Objective To test the hypothesis that marrow stromal cells （MSCs）, when implanted into selfmyocardium in rabbits, can undergo milieu-dependent differentiation and express cardiomyogenic phenotypes and enhance cardiac function of ischemic hearts, through establish a clinically relevant model for autologous MSCs transplantation, Methods Thirteen New Zealand White rabbits were randomly divided into experimental group （n= 7） and control group （n= 6）. In experimental group, autotogous MSCs（3× 106 cells/30μl） labeled with Bromodeoxyuridine （BrdU） were respectively injected into superior, central and inferior sites in the periphery of the myocardial infarct region. Phosphate buffer saline （PBS） was injected into the scar of the control group hearts according to the same procedure used in the experimental group. Four weeks later, the transplanted labeled MSCs were detected by laser scanning confocal microscopy and the cardiac function were examined by echocardiogram and muhichannel physiologic recorder. Results After 4 weeks, transplanted MSCs were demonstrated myogenic differentiation with the expression of α-sarcomeric actin and connexin 43 located in intercalated disk. MSCs increased the number of vessels compared with controls in myocardial ischemia area. MSCs implantation resulted in markedly improved left ventricular contractility[left ventricular ejection fraction （LVEF）： 0. 51 ± 0.07 vs. 0. 43 ± 0.06 ,left ventricular lateral wall motion distance （LVLWMD） ：1. 75±0. 42mm vs. 1.09±0. 28mm, left ventricular systolic wall thickening ratio（LVAT） ：0. 19%±0.05% vs. 0. 11%±0.04%, left ventricular systolic pressure （LVSP）： 113. 1± 6.3mmHg vs. 99, 5 ± 5, lmmHg, left ventricular end diastolic pressure （LVEDP）： 11. 5±2. lmmHg vs, 14, 3 ±3. lmmHg, maximum rate of left ventricular pressure rise （＋dp/dtmax）：4 618. 3±365. 2 mmHg/s vs. 3 268. 1± 436.9 mmHg/s, maximum rate of left ventricular pressure fall （-dp/dtmax） ：3 008.8±346.7 mmHg/s vs. 2 536.9± 380.4 mmHg/s, P〈0.05]. Conclusion Transplanted autologous MSCs are able to undergo differentiation to form myocardial cells and improve the cardiac function of ischemia myocardium effectively. Autologous MSCs transplantation may have significant clinical potential in treatment myocardial ischemia.
Objective To investigate the effect of repairing bone defect with tissue engineered bone seeded with the autologous red bone marrow (ARBM) and wrapped by the pedicled fascial flap and provide experimental foundation for cl inicalappl ication. Methods Thirty-two New Zealand white rabbits (male and/or female) aged 4-5 months old and weighing2.0-2.5 kg were used to make the experimental model of bilateral 2 cm defect of the long bone and the periosteum in the radius. The tissue engineered bone was prepared by seeding the ARBM obtained from the rabbits on the osteoinductive absorbing material containing BMP. The left side of the experimental model underwent the implantation of autologous tissue engineered bone serving as the control group (group A). While the right side was designed as the experimental group (group B), one 5 cm × 3 cm fascial flap pedicled on the nameless blood vessel along with its capillary network adjacent to the bone defect was prepared using microsurgical technology, and the autologous tissue engineered bone wrapped by the fascial flap was used to fill the bone defect. At 4, 8, 12, and 16 weeks after operation, X-ray exam, absorbance (A) value test, gross morphology and histology observation, morphology quantitative analysis of bone in the reparative area, vascular image analysis on the boundary area were conducted. Results X-ray films, gross morphology observation, and histology observation: group B was superior to group A in terms of the growth of blood vessel into the implant, the quantity and the speed of the bone trabecula and the cartilage tissue formation, the development of mature bone structure, the remolding of shaft structure, the reopen of marrow cavity, and the absorbance and degradation of the implant. A value: there was significant difference between two groups 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those three time points in groups A and B (P lt; 0.05). For the ratio of neonatal trabecula area to the total reparative area, there were significant differences between two groups 4, 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those four time points in group B (P lt; 0.05).For the vascular regenerative area in per unit area of the junctional zone, group B was superior to group A 4, 8, 12, and 16 weeks after operation (P lt; 0.05). Conclusion Tissue engineered bone, seeded with the ARBM and wrapped by the pedicled fascial flap, has a sound reparative effect on bone defect due to its dual role of constructing vascularization and inducing membrane guided tissue regeneration.
OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.
Objective
To evaluate the limbs shortening and re-lengthening in the treatment of tibial infectious bone defect and chronic osteomyelitis.
Methods
Between January 2011 and April 2016, 19 cases of tibial infectious bone defect and chronic osteomyelitis were treated with the limbs shortening and re-lengthening technique. There were 13 males and 6 females, aged from 22 to 62 years (mean, 44 years). The causes of injury included traffic accident injury in 16 cases, crush injury in 1 case, and falling from height in 2 cases. One patient was infected after plate internal fixation of closed tibial fracture and 18 patients after external fixation of open tibial fractures (Gustilo type IIIB). The mean previous operation times was 3 times (range, 2-5 times). The time from injury to bone transport operation was 3-11 months (mean, 6.5 months). The bone defect length was 2.0-5.5 cm (mean, 4.3 cm) after debridement. After tibial shortening, limb peripheral blood supply should be checked after release of the tourniquet. Seven wounds were closed directly, 5 were repaired with adjacent skin flap, 5 were repaired with sural neurovascular flap, 1 was repaired with medial head of gastrocnemius muscle flap, and 1 underwent skin grafting. Single arm external fixator or ring type external fixator were used, and completely sawed off between 2 sets of external fixation screws at proximal and distal metaphysis of the tibia. Limb lengthening was performed after 1 week with the speed of 1 mm/d.
Results
All patients were followed up 10-36 months with an average of 14 months. Two cases delayed healing of the wound after operation, and the other wounds healed primarily. Natural healing of the opposite end of the bone were found in 18 cases, and 1 case had nonunion in the opposite end of the bone because of incomplete removal of lesion bone. There were 5 cases of slow growth of the callus, and healed smoothly by " accordion” technology and injecting red bone marrow in 4 cases, and by bone grafting and internal fixation in 1 case. The time of bone lengthening was 1-3 months, the prolongation index was 1.6-2.7 cm/month (2.20 cm/month). The bone healing time was 7-13 months (mean, 11.1 months). According to tibial stem diagnostic criteria Johner-Wruhs score, 9 cases were excellent, 8 cases were good, 2 cases were fair, with an excellent and good rate of 89.5%.
Conclusion
Limbs shortening and re-lengthening is an effective method for the treatment of tibial infectious bone defect and chronic osteomyelitis, with the advantages of improving the immediate alignment of the osteotomy ends, significantly shortening the bone healing time of opposite ends of bone.
【Abstract】 Objective The present study employed both static and dynamic imaging modal ities to study bothintra- and extravascular events attributing to steroid-associated osteonecrosis (ON) using an experimental protocol with a single low-dose l i ppolysaccharide (LPS) injection and subsequently three injections of high-dose methylprednisolone (MPS). Methods Fourteen 28-week-old male New Zealand white rabbits received one intravenous injection of LPS (10 μg/ kg). After 24 hours, three injections of 20 mg/kg of MPS were given intramuscularly at a time interval of 24 hours. Additional 6 rabbits were used as controls. Dynamic MRI was performed on bilateral femora for local intraosseous perfusion before and after LPS injection. Blood samples were collected for haematological examinations before and after LPS injection. Bilateral femora were dissected and decalcified for microCT-based microangiography. ON lesion, intravascular thrombus and extravascular marrow fat cell size were examined histopathologically. Results Intravascular thrombus was observed in all ON rabbits. Extravascular marrow fat cell size was significantly increased in ON rabbits than that of the controls (P lt; 0.05). Compared to basel ine, a significant decrease in ratio of tissue-type-plasminogen-activator/plasminogen-activator inhibitor 1,activated-partial- thromboplatin-time, and a significant increase in ratio of low-density-l ipoprotein/high-density-l ipoprotein were only found in ON rabbits (P lt; 0.05). Dynamic MRI showed a significant decrease in the perfusion index ‘maximum enhancement’ in the ON rabbits (P lt; 0.05) and microCT-based microangiography showed blocked stem vessels in ON samples.Overall, 93% of the rabbits (13/14) developed ON and no rabbits died throughout the experiment period. Conclusion Bothintra- and extravascular events were found attributing to the steroid- associated ON based on our experimental protocol with a single low-dose LPS injection and subsequent three injections of high-dose MPS. Both high ON incidence and no mortal ity in rabbits treated with this inductive protocol suggested its effectiveness for future studies on evaluation of therapeutic efficacy of interventions developed for prevention of steroid-associated ON.
