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Objective To investigate the curative effect and safety of haploidentical allogeneic cytokine-induced killer (CIK)in treatment of advanced hepatocellular carcinoma.Methods The peripheral blood mononuclear cell (PBMC) of the healthy immediate family members of 21 patients with advanced hepatocellular carcinoma (HCC) were collected,induced into haploidentical allogeneic CIK in vitro and transfused to the patients for 4 cycles.The curative effect and safety were assessed.Results The 21 patients were followed up for half a year.The survival rate was 81 % (1 7 /21 ).Among the 21 patients,1 1 cases were with stable disease and 1 0 cases were with progressive disease (including 4 dead cases).Six patients developed fever of different degrees during the treatment and one patient developed rash.The platelet counts of the patients at the fourth cycle after the treatment decreased compared with that before the treatment ,with significance difference (P <0.05).The difference in leukocytes,neutrophils,lymphocytes,hemoglobin,liver function and renal function at the first and fourth cycle after the treatment had no statistical significance (all in P >0.05 ).Conclusions Haploidentical allogeneic CIK in treatment of advanced HCC may effectively improve the quality of life and the adverse reactions are tolerable,which is a relatively safe therapy.
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Objective To evaluate the clinical outcomes of a hovel porous β-tricalcium phosphate (β-TCP)and control allograft for the repair of lacunar bone defects caused by solitary bone cyst curettage.Methods From January 2003 to December 2008,the patients with solitary bone cyst were randomized into an experimental(55 cases)and a control(40 cases)group.The control group received particulate allograft bone as the graft material,and the experimental group received β-TCP.At 1 week,1,2,3,6,12,24,48months after surgery,a new radiographic scoring system was employed to calculate the biodegradation of bone graft and evaluate the influence of multiple factors.Histologic characteristic of the degradation process of β-TCP were also evaluated.Results All the cases were followed up for average 28.4 months.Radiographic semi-quantitative analysis revealed that the biodegradation effieiencies were not significantly difierent between β-TCP and allografts(P=0.424).Degradation percentage of the implanted β-TCP or allograft was higher in younger patients than those in the older ones.Degradation of β-TCP was significantly higher than that of allografts over 3 years after surgery(P=0.04).In the experimental group,β-TCP degradation was greater in the loose packing treatment than that in the dense packing treatment (P=0.03).Histological observation demonstrated that the process of new bone formation accompanied the degradation of β-TCP.Conclusion The interporous β-TCP could be an advantageous alternative to allografts for repair bone defects caused by bone cyst.The clinical application of β-TCP is safe and reliable,which shows better biodegradation and osteogenesis than allografts in long-term follow-up.
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Objective To explore the optimum flow shear stress and mass transport for the construction of tissue-engineered bone.Methods The β-tricalcium phosphate (β-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (HBMMSCs) were cultured in perfusion bioreactor.When the same flow rate was applied,the flow shear stress was separately 1×,2× and 3×.When the same flow shear stress was applied,the flow rates were separately 3 ml/min,6 ml/min and 9 ml/min.Cell proliferation was measured by MTT method.The construction of tissue-engineered bone was evaluated by measuring alkaline phosphatase (AKP) activity,secretion of osteopontin (OP) and osteocalcin (OC),and the mineralization of extracellular matrix (ECM).The flow shear stress and the mass transport were obtained using computational fluid dynamics.Results When the flow rate was same,the most cell proliferation was found in 2× group.The AKP activity and secretion of OC was higher in 2× and 3× groups than in those in 1× group.After 28days,the highest amount of mineralization of ECM was found in 3× group.When the flow shear stress was same,the AKP activity was highest in 6 ml/min group.After 28 days,secretion of OC and formation of mineralized ECM was highest in 3 ml/min group.When the flow rate was same,the flow shear stress was separately 0.004-0.007 Pa,0.009-0.013 Pa and 0.013-0.018 Pa.When the flow shear stress was same,the flow rate was separately 0.267-0.384 mm/s,0.521-0.765 mm/s and 0.765-1.177 mm/s.Conclusion When the tissue-engineered bone was constructed,0.013-0.018 Pa flow shear stress and 0.267-0.384 mm/s mass transport velocity could improve the construction of the tissue-engineered bone in vitro.
