Objective To investigate the behavior of rat calvarial osteoblasts cultured on chitosan-gelatin/hydroxyapatite (CSGel/HA) composite scaffolds. Methods The rat calvarial osteoblasts (the 3rd passage) were seeded at a density of 1.01×106 cells/ml onto the CS-Gel/HA composite scaffolds having porosity 85.20%, 90.40% and 95.80%. Cell number was counted after cultured for 3 days,1 week, 2 weeks and 3 weeks. Cell proliferation, bone-like tissue formation, and mineralization were separately detected by HE, von Kossa histological stainingtechniques. Results The CS-Gel/HA composite scaffolds supported the attachmentof seeded rat calvarial osteoblasts. Cells proliferated faster in scaffold withhigher porosity 90.40% and 95.80% than scaffold with lower porosity 85.20%. The osteoblasts/scaffold constructs were feasible for mineral deposition, and bonelike tissue formation in 3 weeks. Conclusion This study suggests the feasibility of using CS-Gel/HA composite scaffolds for bone 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.
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 study the function of the composite of bone matrix gelatin(BMG) and plaster in the repairing process of bone defects. METHODS Sixteen New Zealand rabbits which were defected in corpus radii were made as implant zone of bone. Sixteen sides of radii were implanted with the composite of BMG and plaster as experimental group. Others were implanted with BMG(8 sides) and bone stored in alcohol(8 sides) as control groups. The repairing process in bone defects were observed by X-ray and histological examination. RESULTS There was an obvious osteogenesis in experimental group. The defects of radii were almost healed at 12th week after operation. There were osteogenesis in both control groups, but the repairing process was slower than that of the experimental group. CONCLUSION The composite of BMG and plaster is a good material for bone transplantation.
Objective To investigate the early diagnostic value of urinary neutrophil gelatinase-associated lipocalin (NGAL) for acute kidney injury (AKI) after acute Stanford type A aortic dissection. Methods From January 2018 to December 2018, the clinical data of 50 patients who underwent open surgery for acute Stanford type A aortic dissection were analyzed in Nanjing First Hospital. Urine specimens were collected before and 2 hours after the aortic dissection surgery. Patients were divided into an AKI group (n=27) and a non-AKI group (n=23) according to the Kidney Disease Improving Global Outcomes criteria. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of urine NGAL. ResultsThe incidence of postoperative AKI was 54.00% (27/50). There was a statistically significant difference between the two groups in serum creatinine concentration at 2 hours after surgery and urinary NGAL concentration before the surgery (P<0.05). The area under ROC curve of preoperative urinary NGAL concentration was 0.626. When cut-off value was 43 ng/mL, the sensitivity was 40.7%, specificity was 95.7%. The area under ROC curve of urinary NGAL concentration at 2 hours after surgery was 0.655, and when the cut-off value was 46.95 ng/mL, the sensitivity was 63.0%, specificity was 78.3%. Conclusion Urine NGAL can predict postoperative AKI in patients with acute Stanford type A aortic dissection, but its value is limited.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
Objective To investigate the effect of a porous calcium phosphate/bone matrix gelatin (BMG) composite cement (hereinafter referred to as the " porous composite cement”) for repairing lumbar vertebral bone defect in a rabbit model. Methods BMG was extracted from adult New Zealand rabbits according to the Urist’s method. Poly (lactic-co-glycolic) acid (PLGA) microsphere was prepared by W/O/W double emulsion method. The porous composite cement was developed by using calcium phosphate cement (CPC) composited with BMG and PLGA microsphere. The physicochemical characterizations of the porous composite cement were assessed by anti-washout property, porosity, and biomechanical experiment, also compared with the CPC. Thirty 2-month-old New Zealand rabbits were used to construct vertebral bone defect at L3 in size of 4 mm×3 mm×3 mm. Then, the bone defect was repaired with porous composite cement (experimental group, n=15) or CPC (control group, n=15). At 4, 8, and 12 weeks after implantation, each bone specimen was assessed by X-ray films for bone fusion, micro-CT for bone mineral density (BMD), bone volume fraction (BVF), trabecular thickness (Tb. Th.), trabecular number (Tb.N.), and trabecular spacing (Tb. Sp.), and histological section with toluidine blue staining for new-born bone formation. Results The study demonstrated well anti-washout property in 2 groups. The porous composite cement has 55.06%±1.18% of porosity and (51.63±6.73) MPa of compressive strength. The CPC has 49.38%±1.75% of porosity and (63.34±3.27) MPa of compressive strength. There were significant differences in porosity and compressive strength between different cements (t=4.254, P=0.006; t=2.476, P=0.034). X-ray films revealed that the zone between the cement and host bone gradually blurred with the time extending. At 12 weeks after implantation, the zone was disappeared in the experimental group, but clear in the control group. There were significant differences in BMD, BVF, Tb. Th., Tb. N., and Tb. Sp. between 2 groups at each time point (P<0.05). Histological observation revealed that there was new-born bone in the cement with the time extending in 2 groups. Among them, bony connection was observed between the new-born bone and the host in the experimental group, which was prior to the control group. Conclusion The porous composite cement has dual bioactivity of osteoinductivity and osteoconductivity, which are effective to promote bone defect healing and reconstruction.
