全骨髓贴壁法培养兔骨髓间充质干细胞体外定向成骨诱导分化及鉴定

背景:流式细胞仪分离法和免疫磁珠分离法对细胞活性影响较大,密度梯度离心法虽然能够获得纯度高的单核细胞,但由于多次离心可造成细胞的大量流失且对细胞活性有一定的影响使其应用值得商榷。目的:采用全骨髓贴壁法分离兔骨髓间充质干细胞进行成骨诱导分化及鉴定。方法:采用全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,倒置显微镜下观察细胞形态学特征。在成骨诱导剂作用下,通过碱性磷酸酶染色试剂盒行碱性磷酸酶染色,Ⅰ型胶原免疫细胞化学染色,VonKossa法及茜素红进行矿化结节染色以及电镜下检测兔骨髓间充质干细胞成骨诱导后的形态结构。结果与结论:经诱导后细胞出现与成骨细胞相似的形态学特征,碱性磷酸酶染色阳性,Ⅰ型胶原免疫细胞化学染色,Von-Kossa法及茜素红矿化结节染色阳性。表明经成骨诱导剂诱导后全骨髓贴壁法体外分离纯化培养的兔骨髓间充质干细胞能向成骨细胞方向分化增殖。

成人骨髓间充质干细胞向成骨细胞定向诱导分化的实验研究

目的研究体外培养的成人骨髓间充质干细胞(MSC)在特定的条件下定向诱导分化为成骨细胞,探讨其作为骨组织工程种子细胞的可行性和应用价值.方法抽取健康成年志愿者骨髓,在含10%胎牛血清的高糖DMEM培养基中采用全骨髓培养法,培养传代后改用含地塞米松(1×10-8mol/L)、β-甘油磷酸钠(10mmol/L)和维生素C(50mg/L)的条件培养基培养,在相差显微镜和电镜下观察细胞形态,免疫组化检测Ⅰ型胶原,Gomori钙钴法进行碱性磷酸酶(AP)染色、vonKossa法进行钙结节染色,同时测定细胞内AP(碱性磷酸酶)含量变化,并进行统计学分析.结果原代和传代培养的细胞具有活跃的增殖能力,诱导培养2～3周后,透射电镜下观察见大量扩张的粗面内质网、高尔基体和线粒体,细胞核较为幼稚.I型胶原染色、AP及钙结节染色等均为强阳性;AP活性明显增强(P＜0.05).结论MSC取材安全方便,易于诱导分化为成骨细胞,有望成为理想的骨组织工程种子细胞来源,本实验方法可作为骨组织工程种子细胞培养的常规方法之一.

Preparation of new tissue engineering bone-CPC/PLGA composite and its application in animal bone defects

To investigate the feasibility of implanting the biocomposite of calcium phosphate cement(CPC)/polylactic acid-polyglycolic acid(PLGA) into animals for bone defects repairing,the biocomposite of CPC/PLGA was prepared and its setting time,compressive strength,elastic modulus,pH values,phase composition of the samples,degradability and biocompatibility in vitro were tested.The above-mentioned composite implanted with bone marrow stromal cells was used to repair defects of the radius in rabbits.Osteogenesis was histomorphologically observed by using an electron-microscope.The results show that compared with the CPC,the physical and chemical properties of CPC/PLGA composite have some differences in which CPC/PLGA composite has better biological properties.The CPC/PLGA composite combined with seed cells is superior to the control in terms of the amount of new bones formed after CPC/PLGA composite is implanted into the rabbits,as well as the speed of repairing bone defects.The results suggest that the constructed CPC/PLGA composite basically meets the requirements of tissue engineering scaffold materials and that the CPC/PLGA composite implanted with bone marrow stromal cells may be a new artificial bone material for repairing bone defects because it can promote the growth of bone tissues.

Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum

The use of periosteum-derived progenitor cells （PCs） combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional （3D） poly-lactic-co-glycolic acid （PLGA） fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type Ⅰ collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction （RT-PCR）. The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules ofneo-bone tissue formation and the presence of matrix mineralization were confirmed by positive yon Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.