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[Objective]To explore the vascularization within spherical porous ?-TCP scaffolds in vivo.[Method]Thirty adult rabbits were selected and divided into 5 groups randomly.Spherical porous ?-TCP scaffolds(the diameter was 2 cm,the pore size was 500～600?m,the interconnection size was 110～120 ?m) were harvested at 1,2,4,8 and 12 weeks after embedded into muscle-fascia lumbodorsalis pouches in each rabbit separately,in order to observe the vascularization by means of morphological and quantificational analysis.[Result]No vessel was detected at one week after surgery,and only a few immature vascular buds could be seen at the 2nd week.The first vascularization peak could be observed at 4 weeks,characterized by the growth of quantity of new blood vessels(P0.05).[Conclusion]The process of vascularization comprises two phases,the quantity increasing in the early stage and the quality enhancing in the later stage.The special architecture and biodegradation characters of scaffolds could influence the vascularization.
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We conducted studies to confirm the hypothesis that the cellular damage occurring around implanted biphasic bioceramics could be related to a micro-particles release because of an insufficient sintering. An in vitro cytotoxicity study was performed on four biphasic ceramic (BCP) samples. Without the treatment of extraction medium, a cytotoxicity was observed, although after centrifugation this cytotoxicity disappeared in all samples. (2) Micro-particles of HA, beta-TCP and 40%beta-TCP/60%HA mixture were used for a cell inhibition study. A decrease of cell viability was observed with the increase in particles concentration. At 10000 particles/ cell, the viability and proliferation were completely inhibited. (3) HA, beta-TCP and BCP ceramic granules were implanted in rabbit femoral cavities for 12 weeks. No degradation of HA granules was observed. The degradation was higher for beta-TCP (40%) than for BCP (5%). On the other hand, new bone formation was significantly higher for beta-TCP (21%) and HA (18%) than for BCP (12%). Much more micro-particles were formed around BCP granules than around beta-TCP, and were phagocytosed by macrophages. The release of ceramic micro-particles could be related to the sintering process. BCP ceramics have to be sintered at only 1160 degrees C. Consequently, HA microparticles of BCP ceramic are incompletely sintered and easily released after immersion or implantation. The microparticles could be at the origin of local inflammation and cell damage and could perhaps modify osteogenesis. Particular attention must be paid to this problem with regard to BCP ceramics because of the sintering difficulties of this bioceramic.
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Biocompatible Materials , Chemistry , Calcium Phosphates , Chemistry , Cells, Cultured , Ceramics , Chemistry , Fibroblasts , Cell Biology , Hydroxyapatites , Chemistry , Materials Testing , Particle Size , Prostheses and ImplantsABSTRACT
Tissue-engineering bone with porous β-tricalcium phosphate (β-TCP) ceramic and autologous bone marrow mesenchymal stem cells (MSC) was constructed and the effect of this composite on healing of segmental bone defects was investigated. 10-15 ml bone marrow aspirates were harvested from the iliac crestof sheep, and enriched for MSC by density gradient centrifugation over a Percoll cushion (1. 073 g/ml). After cultured and proliferated, tissue-engineering bones were constructed with these cells seeded onto porous β-TCP, and then the constructs were implanted in 8 sheep left metatarsus defect (25 mm in length) as experimental group. Porous β-TCP only were implanted to bridge same size and position defects in 8 sheep as control group, and 25 mm segmental bone defects of left metatarsus were left empty in 4 sheep as blank group. Sheep were sacrificed on the 6th, 12th, and 24th week postoperatively and the implants samples were examined by radiograph, histology, and biomechanical test. The 4 sheep in blank group were sacrificed on the 24th week postoperatively. The results showed that new bone tissues were observed either radiographic or histologically at the defects of experimental group as early as 6th week postoperatively, but not in control group, and osteoid tissue, woven bone and lamellar bone occurred earlier than in control group in which the bone defects were repaired in "creep substitution" way, because of the new bone formed in direct manner without progression through a cartilaginous intermediate. At the 24th week, radiographs and biomechanical test revealed an almost complete repair of the defect of experimental group, only partly in control group. The bone defects in blank group were non-healing at the 24th week. It was concluded that engineering bones constructed with porous β-TCP and autologous MSC were capable of repairing segmental bone defects in sheep metatarsus beyond "creep substitution" way and making it healed earlier. Porous β-TCP being constituted with autologous MSC may be a good option in healing critical segmental bonedefects in clinical practice and provide insight for future clinical repair of segmental defect.