This study aimed to characterize and magnetic resonance imaging (MRI) track the mesenchymal stem cells labeled with polylysine-coated superparamagnetic iron oxide (PLL-SPIO). Rat bone marrow derived mesenchymal stem cells (rMSCs) were labeled with 25, 50 and 100 μg/mL PLL-SPIO for 24 hours. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity and differentiation potential. Electron microscopic observations and Prussian blue staining revealed that 75%-100% of cells were labeled with iron particles. PLL-SPIO did not show any cytotoxicity up to 100 μg/mL concentration. Both 25 μg/mL and 50 μg/mL PLL-SPIO labeled stem cells did not exhibit any significant alterations in the adipo/osteo/chondrogenic differentiation potential compared to unlabeled control cells. The lower concentration of 25 μg/mL iron labeled cells emitted an obvious dark signal in T1W, T2WI and T2*WI MR image. The novel PLL-SPIO enables to label and track rMSCs for in vitro MRI without cellular alteration. Therefore PLL-SPIO may potentially become a better MR contrast agent especially in tracking the transplanted stem cells and other cells without compromising cell functional quality.
Objective To optimize the culture conditions of the human bone marrow mesenchymal stem cells(hMSCs). Methods The influence of the primary culture method, planting density, the time of the first medium changing , culture medium and serum concentration on growth of the hMSCs were analyzed. Results When the other conditions were the same, the density gradient isolation was better than whole-marrow isolation;2.5×105 cells/cm2 was the best planting density;the best first medium changing was the fifth day at primary culture, DMEM medium was better than α-MEM, serum C was the best of four serums compared, 10% was the suitable serum concentration. The hMSCs under the optimal conditions could expand over 15 passages, remaining their normal modality and differentiation potentials. Conclusion The optimal culture condition of the hMSCs is established and it is a new investigation on application of hMSCs to tissue engineering.
Objective To investigate the possibility of sheep joint cartilage defect repair with tissue engineered cartilage constructed by using porous bioceramics as scaffold and TGF-β induced autologous bone marrow derived mesenchymal stem cells(MSCs) as seed cell. Methods In the experimental group(n=12), autologous MSCs were isolated and expanded in vitro and then implanted into the pre molded porous β-TCP; the cell β-TCP complex was implanted into sheep right humeral cartilage defect. The defects in β-TCP (n= 12) group were repaired by B-TCP only, while defects in the control group (n= 4) were left un-repaired. Samples were extracted 12 and 24 weeks after operation for histological, histochemical and immunohistochemical analysis. Results In the experimental group, cartilage-like tissue formation could be seen on the surface of the implants. Microscopic analysis demonstrated obvious degradation of B-TCP and extensive new cartilage formation 12 weeks after operation, containing rich extracellularmatrix. The cells were stained positively with type II collagen. The bioceramics had almo st completely been degraded and abundant cartilage formation could be seen in the whole defects 24 weeks later. In the B-TCP group, marginal cartilage ingrowth could be seen 12 weeks after operation and the number of chondrocytes increasedmarkedly after 24wee s. However, no cartilage can be found in the middle of the material. In the control group, only a small quantity of new cartilage formation could be seenalong the margin of defects. Conclusion It is feasible to generate tissue engineered cartilage with porous B-TCP and auto logousM SCs for cartilage defect repair.