Application of CT perfusion imaging in detection of tumor angiogenesis in osteosarcoma

Objective： The aim of the study was to explore the application of 64-slice spiral computed tomography perfusion imaging （CTPI） in evaluating angiogenesis in human osteosarcoma. Methods： Twenty-six patients （18 males and 8 females ranging from 9 to 56 years old, with an average of 19 years） with osteosarcoma underwent 64-slice spiral CTPI. We analyzed the correlations of CTPI parameters including blood flow （BF）, blood volume （BV）, time to peak （TTP）, and permeability surface （PS） with the expression of markers of angiogenesis. Statistical analysis was performed with paired-samples t test, and Pearson correlation analysis was employed to investigate the correlations of CTPI parameters with microvessel density （MVD）. Results： Mean BF, BV, TTP, and PS values of osteosarcoma group were （46.6 ± 25.1） mL/100 g/min, （61.8 ± 29.5） mL/100 g, （122.9 ± 26.2） seconds, and （44.5 ± 14.6） mL/100 g/min, respectively. Those in the normal muscle group were （5.2 ± 6.6） mL/100 g/min, （9.6 ± 7.3） mL/100 g, （115.5 ± 33.1） seconds and （17.0 ± 29.3） mL/100 g/min, respectively. Osteosarcoma group showed higher BF, BV and PS compared with the normal muscle group （P = 0.000, P = 0.000, and P = 0.000, respectively）. However, no significant difference was found in TTP between osteosarcoma tissue and normal adjacent muscle tissue （P = 0.273）. BF, BV, and PS were positively correlated with MVD （r = 0.83, P = 0.000; r = 0.87, P = 0.000; and r = 0.63, P = 0.001, respectively）. No correlation was found between TTP and MVD （r = –0.02, P = 0.93）. Conclusion： CTPI is useful for assessing tumor vascularity of osteosarcoma and CTPI parameters are positively correlated with MVD.

Synthesis of FER zeolite with piperidine as structure-directing agent and its catalytic application

The synthesis of ferrierite(FER)zeolite using piperidine as an organic structure‐directing agent was investigated.X‐ray diffraction,X‐ray fluorescence,N2‐adsorption,and scanning electron microscopy were used to characterize the crystal phases,textural properties,and particle morphologies of the zeolite samples.The crystallization behavior of the FER zeolite was found to be directly related to crystallization temperature.At150?C,pure FER phase was observed throughout crystallization.At160–170?C,MWW phase appeared first and gradually transformed into FER phase over time,indicating that the FER phase was thermodynamically favored.In the piperidine‐Na2O‐H2O synthetic system,alkalinity proved to be the crucial factor determining the size and textural properties of FER zeolite.Furthermore,the obtained FER samples exhibited good catalytic performance in the skeletal isomerization of1‐butene.

Study on biocompatibility of PDLLA/HA/DBM with co-cultured human osteoblasts in vitro

Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDLLA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen Ⅰ expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of biomaterial and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.

Recent Progress in Cartilage Tissue Engineering--Our Experience and Future Directions

Given the limited spontaneous repair that follows cartilage injury, demand is growing for tissue engi- neering approaches for cartilage regeneration. There are two major applications for tissue-engineered cartilage. One is in orthopedic surgery, in which the engineered cartilage is usually used to repair cartilage defects or loss in an articular joint or meniscus in order to restore the joint function. The other is for head and neck reconstruction, in which the engineered cartilage is usually applied to repair cartilage defects or loss in an auricle, trachea, nose, larynx, or eyelid. The challenges faced by the engineered car- tilage for one application are quite different from those faced by the engineered cartilage for the other application. As a result, the emphases of the engineering strategies to generate cartilage are usually quite different for each application. The statuses of preclinical animal investigations and of the clinical translation of engineered cartilage are also at different levels for each application. The aim of this review is to provide an opinion piece on the challenges, current developments, and future directions for cartilage engineering for both applications.

