin Medical University (from Oct. 2016 to Apr. 2017). The differential expression of lncRNA of tissues from AgP patients (n=20) and healthy volunteers (n=20) were examined via microarray assay. Bioinformatics was applied to analyze the expression data of lncRNA and correlative mRNA. Two lncRNAs (lncRNA-TNFRSF13C and lncRNA-API5) were chosen to verify the microarray results by using real time quantitative polymerase chain reaction (PCR) in the other gingival tissues. Results: Compared with the result of healthy gingival tissues, totally 8 632 lncRNAs were differentially expressed in tissues from AgP patients. From these data, 1 986 lncRNAs were significantly upregulated while 6 646 lncRNAs were downregulated, amongst which 48 lncRNAs were upregulated (>10 times) (P<0.05), 14 lncRNAs were downregulated (>10 times) (P<0.05). Furthermore, totally 5 519 correlative mRNAs were differentially expressed, amongst which 1 676 mRNAs were upregulated (≥2 times, P<0.05) and 3 843 mRNAs were downregulated≤0.5 (P<0.05). The selected lncRNA-TNFRSF13C and lncRNA-API5 were up-regulated in AgP (P<0.05), which confirmed the results of microarray. From bioinformatics, differential expression lncRNAs were in association with many signal pathways including toll-like receptor signaling pathway, cell cycle and apoptosis pathway, and tumor necrosis factor receptor superfamily pathway. Conclusions: LncRNA may be involved in the pathogenesis of AgP through various pathways, which need to be further explored.

Expression and pathological relevance of inducible nitric oxide synthase in osteosarcoma of the jaws.

The aim of the present study was to examine the expression of inducible nitric oxide synthase (iNOS) in osteosarcoma of the jaw, and its relationship with tumour angiogenesis and clinicopathological characteristics. Streptavidin peroxidase immunohistochemical staining was used to detect the expression level of iNOS and CD34 in paraffin-embedded samples from 25 patients. Osteosarcoma of the jaw was associated with overexpression of iNOS, which correlated with tumour microvessel density (MVD). iNOS expression correlated with the size, pathological grade and clinical stage of the osteosarcoma, and also with clinicopathological characteristics such as primary occurrence or recurrence of tumours. There was no correlation with metastasis. iNOS may promote tumour angiogenesis in osteosarcoma of the jaw, and so may represent an important target in anti-tumour therapy.

Mandibular reconstruction with the vascularized fibula flap: comparison of virtual planning surgery and conventional surgery.

This study evaluated the accuracy of mandibular reconstruction and assessed clinical outcomes in both virtual planning and conventional surgery patients. ProPlan CMF surgical planning software was used preoperatively in the virtual planning group. In the virtual planning group, fibula flaps were harvested and osteotomized, and the mandibles were resected and reconstructed assisted by the prefabricated cutting guides and templates. The main outcome measures included the operative time, postoperative computed tomography (CT) scans, facial appearance, and occlusal function. The ischemia time and total operation time were shorter in the virtual planning group than in the conventional surgery group. High precision with the use of the cutting guides and templates was found for both the fibula and mandible, and a good fit was noted among the pre-bent plate, mandible, and fibula segments in the virtual planning group. Postoperative CT scans also showed excellent mandibular contours of the fibula flaps in accordance with virtual plans in the virtual planning group. This study demonstrated that virtual surgical planning was able to achieve more accurate mandibular reconstruction than conventional surgery. The use of prefabricated cutting guides and plates makes fibula flap moulding and placement easier, minimizes the operating time, and improves clinical outcomes.

Enhancement of the mitogenic effect by artificial juxtacrine stimulation using immobilized EGF.

Mouse epidermal growth factor (EGF) was covalently conjugated with the water-soluble polymer, poly(acrylic acid) (EGF-PAA), or with the water-insoluble polymer, surface-hydrolyzed poly(methyl methacrylate) (EGF-PMMA). Immobilized EGF (EGF-PMMA) stimulated DNA synthesis in Chinese hamster ovary cells overexpressing EGF receptors in amounts that were 5 to 10% of those of free EGF required for comparable effects. In addition, the maximal mitogenic effect of EGF-PMMA was greater than that of unconjugated EGF or EGF-PAA. EGF, EGF-PAA, and EGF-PMMA induced the autophosphorylation of EGF receptors and the stimulation of mitogen-activated protein kinase. However, whereas the onset of these effects was delayed with EGF-PMMA, they persisted for much longer than those of EGF and EGF-PAA. Unlike EGF and EGF-PAA, EGF-PMMA was not associated with cells after their removal from culture and did not induce receptor internalization. Culturing cells with PMMA-immobilized EGF thus represents a model system for studying "juxtacrine" stimulation of cells by membrane-bound growth factors.

