ObjectiveTo explore the correlation of serum neutrophil gelatinase-associated lipocalin (sNGAL) with inflammatory response in patients with community-acquired pneumonia (CAP) and assess the diagnostic value of sNGAL for severe CAP (SCAP).MethodsFrom January 2018 to June 2019, a total of 85 patients with CAP were enrolled in this study. Age, length of hospital stay, the levels of serum creatinine, blood urea nitrogen, white blood cell count,C-reactive protein (CRP), interleukin-6 (IL-6), and procalcitonin, and CURB-65 score were compared between patients with SCAP (n=34) and patients without SCAP (n=51). The correlations of sNGAL with serum creatinine, blood urea nitrogen, white blood cell count, CRP, IL-6, procalcitonin, and CURB-65 score were assessed with Spearman’s correlation analysis. The area under the receiver operating characteristic (ROC) curve for sNGAL diagnosing SCAP was examined. ResultsCompared with patients without SCAP, SCAP patients demonstrated older age, longer hospital stay, higher serum CRP and IL-6 concentritions, and higher CURB-65 score (P<0.05). The Spearman’s correlation test showed that sNGAL was positively correlated with serum CRP, IL-6, PCT and CURB-65 score (rs=0.472, 0.504, 0.388, and 0.405, respectively; P<0.01). According to ROC analysis, the area under curve of sNGAL for diagnosing SCAP were 0.816, with a sensitivity of 76.56% and a specificity of 74.4% when the cut-off value was 171.0 ng/mL.ConclusionssNGAL concentration is positively correlated with the serverity of CAP. It can be regarded as a reliable indicator for diagnosis of SCAP in patients with CAP.
Objective To introduce an injectable andin situ gelling gelatin hydrogel, and to explore the possibility as a carrier for demineralized bone matrix (DBM) powder delivery. Methods First, thiolated gelatin was prepared and the thiol content was determined by Ellman method, and then the injectable andin situ gelling gelatin hydrogel (Gel) was formed by crosslinking of the thiolated gelatin and poly (ethylene oxide) diacrylate and the gelation time was determined by inverted method. Finally, the DBM-Gel composite was prepared by mixing Gel and DBM powder. The cytotoxicity was tested by live/dead staining and Alamar blue assay of the encapsulated cells in the DBM-Gel. Forin vitro cell induction, C2C12 cells were firstly incubated onto the surface of the DBM and then the composite was prepared. The experiment included two groups: DBM-Gel and DBM. The alkaline phosphatase (ALP) activity was determined at 1, 3, 5,and 7 days after culture.In vivo osteoinductivity was evaluated using ectopic bone formation model of nude rats. Histological observation and the ALP activity was measured in DBM-Gel and DBM groups at 4 weeks after implantation. Results The thiol content in the thiolated gelatin was (0.51±0.03) mmol/g determined by Ellman method. The gelation time of the hydrogel was (6±1) minutes. DBM powder can be mixed with the hydrogel and injected into the implantation site within the gelation time. The cells in the DBM-Gel exhibited spreading morphology and connected each other in part with increasing culture time. The viability of the cells was 95.4%±1.9%, 97.3%±1.3%, and 96.1%±1.6% at 1, 3, and 7 days after culture, respectively. The relative proliferation was 1.0±0.0, 1.1±0.1, 1.5±0.1, and 1.6±0.1 at 1, 3, 5, and 7 days after culture respectively.In vitro induction showed that the ALP activity of the DBM-Gel group was similar to that of the DBM group, showing no significant difference (P>0.05). With increasing culture time, the ALP activities in both groups increased gradually and the activity at 5 and 7 days was significantly higher than that at 1 and 3 days (P<0.05), while there was no significant difference between at 1 and 3 days, and between 5 and 7 days (P>0.05). At 4 weeks after implantationin vivo, new bone and cartilage were observed, but no bone marrow formation in DBM-Gel group; in DBM group, new bone, new cartilage, and bone marrow formation were observed. The histological osteoinduction scores of DBM-Gel and DBM groups were 4.0 and 4.5, respectively. The ALP activities of DBM-Gel and DBM groups were respectively (119.4±22.7) and (146.7±13.0) μmol/mg protein/min, showing no significant difference (t=–2.085,P=0.082). Conclusion The injectable andin situ gelling gelatin hydrogel for delivery of DBM is feasible.