OBJECTIVE: To explore the possibility of repair long segmental bone defects and preventing infection with cefazolin loaded bone matrix gelatin (C-BMG). METHODS: C-BMG was made from putting cefazolin into BMG by vacuum adsorption and freeze-drying techniques. The sustaining period of effective drug concentration in vitro and in vivo was detected by inhabition bacteria, and the drug concentration in local tissues (bone and muscle) and plasma after implantation of C-BMG was examined by high performance liquid chromatography(HPLC). RESULTS: The effective inhibition time to staphylococcus aureus of C-BMG was 22 days in vitro, while 14 days in vivo. The drug concentration in local tissues(bone and muscle) were higher than that of plasma, and the drug concentration in local tissues was higher in early stage, later it kept stable low drug release. It suggested that C-BMG had excellent ability to repair segmental long bone defects. CONCLUSION: C-BMG can gradually release cefazolin with effective drug concentration and has excellent ability to repair segmental long bone defects. It may be a novel method to repair segmental long bone defects and prevent infection after the operation.
ObjectiveTo investigate the diagnostic value of serum neutrophil gelatinase-associated lipocalin (NGAL) for early acute kidney injury (AKI) after tetralogy of Fallot (TOF) surgery. MethodsWe retropectively analyzed the clinical data of 113 patients underwent TOF surgery in our hospital bewteen April 2012 and April 2014. There were 67 males and 46 females at the average age of 8.28±4.75 months ranging from 5 months to 18 months. According to the different clinical manifestation of AKI, those patients were devided into a group A, group B, and group C. In the group A, there were 78 patients with 43 males and 35 females at the mean age of 8.18±3.72 months. In the group B, there were 20 patients with 12 males and 8 females at the mean age of 8.25±1.27 months. In the group C, there were 15 patients with 12 males and 3 females at the mean age of 8.09±2.92 months. We collected the blood in different time before and after the operation. At the same time, we carried on one-way analysis of variance to detect the differences among the three groups. ResultsThere was no statistical difference in the level of serum NGAL among the 3 groups before operation. Compared to pre-operation, there was no statistical difference in the level of serum NGAL among the different time of the group A (P>0.05). There was oliguria and potassium increased in the group B. After strengthening cardiac and lightening heart load, urine volume recovered. There was a transient rise in serum NGAL and the summit is 199.90±49.44 ng/ml at the 8th hour. Compared with that before operation, there was a statistical difference. After 12 hours, the serum NGAL decreased to the normal level. The serum NGAL levle of Group C had constantly increased and there was a statistical difference compared with that before the surgery. After the treatment of peritoneal dialysis, the serum NGAL returned to the normal level. The area under receiver operating characteristic (ROC) curve of serum NGAL in the group C was 0.881 (95%CI:0.73-1.00, P<0.05). ConclusionThe detection of serum NGAL level can be valuable for early diagnosis and treatment for AKI after TOF surgery.
Objective To fabricate a novel porous bioactivecomposite biomaterial consisting of poly lactic acid (PLA)bone matrix gelatin(BMG) by using the supercritical carbon dioxide fluid technique (SC-CO2) and to evaluate its osteoinductive activity. Methods The cortical bones selected from healthy adult donors were processed into BMG by the defatting, demineralizing, and deproteinizing processes. PLA and BMG were mixed at a volume radio of 3∶1; then, the PLA-BMG mixed material and the pure PLA material were respectively placed in the supercritical carbon dioxide reaction kettles, and were respectively added by the NaCl particles 100200 μm in diameter for theporosity of the materials so that the porous PLA-BMG composite material and the porous PLA composite material could be formed. The mouse osteoblastlike MC3T3-E1 cells were cultured in the dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum. Then, 20 μl of the MC3T3E1 cell suspensions containing 2 ×106 cells /ml were delivered into the culturing plate (24 wells/plate) made of the different materials, which were co-cultured for 2 weeks. In the PLA-BMG group, 100 μg of the crushed PLA-BMG material was contained in each well; in the PLA group, 100 μg of the crushed PLA material was containedin each well; and in the DMEM group, only DMEM was contained, which served as the control group. There were 6 wells in each group. The quantitative analysis onthe calcification area was performed by the staining of the alizarin red S. Theco-cultured cells were harvested and lysated in 1 ml of 0.2% Nonidet P-40 by the ultrasonic lysating technique. Then, the ALP activity and the Ca content were measured according to the illuminations of the reagent kits. Results The porous PLABMG composite material showed a good homological porosity with a pore diameter of 50-150 μm and a good connectivity between the pores. The ALP activity, the Ca content, and the calcification area were significantly greater in the PLABMG group than in the PLA group and the control group (325.59±70.40 U/gprot, 3.51±1.64 mmol/gprot, 42.98±4.44% vs. 63.62±30.01 U/gprot, 1.04±0.21 mmol/gprot, 9.55±1.94%, and 2.40±1.47 U/gprot, 0.70±0.24 mmol/gprot, 0.86±0.41%; Plt;0.05). Meanwhile, there was a statistically significant difference between the PLA group and the control group in the ALP activity and the calcification area (Plt;0.05). Conclusion The porous PLABMG composite material prepared by the use of SC-CO2 has a good steoinductive activity and can be used as a promising bone biomaterial and a bone tissue engineered scaffold.