The Experimental Study on Constructing the Tissue Engineered Myocardium-like Tissue in vitro with Bone Mesenchymal Stem Cells

Objective:Unlike other tissues,myocardium has not substitute whick can be used to repair damaged cardiac tissue.This paper proposes engineering 3-D myocardium-like tissue constructs in vitro with bone mesenchymal stem cells(BMSCs) of infant and poly-lactic-co-glycolic acid(PLGA)in vitro.Methods:Bone marrow was obtained from the sternal marrow cavum outflow of infant with congenital heart disease (CHD)undergoing cardiac operation.BMSCs were obtained by density gradient centrifugation.The cells in passages two were induced in DMED with 10 umol/L 5- Azacytidine(5-Aza)for 24 h.When the induced BMSCS were cultured nearly into filled,the cells were planted in the scaffold of PLGA in 5.5×106 cells/cm2.The cell- scaffold complex has been cultured in the shake cultivation for 1 week,then the complex has been planted in the dorse of the nude mouse.When the experiment had been finished,the histology,immunology,real time PCR and so on were done.Results: The BMSCs of infant with congenital heart disease have the properties of the stable growth and the rapid proliferation.The immunohistochemistry showed that tissue engineered myocardium constructed in vitro expressed some cardiac related proteins such asα-actin,Cx-43,Desmine,cTNI and so on.The transparent myofilaments,gap junctions and intercalated disk-like structure formation could be observed in the 3D tissue-like constructs by transmission electron microscope(TEM).The engineered myocardium-like tissue had the auto-myocardial property as assessed by real time- PCR and so on.Conclusion:The engineered myocardial tissue-like constructs could be built with infant BMSCs and PLGA in vitro.Our results may provide the first step on the long road toward engineering myocardial material for repairing the defect or augmenting the tract in CHD,such as ventricular septal defect,tetralogy of Fallot and so on.

The Stability of Logistic Model with Random Impulse

This paper is devoted to studying the stability of Logistic model with random impulse by using the theory of Markov skeleton processes and a convenient condition for Logistic model with random impulse to be stable is given.

A first order system model of fracture healing

A first order system model is proposed for simulating the influence of stress stimulation on fracture strength during fracture healing. To validate the model, the diaphyses of bilateral tibiae in 70 New Zealand rabbits were osteotomized and fixed with rigid plates and stress-relaxation plates, respectively. Stress shielding rate and ultimate bending strength of the healing bone were measured at 2 to 48 weeks postoperatively. Ratios of stress stimulation and fracture strength of the healing bone to those of intact bone were taken as the system input and output. The assumed first order system model can approximate the experimental data on fracture strength from the input of stress stimulation over time, both for the rigid plate group and the stress-relaxation plate group, with different system parameters of time constant and gain. The fitting curve indicates that the effect of mechanical stimulus occurs mainly in late stages of healing. First order system can model the stress adaptation process of fracture healing. This approach presents a simple bio-mathematical model of the relationship between stress stimulation and fracture strength, and has the potential to optimize planning of functional exercises and conduct parametric studies.

Tethering of Gly-Arg-Gly-Asp-Ser-Pro-Lys Peptides on Mg-Doped Hydroxyapatite

Stem cell homing, namely the recruitment of mesenchymal stem cells （MSCs） to injured tissues, is highly effective for bone regeneration in vivo. In order to explore whether the incorporation of mimetic peptide sequences on magnesium-doped （Mg-doped） hydroxyapatite （HA） may regulate the homing of MSCs, and thus induce cell migration to a specific site, we covalently functionalized MgHA disks with two chemotactic/haptotactic factors： either the fibronectin fragment III1-C human （FF III1-C）, or the peptide sequence Gly-Arg-Gly-Asp-Ser-Pro-Lys, a fibronectin analog that is able to bind to integrin trans- membrane receptors. Preliminary biological evaluation of MSC viability, analyzed by 3-（4,5-dimethyl- thiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） test, suggested that stem cells migrate to the MgHA disks in resoonse to the grafted haototaxis stimuli.