Objective To develop a diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity and provide a new avenue for alleviating the inflammatory response and enhancing cartilage regeneration in vivo. Methods Diclofenac sodium was homogeneously mixed with gelatin to prepare a diclofenac sodium-loaded porous gelatin scaffold by freeze-drying method as the experimental group, and a pristine porous gelatin scaffold was served as a control group. The general morphology of the scaffold was observed, the pore size of the scaffold was measured by scanning electron microscopy, the porosity of the scaffold was calculated by drainage method, the loading of diclofenac sodium into the gelatin scaffold was detected by fourier transform infrared spectrometer and X-ray diffraction examinations, and the release kinetics of diclofenac sodium from gelatin scaffold was tested using an in vitro release assay. The two scaffolds were co-cultured with lipopolysaccharide-predisposed RAW264.7 in vitro, and the expressions of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immuno sorbent assay, and Western blot, to detect the in vitro anti-inflammatory effect of the drug-loaded scaffold. Thereafter, the second generation chondrocytes of New Zealand white rabbits were inoculated on the two groups of scaffolds for in vitro culture, and the cytocompatibility of the scaffold was tested by live/dead staining and cell counting kit 8 assay, the feasibility of in vitro cartilage regeneration of the scaffold was evaluated via gross observation, HE staining, Safranin-O staining, and immunohistochemical collagen type Ⅱ staining, as well as biochemical quantitative analyses. Finally, the two groups of chondrocyte-scaffolds were implanted subcutaneously into New Zealand white rabbits, and after 4 weeks, the general observation, HE staining, safranin O staining, immunohistochemical collagen type Ⅱ staining, and biochemical quantitative analyses were performed to verify the cartilage regeneration in vivo, and the expression of inflammation-related genes CD3 and CD68 was detected by RT-PCR to comprehensively evaluate the anti-inflammatory performance of the scaffolds in vivo. Results The two scaffolds exhibited similar gross, microporous structure, pore size, and porosity, showing no significant difference (P>0.05). Diclofenac sodium was successfully loaded into gelatin scaffold. Data from in vitro anti-inflammatory assay suggested that diclofenac sodium-loaded gelatin scaffold showed alleviated gene and protein expressions of IL-1β and TNF-α when compared with gelatin scaffold (P<0.05). The evaluation of cartilage regeneration in vitro showed that the number of living cells increased significantly with the extension of culture time, and there was no significant difference between the two groups at each time point (P>0.05). White cartilage-like tissue was regenerated from the scaffolds in both groups, histological observation showed typical cartilage lacuna structure and specific cartilage extracellular matrix secretion. There was no significant difference in the content of cartilage-specific glycosaminoglycan (GAG) and collagen type Ⅱ between the two groups (P>0.05). In vivo experiments showed that the samples in the experimental group had porcelain white cartilage like morphology, histologic staining showed obvious cartilage lacuna structure and cartilage specific extracellular matrix, the contents of GAG and collagen type Ⅱ were significantly higher than those in the control group, and the protein and mRNA expressions of CD3 and CD68 were significantly lower than those in the control group, with significant differences (P<0.05). ConclusionThe diclofenac sodium-loaded gelatin scaffold presents suitable pore size, porosity, and cytocompatibility, as well as exhibited satisfactory anti-inflammatory ability, providing a reliable scheme for alleviating the inflammatory reaction of regenerated cartilage tissue after in vivo implantation and promoting cartilage regeneration in vivo.
ObjectiveTo study the expression of lipid associated with neutrophil gelatinase associated lipocalin (NGAL) in nude mice orthotopic pancreatic cancer tissues and the relationship between the occurred and development of pancreatic cancer.
MethodsThe expressions of NGAL mRNA and protein of pancreatic cancer tissues and their adjacent tissues, and normal pancreatic tissues in nude mice were detected by using RT-PCR and immunohistochemical methods.
ResultsThe expressions of NGAL mRNA in pancreatic cancer tissues and adjacent tissues were significantly higher than that in normal pancreatic tissues (P < 0.05), and the expression of NGAL mRNA in pancreatic carcinoma tissues was significantly higher than that in para carcinoma tissues (P < 0.05). The strong positive expression rate of NGAL protein in pancreatic carcinoma tissues was significantly higher than thoes in para carcinoma tissues and normal pancreatic tissues (P < 0.05).
