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 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.
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.
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.
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.
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.
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 investigate the effect of rhBMP-2 combined with porous CPC on spine fusion in rabbits. Methods rhBMP-2 (1 mg) was loaded with 1 g CPC and 6.0 cm × 2.0 cm × 0.5 cm absorbable gelatin sponge (AGS), respectively, and thereafter frozen to prepare the biomaterial of rhBMP-2/CPC and rhBMP-2/AGS. Forty-five 24-week-old New Zealand rabbits (weight 2.5-3.5 kg) were randomly divided into 3 groups: group A (n=17), group B (n=11) and group C (n=17).With the exposure and removal of L5, 6 transverse process’s posterior bone cortex in all the rabbits, the corresponding cancellous bones were exposed and the posterior bilateral intertransverse bone grafting of L5, 6 were performed on the three groups, then the rhBMP-2/CPC, rhBMP-2/AGS and CPC was implanted into the rabbits of group A, B and C, respectively. Gross observation, histology assay and image examination were conducted 4, 8 and 24 weeks after operation. Results Decalcified hard tissue section demonstrated obvious callus connections in group A, small pieces of callus in group B, and fibrous connection and few cartilage in group C at 4 and 8 weeks after operation. By Kacena measurement standard, the score of group A, B and C at 4 weeks after operation was (7.30 ± 0.76), (3.68 ± 1.60) and (1.75 ± 0.54) points, respectively, and their score at 8 weeks after operation were (8.32 ± 1.11), (3.75 ± 1.23) and (1.47 ± 0.23) points, respectively, indicating there were significant differences between group A and group B as well as between group A and group C at different time points (P lt; 0.05). Undecalcified hard tissue section demonstrated that there was cancellous bone-l ike tissue regeneration in group A, and fiber connection around the implants and l ittle ossification in group C at 4 and 8 weeks after operation. By three dimensions reconstructed CT, group A, B and C scored (2.50 ± 0.57), (1.00 ± 0.00) and (1.00 ± 0.00) points respectively, indicating there was a significant difference between group C and groups A and B as well as between group A and group B (P lt; 0.05). Conclusion As a carrier of rhBMP-2, the CPC is capable of promoting spine bone fusion in rabbits and is a new type of artificial bone repair material.
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 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.