A force feedback master finger in exoskeleton type

In order to eliminate the drawbacks of conventional force feedback gloves, a new type of master fin- ger has been developed. By utilizing three ＂four-bar mechanism joint＂ in series and wire coupling mecha- nism, the master finger transmission ratio is kept exactly 1：1.4：1 in the whole movement range and it can make active motions in both extension and flexion directions. Additionally, to assure faster data transmission and near zero delay in the master-slave operation, a digital signal processing/field programmable gate array （DSP/FPGA-FPGA） structure with 200μs cycle time is designed. The operating modes of the master finger can be contact or non-contact, which depends on the motion states of a slave finger, free motion or constrained motion. The position control employed in non-contact mode ensures unconstrained motion and the force control adopted in contact mode guarantees natural contact sensation. To evaluate the performances of the master finger, an experiment between the master finger and a DLR/HTT dexterous finger is conducted. The results demonstrate that this new type master finger can augment telepresence.

Microstructure and Mechanical Properties of Calcium Phosphate Cement/gelatine Composite Scaffold with Oriented Pore Structure for Bone Tissue Engineering

In this study,the macroporous calcium phosphate cement with oriented pore structure was prepared by freeze casting.SEM observation showed that the macropores in the porous calcium phosphate cement were interconnected aligned along the ice growth direction.The porosity of the as-prepared porous CPC was measured to be 87.6% by Archimede's principle.XRD patterns of specimens showed that poorly crystallized hydroxyapatite was the main phase present in the hydrated porous calcium phosphate cement.To improve the mechanical properties of the CPC scaffold,the 15% gelatine solution was infiltrated into the pores under vacuum and then the samples were freeze dried to form the CPC/gelatine composite scaffolds.After reinforced with gelatine,the compressive strength of CPC/gelatin composite increased to 5.12 MPa,around 50 times greater than that of the unreinforced macroporous CPC scaffold,which was only 0.1 MPa.And the toughness of the scaffold has been greatly improved via the gelatine reinforcement with a much greater fracture strain.SEM examination of the specimens indicated good bonding between the cement and gelatine.In conclusion,the calcium phosphate cement/gelatine composite with oriented pore structure prepared in this study might be a potential scaffold for bone tissue engineering.

A new analytical algorithm for computing probability distribution of project completion time

An analytical algorithm was presented for the exact computation of the probability distribution of the project completion time in stochastic networks,where the activity durations are mutually independent and continuously distributed random variables. Firstly,stochastic activity networks were modeled as continuous-time Markov process with a single absorbing state by the well-know method of supplementary variables and the time changed from the initial state to absorbing state is equal to the project completion time.Then,the Markov process was regarded as a special case of Markov skeleton process.By taking advantage of the backward equations of Markov skeleton processes,a backward algorithm was proposed to compute the probability distribution of the project completion time.Finally,a numerical example was solved to demonstrate the performance of the proposed methodology.The results show that the proposed algorithm is capable of computing the exact distribution function of the project completion time,and the expectation and variance are obtained.

Analysis of Fatigue Fracture with Scanning Electron Microscopy of Valve Spring Steel

Valve spring steel for engines, belonging to the class of super clean steel, must be supported due to their application, high numbers of cycles in fatigue and cannot suffer any type of failure, which would be catastrophic for the vehicle. From these considerations, it was tested in axial fatigue, a test that can detect possible internal defects in their structure, caused by inclusions, a class of valve spring steel, where it aimed to discover the values of their fatigue life, followed by an analysis ofmicrostructural fracture surface by SEM （scanning electron microscopy）. It was proved, after testing, the specimens tested broke up a number of cycles always compatible with the life work of a valve spring and that fractures always occurred by surface defects in the specimens.

Design of comprehensive test system for detecting overlying strata mining-induced fractures on surface with radon gas

Based on radon gas properties and its existing projects applications, we firstly attempted to apply geo- physical and chemical properties of radon gas in the field of mining engineering, and imported radioac- tive measurement method to detect the development process of the overlying strata mining-induced fractures and their contained water quality in underground coal mining, which not only innovates a more simple-fast-reliable detection method, but also further expands the applications of radon gas detection technology in mining field. A 3D simulation design of comprehensive testing system for detecting strata mining-induced fractures on surface with radon gas (CTSR) was carried out by using a large-scale 3D solid model design software Pro/Engineer (Pro/E), which overcame three main disadvantages of ''static design thought, 2D planar design and heavy workload for remodification design'' on exiting design for mining engineering test systems. Meanwhile, based on the simulation design results of Pro/E software, the sta- bility of the jack-screw pressure bar for the key component in CTSR was checked with a material mechan- ics theory, which provided a reliable basis for materials selection during the latter machining process.

