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 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 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 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 evaluate the biomechanicalproperties and structuralcharacteristics of various composites of partially decalcified allogenic bone matrix gelatin and bone cement at different ratios. Methods According to Urist method, partially decalcified allogenic bone matrix gelatin was prepared and mixedwith bone cement at different ratios of 0, 400, 500, and 600mg/g. Then the comparisons of these composites were performed in microstructure, ultimate compression strength and ultimate bending strength properties. Results The electronic microscope showed that the bone particles and bone cement were distributed evenly in the composite, irregularly connecting by multiple points; with the increase ofbone particles and decrease of bone cement in the composite, there were more and more natural crevices, varying from 100 μm to 400 μm in width, in the biomaterials. Of all the composites with the ratios of 0, 400,500, and 600 mg/g, the measurements of ultimate compression strength were (71.7±2.0) MPa, (46.9±3.3) MPa, (39.8±4.1) MPa, and (32.2±3.4) MPa, respectively; and the measurements ofultimate bending strength were (65.0±3.4) MPa, (38.2±4.0) MPa, (33.1±4.3) MPa and (25.3±4.6) MPa, respectively. Conclusion The compositeof partially decalcified allogenic bone matrix gelatin and bone cement has a good biomechanical property and could be easily fabricated and re-shaped, which make it available to be used clinically as an idea bone graft biomaterial.
OBJECTIVE: To prepare chitosan-gelatin/hydroxyapatite (CS-Gel/HA) composite scaffolds, and to investigate the influence of components and preparing conditions to their micromorphology. METHODS: The CS-Gel/HA composite scaffolds were prepared by phase-separation method. Micromorphology and porosity were detected by using scanning electron microscope and liquid displacement method respectively. RESULTS: Porous CS-Gel/HA composite scaffolds could be prepared by phase-separation method, and their density and porosity could be controlled by adjusting components and quenching temperature. CONCLUSION: The study suggests the feasibility of using CS-Gel/HA composite scaffolds for the transplantation of autogenous osteoblasts to regenerate bone tissue.
Although the recent studies have concerned the pathogenesis and therapeutic strategies of acute kidney injury (AKI), the mortality of AKI is still terribly high, and it is still one of the most important death factors in the intensive care unit. There is no doubt that early verdict of AKI, is good for a more aggressive treatment and can promise an improved prognosis for AKI patients. Serum creatinine level, serving as the gold standard for diagnosis of kidney injury, cannot meet current clinical work in its sensitivity and specificity of diagnosis of early AKI. Over the past decades, researchers worked to find and verify novel AKI biomarkers, including neutrophil gelatinase associated lipocalin, interleukin-18, kidney injury molecule-1 and cystatin-C, which were proved to be the potential reliable predictor of AKI development and prognosis, and were of great importance to the early diagnosis and clinical monitoring of AKI. This paper reviews the main studies on these novel prognostic predictors of AKI over the decades and evaluates their roles and limitations in early diagnosis and clinical prognosis prediction.
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 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.