| The segmental defect of the long tubular bone is usually caused byhigh-energy trauma, bone tumor, infection and other, which is one of the mostintractable complications in the orthopedics. While the range of the bone defectis often beyond the capability of healing, and this will eventually result in limblength discrepancy, deformation and dysfunction. Many methods had been triedto bright the defect and reconstruct the anatomic and mechanical integrity of thebone, such as allograft, autograft and artificial materials graft, but theirapplication were limited by many shortcomings and the prognosis wasunsatisfactory until now. With the development of the biology and engineering,the bioengineering is deemed a promising treatment for the segmental bonedefect. This method contains two essential ingredients that are the osteogeniccells and the scaffolds which held the property of osteoinduction andosteoconduction. The core technology of this method is to efficiently combinethe cells with the biomaterials, and exerts biological function by amplifying, differentiating and ex-secreting extracellular matrix. To simulate cell-friendlyenvironment and enhance the efficiency of fabricating, the bioreactor is the mostimportant apparatus in bone tissue engineering process. Until now, a variety ofbioreactors had been built which mainly for small volume and simple shapescaffolds. In this study, to validate the design and application of a new pattern ofperfusion bioreactor for fabricating large volume bio-engineering bone, massiveβ-TCP ceramic cylinders combined with rabbit bone marrow stem cells werecultured in the bioreactor and a series of test were made.1. The Design and Building of a Novel Composite Perfusion BioreactorObjective To build a novel perfusion bioreactor in which the intermittentlow-pressure was imposed in perfusion cassette and study its influence to theperfusion features of the porous β-TCP ceramic cylinders. Methods The newperfusion bioreactor take commercially available Berzelius beakers as perfusioncassettes which are assembled in an aluminum alloy frame and contains othervital components such as vacuum pump which can create intermittent lowpressure in the perfusion cassette (-0.01Mpa，0.5Hz) and controlled by thedigital controller. To evaluate its influence to the perfusion features of the longsegmental porous β-TCP ceramic cylinders, two columns of scaffolds dyed withimpermanent pigment particles were separately perfused under atmosphericpressure and intermittent low pressure at different perfusion rate. Results In theatmospheric environment, the higher of the perfusion rate the more completelyelution of the pigment particles, but there are still residuals in the upper half ofthe ceramic cylinders. But even at low perfusion rates, the pigment particles inthe intermittent low pressure group were more uniformly washed out from thescaffolds. Conclusion The intermittent low pressure in the cassette willremarkably influence the distribution of the liquid flow in the large porous β-TCP ceramic cylinders, even at low perfusion rate the perfusion of thescaffolds is more uniformly, and avoid the mechanical impairment of the highperfusion rate. 2. The Method of Combining BMSCS to the Large Volume Porous β-TCPCeramic CylindersObjective To study the appropriate method of combining the rabbit BMSCs tothe scaffold in vitro. Methods The femur of the tenth-month-old New Zealandrabbit were taken out and the BMSCs were collected and isolated by adherentmethod. The2nd generation were concentrated and resuspended at threedifferent cell density:1×107/ml、5×106/ml and1×106/ml and were divided asgroup A, B and C, then they were respectively dripped into the one-end-sealedtract of the hollow porous β-TCP ceramic cylinder with the volume of0.1,0.2and1.0ml and the time consumption were recorded. After2hours incubation,the cells seeped from the scaffolds were collected and counted，then theefficiency of combination in each group was calculated. Results Among thethree groups, the group A and B took the least time (2.13±0.13min,2.07±0.16min), otherwise the group C is time-consuming. The group C wasmost effectively in combination (64.33±2.20%), in contrast the group B and Chad inferior efficiency (54.00±1.67%,54.17±2.90%). Conclusion whencombining the BMSCs to the porous β-TCP ceramic cylinder by dripping of cellsuspension, the most efficiently way is0.2ml cell suspension with concentrationof5×106/ml. 3. The Incubation of Cells/scaffold Compounds in the Novel PerfusionBioreactorObjective To study the feasibility and validity of the novel perfusion bioreactorthe cells/scaffold compounds were incubated in it and the markers of themetabolism and differentiation were analyzed. Methods After the combinationof BMSCs to the porous β-TCP ceramic cylinder, the compounds wereincubated in static condition, atmospheric pressure perfusion and intermittentlow-pressure perfusion respectively as control group, experimental group-Ⅰandexperimental group-Ⅱ. Each group was cultured for1week,2weeks,3weeksand4weeks and the specimens were regularly taken to detect the daily glucoseconsumption, the specific activity of alkaline phosphatase, the level of theosteopotin. The cellular activity and the hard tissue slicing were examined at theconclusion of the culture. Results The daily glucose consumptions and MTTof the experimental group Ⅱ were both highest at the every check point (P<0.05)compared to the other groups. This group also had the highest level of specificactivity of alkaline phosphatase and osteopotin, but the peak of both markerswere postponed than the other two groups. The hard tissue slicing demonstratedthat in the experimental group-Ⅱthe cell distribution was more uniformly andthe rate of pore occupation in core of the scaffold was higher than experimentalgroup-Ⅰ. Conclusion The building of this novel perfusion bioreactor isconveniently and its application is practical. The principle of the design can beapplied in fabricating active tissue engineering bone with more volume andmore complicated configuration. |
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