ConclusionsNGAL is highly expressed in pancreatic cancer tissues, and NGAL may be an important regulatory factor in the development of pancreatic cancer.
Objective To investigate the biocompatibility of type I collagen scaffold with rat bone marrow mesenchymal stem cell (BMSCs) and its role on proliferation and differentiation of BMSCs so as to explore the feasibility of collagen scaffold as neural tissue engineering scaffold. Methods Type I collagen was used fabricate collagen scaffold. BMSCs were isolated by density gradient centrifugation. The 5th passage cells were used to prepare the collagen scaffold-BMSCs complex. The morphology of collagen scaffold and BMSCs was observed by scanning electron microscope (SEM) and HE staining. The cell proliferation was measured by MTT assay at 1, 3, 5, and 7 days after culturein vitro. After cultured on collagen scaffold for 24 hours, the growth and adhesion of green fluorescent protein positive (GFP+) BMSCs were observed by confocal microscopy and live cell imaging. Results The confocal microscopy and live cell imaging results showed that GFP+ BMSCs uniformly distributed in the collagen scaffold; cells were fusiform shaped, and cell process or junctions between the cells formed in some cells, indicating good cell growth in the collagen scaffold. Collagen scoffold had porous fiber structure under SEM; BMSCs could adhered to the scaffold, with good cell morphology. The absorbance (A) value of BMSCs on collagen scaffold at 5 and 7 days after culture was significantly higher than that of purely-cultured BMSCs (t=4.472,P=0.011;t=4.819,P=0.009). HE staining showed that collagen scaffold presented a homogeneous, light-pink filament like structure under light microscope. BMSCs on the collagen scaffold distributed uniformly at 24 hours; cell displayed various forms, and some cells extended multiple processes at 7 days, showing neuron-like cell morphology. Conclusion Gelatinous collagen scaffold is easy to prepare and has superior biocompatibility. It is a promising scaffold for neural tissue engineering.
Objective To explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freezedrying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at thedensity of 2×104/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semiquantitative RT-PCR with housekeeping gene β-tubulin as internalstandard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected tothe detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition andwas treated with the same as group A. Besides these 2 groups, another 2 rhBMP2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex. ResultsSEM showed that cells attached closely to the complex and grew well. In group A, the expression level(1.28±0.17)of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56±0.09) of the control group A, being statistically -significantly different(P＜0.05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells. Conclusion The complexmade of rhBMP-2 and chitosan-gelatin scaffolds has b osteogenesis ability in vitro.
Abstract To observe the effect of exogenous high molecular weight nerve growth factor (HMW-NGF) mixed with bletilia striata gelatin (BSG) in the promotion of healing, the experiment was performed as follow: (1) In serumfree medium, the normal saline, BSG, HMWNGF, and BSG+HMW-NGF were added separately, and then, the chick embryo root ganglions (DRGs) were cultivated in the above prepared media and the axonal growth was observed. (2) 40 SD rats were divided into 4 groups. A wound of 2cm×2cm was made on the back of every rat. No treatment was given in group one. In other groups, BSG, HMW-NGF, andBSG+HMW-NGF were given separately to the wounds once daily. After 3 and 10 days, the wound area of every rat was measured, cells in the wounds were observed under light microscope and were calculated, and the time of healing was recorded. The results showed that BSG, HMW-NGF, especially BSG+HMW-NGF could promote wound healing.