Monosynaptic connections between neurons of trigeminal mesencephalic nucleus and jaw-closing motoneurons in the rat: an intracellular horseradish peroxidase labelling study.

In order to confirm the monosynaptic connections of muscle spindle-mediated jaw stretch reflexes, 8 neurons of trigeminal mesencephalic nucleus innervating masseteric muscle spindles were identified electrophysiologically and stained intracellularly with horseradish peroxidase. These axon terminals projected to ipsilateral dorsal and dorsolateral divisions of trigeminal motor nucleus and extensive premotor areas. Under electron microscope, labeled terminals made monosynaptic contacts predominantly with dendrites in the jaw-closing motoneuron pools. One labeled and many non-labeled terminals were frequently observed to converge simultaneously on one dendrite in the area. However, it was of particular interest that 28% of the labeled terminals constituted the intermediate component of axo-axodendritic synaptic triads. The present study confirmed, for the first time, monosynaptic connections between jaw-closing muscle spindle afferents and jaw-closing motoneurons. These findings also provided ultrastructural evidence for the monosynaptic excitation of muscle spindle-mediated jaw stretch reflexes which received presynaptic and postsynaptic inhibitions of the premotor neurons from other sources.

A quality assurance phantom for IMRT dose verification.

This paper investigates a quality assurance (QA) phantom specially designed to verify the accuracy of dose distributions and monitor units (MU) calculated by clinical treatment planning optimization systems and by the Monte Carlo method for intensity-modulated radiotherapy (IMRT). The QA phantom is a PMMA cylinder of 30 cm diameter and 40 cm length with various bone and lung inserts. A procedure (and formalism) has been developed to measure the absolute dose to water in the PMMA phantom. Another cylindrical phantom of the same dimensions, but made of water, was used to confirm the results obtained with the PMMA phantom. The PMMA phantom was irradiated by 4, 6 and 15 MV photon beams and the dose was measured using an ionization chamber and compared to the results calculated by a commercial inverse planning system (CORVUS, NOMOS, Sewickley, PA) and by the Monte Carlo method. The results show that the dose distributions calculated by both CORVUS and Monte Carlo agreed to within 2% of dose maximum with measured results in the uniform PMMA phantom for both open and intensity-modulated fields. Similar agreement was obtained between Monte Carlo calculations and measured results with the bone and lung heterogeneity inside the PMMA phantom while the CORVUS results were 4% different. The QA phantom has been integrated as a routine QA procedure for the patient's IMRT dose verification at Stanford since 1999.

The inhibitory effect of Chinese tea and its polyphenols on in vitro and in vivo N-nitrosation.

The objective of this study is to evaluate the possible role of Chinese tea as a natural inhibitor of N-nitrosation, and to compare the relative inhibitory potency of various kinds of Chinese tea in vitro and in vivo. Studies on the inhibitory effect of 145 samples of Chinese tea on the formation of N-nitrosomorpholine (NMOR) showed that the inhibitory potency of the 7 types of Chinese tea differed greatly, the average blocking rate of green tea (89.04%, n = 60), crush, tear, and curl (CTC) black tea (86.99%, n = 13), brick tea (85.40%, n = 7), jasmine tea (84.99%, n = 21), Oolong tea (82.37%, n = 9), sun-dried tea (61.99%, n = 13) and Chinese Gongfu black tea (54.98%, n = 22), were positively correlated with their polyphenols contents. The inhibitory effect of green tea and black tea on endogenous N-nitrosation was also confirmed in 3 human volunteer experiments. Drinking tea after meal had a greater effect than drinking it before meal. It is concluded that Chinese tea can exert a relatively strong inhibitory potency for N-Nitroso compounds (NOC) formation both in vitro and in humans, and the active constituents may be related to their polyphenols contents, especially the tea catechin derivatives. The results also demonstrated that the amounts of NMOR formed in vitro depended on the molecular structure of tea catechin derivatives and their molar ratios to nitrite.

[Cholangioplasty by using a patch of bovine pericardium in the treatment of stricture of extrahepatic bile duct].

From Sept. 1989 to Jan. 1994, processed fresh bovine pericardium was used for the repairment of stricture of extrahepatic bile duct in 6 cases (7 times). All patients have been followed up for 8 approximately 52 months (average 24 months). One case reoperated on for recurrent jaundice on 26th month postoperation. All cases are alive and in good health, without any clinical symptom.