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Tissue-engineering bone with porous ,betatricalcium phosphate (3-TCP) ceramic and autologous bone marrow mesenchymal stem cells (MSC) was constructed and the effect of this composite on healing of segmental bone defects was investigated. 10-15 ml bone marrow aspirates were harvested from the iliac crest of sheep, and enriched for MSC by density gradient centrifugation over a Percoll cushion (1. 073 g/ml). After cultured and proliferated, tissue-engineering bones were constructed with these,cellS seeded onto porous f-TCP, and then the constructs were implanted in 8 sheep left metatarsus defect (25 mm in length) as experimental group. Porous ,-TCP only were implanted to bridge same size and position defects in 8 sheep as control group, and 25 mm segmental bone defects of left metatarsus were left empty in 4 sheep as blank group. Sheep were sacrificed on the 6th, 12th, and 24th week postoperatively and the implants samples were examined by radiograph, histology, and biomechanical test. The 4 sheep in blank group were sacrificed on the 24th week postoperatively. The results showed that new bone tissues were observed either radiographic or histologically at the defects of experimental group as early as 6th week postoperatively, but not in control group, and osteoid tissue, woven bone and lamellar bone occurred earlier than in control group in which the bone defects were repaired in "creep substitution" way, because of the new bone formed in direct manner without progression through a cartilaginous intermediate. At the 24th week, radiographs and biomechanical test revealed an almost complete repair of the defect of experimental group, only partly in control group. The bone defects in blank group were non-healing at the 24th week. It was concluded that engineering bones constructed with porous -TCP and autologous MSC were capable of repairing segmental bone defects in sheep metatarsus beyond "creep substitution" way and making it healed earlier. Porous ,-TCP being constituted with autologous MSC may be a good option in healing critical segmental bone defects in clinical practice and provide insight for future clinical repair of segmental defect.
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Bone Marrow Cells/cytology , Calcium Phosphates/pharmacology , Cells, Cultured , Fractures, Bone/therapy , Implants, Experimental , Mesenchymal Stem Cells/cytology , Metatarsus/injuries , Porosity , Sheep , Tissue EngineeringABSTRACT
<p><b>OBJECTIVE</b>To evaluate the feasibility of growing tissue-engineered cartilage using chondrocytes seeded onto a biodegradable porous bioceramic, the beta-tricalcium phosphate (beta-TCP).</p><p><b>METHODS</b>A porous bioceramic template of beta-TCP was created in the shape of a disc. Chondrocytes isolated from rabbit articular cartilage were seeded on the beta-TCP template and then kept in rotatory cell culture system (RCCS) for 1 week prior to subcutaneous transplantation into athymic mice. The three-dimensional structure was well-maintained 16 weeks after implantation. After 4, 8, 16 weeks, the specimens were harvested and examined macroscopically, histologically and immunohistochemically.</p><p><b>RESULTS</b>Gross morphological and histological analysis of the specimens from the chondrocyte-beta-TCP complex demonstrated new cartilage construction. The overall configuration of the experimental specimens closely resembled the structure of beta-TCP template.</p><p><b>CONCLUSION</b>These findings suggest that porous bioceramic (beta-TCP) is a good "matrix" for chondrocyte, and can be used for cartilage engineering.</p>
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Animals , Female , Mice , Calcium Phosphates , Pharmacology , Cartilage , Transplantation , DNA , Glycosaminoglycans , Immunohistochemistry , Mice, Nude , Tissue EngineeringABSTRACT