Objective To investigate the effect of “two-phase” tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells(MSCs) and allogeneic bone matrix gelatin(BMG) in repairing articular cartilage defects. Methods Thirty-twoNew Zealand white rabbits were involved in the experiment. “Two-phase” allogeneic BMG scaffold (one side of porous cancellous bone and the other side of cortical bone; 3 mm both in diameter and in thickness) was prepared from iliac bone and limb bone of 5 rabbits by sequentially chemical method. The MSCs wereseparated from 18 New Zealand white rabbits and induced to express chondrocyticphenotype. The chondrocyte precursor cells were seeded onto “two-phase” allogeneic BMG to construct tissue engineering cartilage. Masson’s trichrome staining, PAS staining and scanning electronic microscopic observation were carried out at 1, 3 and 5 weeks. The defects of full thickness articular cartilage(3 mm both in diameter and in depth) were made at both sides of femoral medial condyles in 27 rabbits(including 18 of separated MSCs and the remaining 9). The defects were repaired with the tissue engineered cartilage at the right side (group A, n=18), with BMG at the left side(group B, n=18), and without any implant at both sides in the remaining 9 rabbits as a control( group C, n=18). After 1, 3 and6 months, the 6 specimens of femoral condyles were harvested in 3 groups, respectively. Gross observation, Masson’s trichrome and Alcian blue staining, modified Wakitani scoring and in situ hybridization of collagen type Ⅱ were carried out to assess the repair efficacy of tissue engineered cartilage. Results The “two-phase” BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part, the pore size ranged 100-800 μm, in which the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage. In cortical part, the pore size ranged 10-40 μm, on which the cells arranged in a layer and formed the hard part of subchondral bone. After 1 month of transplantation, the cartilage and subchondral bone were regenerated in group A; during observation, the regenerated cartilage graduallythinned, but defect was repaired and the structure of the articular surface ansubchondral bone was in integrity. In groups B and C, defects were not repaired, the surrounding cartilage of defect was abrased. According to the modified Wakitani scoring, the indexes in group A were significantly higher than those in group B and C(Plt;0.01) except the thickness of cartilage at 6 months. The positive cell rate of in situ hybridization for collagen type Ⅱ in group A was also higher than those in groups B and C(Plt;0.01). Conclusion “Two-phase” allogeneic BMG is a prospective scaffold for tissue engineered cartilage,which combines with autologous chondrocyte precursor cells induced from MSCs toconstruct the tissue engineering cartilage. The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.
Objective To evaluate the biocompatibil ity of a new nano TCP/ gelatin / velvet antler polypeptide material. Methods The nano TCP/ gelatin / velvet antler polypeptide material was prepared, and the morphous was observed by scanning electron microscope. L929 and NIH/3T3 cell l ines were cultured conventionally. Acute toxicity test, hemolysis test, cell prol iferation and cytotoxicity test were used to evaluate the biocompatibil ity of the material. Results The compositemicrosphere material was about 10 μm in diamerter and had good spherical geometry, high monodispersity with nanometer size holes on the surface. Toxic symptoms such as hyperspasmia, palsy and death did not appear during the observing stage in acute toxicity test. Maximum hemolysis rate of the material was less than 5% which met the requirement of hemolysis test standard as a medical material. Different concentrations of the materials leaching l iquor could enhance the prol iferation of NIH/3T3 cells, which showed the good biologic activity. Toxicity grade was 0, and the material was no cytotoxic. Conclusion Nano TCP/ gelatin / velvet antler polypeptide material has good biocompatibil ity.
The purpose of this study is to investigate the effect of superparamagnetic chitosan FGF-2 gelatin microspheres (SPCFGM) on the proliferation and differentiation of mouse mesenchymal stem cells. The superparamagnetic iron oxide chitosan nanoparticles (SPIOCNs) were synthesized by means of chemical co-precipitation, combined with FGF-2. Then The SPCFGM and superparamagnetic chitosan gelatin microspheres (SPCGM) were prepared by means of crosslinking-emulsion. The properties of SPCFGM and SPIONs were measured by laser diffraction particle size analyser and transmisson electron microscopy. The SPCFGM were measured for drug loading capacity, encapsulation efficiency and release pharmaceutical properties in vitro. The C3H10 cells were grouped according to the different ingredients being added to the culture medium: SPCFGM group, SPCGM group and DMEM as control group. Cell apoptosis was analyzed by DAPI staining. The protein expression level of FGF-2 was determined by Western blot. The proliferation activity and cell cycle phase of C3H10 were examined by CCK8 and flow cytometry. The results demonstrated that both of the SPIOCNs and SPCFGM were exhibited structure of spherical crystallization with a diameter of (25±9) nm and (140±12) μm, respectively. There were no apoptosis cells in the three group cells. Both the protein expression level of FGF-2 and cell proliferation activity increased significantly in the SPCFGM group cells(P<0.05). The SPCFGM is successfully constructed and it can controlled-release FGF-2, remained the biological activity of FGF-2, which can promote proliferation activity of C3H10 cells, and are non-toxic to the cell.