Multiarm cationic star polymers by atom transfer radical polymerization from ß-cyclodextrin cores: influence of arm number and length on gene delivery.

Controlled ß-cyclodextrin (ß-CD) core-based cationic star polymers have attracted considerable attention as non-viral gene carriers. Atom transfer radical polymerization (ATRP) could be readily used to produce the star-shaped polymers. The precise control of the number of initiation sites on the multifunctional core was of crucial importance to the investigation of the structure-property relationship of the functional star gene carriers. Herein, the controlled multiarm star polymers consisting of a ß-CD core and various arm lengths of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) were prepared via ATRP from the chloroacetylated ß-CD with well-designed initiation sites. Generally, these star polycations can condense plasmid DNA into 100-150 nm nanoparticles with positive zeta potentials of 30-40 mV at N/P ratios (star polymer to DNA ratios) of 17 or higher. The effects of arm numbers and lengths on gene delivery were investigated in detail. With a fixed length of the PDMAEMA arm, the fewer the number of arms, the lower the toxicity. The star polycations with suitable arm numbers possess the best transfection ability. On the other hand, with the fixed molecular weights, the shorter the arms, the lower the toxicity. The polymers with 21 arms possess the lowest transfection efficiency.

Anti-fouling surfaces by combined molecular self-assembly and surface-initiated ATRP for micropatterning active proteins.

A simple method by combined molecular self assembly and surface-initiated atom transfer radical polymerization (SI-ATRP) was proposed to prepare a biologically inert surface for micropatterning active proteins. The MPEG microdomains having a short terminal poly(ethylene glycol) (PEG) unit were prepared by self assembly of 2-(methyoxy(polyethylenoxy) propyl)trimethoxy silane (MPEG-silane). The remaining local regions or poly(poly(ethylene glycol)methyl ether methacrylate-co-glycidyl methacrylate) (P(PEGMEMA-co-GMA)) microdomains were produced via SI-ATRP of PEGMEMA and GMA comonomers. The epoxy groups of the P(PEGMEMA-co-GMA) microdomains were used directly for covalent coupling of an active protein (human immunoglobulin or IgG) via the ring-opening reaction to produce the IgG-coupled microdomains. The IgG-coupled microdomains interact only and specifically with target anti-IgG, while the other antifouling microregions from self-assembled monolayers with short terminal PEG units effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.

In vitro behavior of osteoblast-like cells on PLLA films with a biomimetic apatite or apatite/collagen composite coating.

To investigate the methods to improve the cell-material interaction of devices or tissue engineering scaffolds made of poly(L-lactic acid) (PLLA) polymer, apatite and apatite/collagen composite coatings were formed on PLLA films within 24 h through accelerated biomimetic processes. In vitro investigation using Saos-2 osteoblast-like cells through cell culture was conducted to assess the biological performance of these biomimetic coatings. The cell morphology on three types of surfaces, viz., PLLA film, PLLA film with the apatite coating, and PLLA film with the apatite/collagen composite coating, was studied using scanning electron microscopy (SEM). Cell viability was estimated using the MTT assay. The differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. The results obtained indicated that the biomimetic apatite and apatite/collagen composite coatings could significantly enhance the proliferation and differentiation of osteoblast-like cells. The apatite/collagen composite coating appears to be promising for the surface modification of PLLA-based devices with much improved interactions with osteoblastic cells.

Enhancement of artificial juxtacrine stimulation of insulin by co-immobilization with adhesion factors.

Insulin was co-immobilized with a cell adhesion factor--fibronectin or polyallylamine--on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. While insulin immobilization did not affect cell adhesion, insulin immobilized on fibronectin-immobilized film reduced the adhesion. Addition of the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited cell adhesion onto fibronectin-immobilized films while cell adhesion onto polyallylamine-immobilized films was not inhibited by RGDS. Small amounts of immobilized insulin (1 to 10% of the amount of free insulin required to achieve cell growth acceleration) were sufficient to stimulate cell proliferation. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. In addition, co-immobilization with the adhesion factor remarkably enhanced the mitogenic effect. The phosphorylation of the receptor with free insulin attained the maximum degree very rapidly but ceased quickly. On the other hand, the receptor phosphorylation with immobilized insulin was accompanied by a longer induction period and lasted a longer period of time than that with free insulin. Insulin co-immobilization on fibronectin or polyallylamineimmobilized films reduced the induction period by enhancement of cell adhesion. The early and long-lasting receptor activation might have been caused by the greater mitogenic effect of co-immobilized biosignaling polypeptides.