Microscopic Inspection on Developmental Process of Myeloma Cells

The developmental process of Myeloma cells under a digital optical microscope has been inspected and monitored by using time-lapsed recording technique. Myeloma cells were cultured in medium contained 20% and 50% of Fetal Bovine Serum （FBS）, respectively. Inspection and monitoring for 6 hours showed the effect of the FBS to mobility, proliferation rate, and development of cell cycle phases of Myeloma cells. Using time-lapsed data, the speed of cells was 3.5-6.0 pm/s when using FBS 20% and increased to 5.0-8.0 p.m/s when using FBS 50%. The rate of cells decreases from 2 cells/hr when using FBS 20% to 1/6 cells/br when using FBS 50%. The cells division process is signified by the change in gray level and it took every 50-70 minutes.

Time Course of Changes in Trabecular Bone Microstructure in Rats with Spinal Cord Injury

The study aimed to examine changes in trabecular bone microstructure （TBMS） during the period of 5 weeks after the injury in a rat model of spinal cord injury （SCI）. Eight-week-old male Wistar rats underwent surgical transection of the lower thoracic spinal cord （SCI, n = 16） or sham operation （SHAM, n = 14）. TBMS （tissue volume, bone volume, bone volume fraction, trabecular thickness, width, number, separation, connectivity density, and trabecular bone pattern factor）, assessed using a micro-computed tomography, was deteriorated 1, 3 and 5 weeks after SCI. In addition, both bone mass and serum biochemical parameters were determined. Dry bone weight, ash weight, bone mineral content （BMC）, and BMC/tissue-volume were significantly lower in the SCI group than in the SHAM group throughout the experimental period. Serum inorganic phosphate and alkaline phosphatase levels were significantly lower in the SCI group than in the SHAM group 1 week after the surgery. SCI resulted in rapid deterioration of both bone mass and microstructure. These changes appeared as early as 1 week after SCI. Based on the authors＇ results, it should be noted that in SCI patients, interventions for preventing bone loss should start as soon as possible after the injury.

Repair of articular cartilage defects in rabbits through tissue-engineered cartilage constructed with chitosan hydrogel and chondrocytes

Objective： In our previous work, we prepared a type of chitosan hydrogel with excellent biocompatibility. In this study, tissue-engineered cartilage constructed with this chitosan hydrogel and costal chondrocytes was used to repair the articular cartilage defects. Methods： Chitosan hydrogels were prepared with a crosslinker formed by combining 1,6-diisocyanatohexane and polyethylene glycol. Chitosan hydrogel scaffold was seeded with rabbit chondrocytes that had been cultured for one week in vitro to form the preliminary tissue-engineered cartilage. This preliminary tissue-engineered cartilage was then transplanted into the defective rabbit articular cartilage. There were three treatment groups： the experimental group received preliminary tissue-engineered cartilage; the blank group received pure chitosan hydrogels; and, the control group had received no implantation. The knee joints were harvested at predetermined time. The repaired cartilage was analyzed through gross morphology, histologically and immunohistochemically. The repairs were scored according to the international cartilage repair society （ICRS） standard. Results： The gross morphology results suggested that the defects were repaired completely in the experimental group after twelve weeks. The regenerated tissue connected closely with subchondral bone and the boundary with normal tissue was fuzzy. The cartilage lacuna in the regenerated tissue was similar to normal cartilage lacuna. The results of ICRS gross and histological grading showed that there were significant differences among the three groups （P〈0.05）. Conclusions： Chondrocytes implanted in the scaffold can adhere, proliferate, and secrete extracellular matrix. The novel tissue-engineered cartilage constructed in our research can completely repair the structure of damaged articular cartilage.