<p><b>OBJECTIVE</b>To explore use of retroviral vector in gene therapy of hepatitis B.</p><p><b>METHODS</b>The recombinant vector Plxsn-HBs was constructed by inserting HBV S gene into pLXSN. The pseudovirus, which was produced from PA317 after transferring with pLXSN-HBs by electroporation, were frozen at different temperature. The activities of the pseudovirus to infect eukaryotic cells and express antigen were determined by comparing the numbers of G418-resistant clones and assaying HBsAg in the supernatant of the cells with ELISA after infection HepG2, NIH3T3 and 293 cells.</p><p><b>RESULTS</b>It was hard to find changes in HBsAg amount at different intervals and different temperatures. G418 resistant clones, however, were variable. When frozen at -20 degrees C, the numbers of clones were half less than that of the beginning after 6 months, few clones were formed after 12 months, and no clone was found after 24 months. When frozen at -40 degrees C, the numbers of clones were 121, 332 and 89 42, 137 and 43 for HepG2, NIH3T3 and 293 cell lines at 12 and 24 months, respectively. When frozen at -70 degrees C, the numbers of clones were 159 463 and 112 for HepG2, NIH3T3 and 293 cell lines at 24 months, respectively. There was no statistical difference compared to that of zero months.</p><p><b>CONCLUSIONS</b>The activity of the peseudovirus to infect eukaryotic cells and expressed antigen was not changed after 2 years frozen at -70 degrees C.</p>
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Cell Line , Genetic Vectors , Hepatitis B Surface Antigens , Genetics , Retroviridae , Genetics , Temperature , TransfectionABSTRACT
【Objective】 To study the relationship of pathologic changes and serologic markers of fibrosis in patients with viral hepatitis. 【Me thods】 Liver s pecimens were obtained by percutaneous needle biopsy under color Doppler ultras ound guidance in 299 patients with viral hepatitis. The specimens were stained b y hematoxylin and eosin (HE), Gordon and Sweet's reticulum methods (RT), in orde r to determine the degree and the stage of pathologic changes with microscopy. H yaluronic acid(HA), collagen type Ⅳ(Ⅳ-C) and human precollagen type Ⅲ（HPCⅢ ）as serum fibrous markers were detected by radioimmunoassay. 【Results】 The se rum levels serologic markers were slightly increased in 97 patients with mild ch ronic hepatitis, moderately increased in 126 patients with moderate chronic hepa titis, and significantly increased in 29 severe cases and 47 subjects with cirrh osis. Both the grade of inflammatory activity and the stage of fibrosis were clo sely related to the levels of serum fibrous markers. 【Conclusion】 Chronic vira l hepatitis pathologic feature and levels of serum markers of fibrosis change al ong with clinical process of patients. The combination of liver biopsy and detec tion of serum markers of fibrosis might be highly valuable for the diagnosis.
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AIM:To compare the cloning efficacy of full-length HBV genome amplified by single fragment PCR and two fragment PCR for choosing the suitable method for full-length HBV genome cloning.METHODS:To amplify the full-length HBV genome from 85 sera sample of HBV patients,single fragment PCR and two fragment PCR were conducted.The products were cloned into the vector and sequenced after identified with double enzyme digestion.At the same time,the titers of 85 samples were detected by real-time PCR.RESULTS:Compared with two fragment PCR,single fragment PCR requested higher level of sera HBV DNA for successful amplification of full-length HBV genome,and the efficacy of single fragment PCR was lower than that of two fragments PCR(P