Development Of Intelligent Bioreactor Of Tissue-engineered And Research Compressive Stress On Bone Tissue Engineering In The Biological Role Of MSCs

Background and objection: The sustaining development and the technique maturation of the bioreactor, the key equipment to culture tissue engineered bone, offered a wider technical platform to cultivate tissue engineered bone in vitro. However, the bioreactors used in cultivation of tissue engineered bone in vitro at present have their limitations, for example,①the low level of intelligence and automation, culture still a large number of manual operations, easy to pollution;②the lack of feedback and regulatory function of stimulus, leading to instability stimulus;③simple stimuli imposed, bad bionic;④More than the need for auxiliary equipment such as incubator. Shortcomings and deficiencies of Current bioreactor of tissue engineering result in the bioreactor can not adapt to the demand for industrialization and commercial production. Can not meet the high level of scientific research on the factors to impose the requirements of precise control. At the same time, it is difficult to construct in vitro tissue-engineered bone biomimetic culture environment.A intelligentized bioreactor of tissue-engineered was developed in this research. The objection of this research was to estimate the performance ourselves developed bioreactor system, culture tissue engineered bone with the system, evaluate the effect of press stress on the cultivated engineered bone, study the optimal mechanics condition to culture engineered bone in vitro, and offer important experimental data bases for further research on the mechanism of proliferation and differentiation of mesenchymal stem cells(MSCs) promoted by press stress.Method:1,Based on the concept of modern bionics design intelligent bioreactor of tissue-engineered. Application of semi-permeable membrane hollow fiber product gas-liquid and liquid-liquid switch integration. Application of highly sensitive receptors and the PLC control system for the production of intelligent feedback system. Application of medical polymer materials and finishing techniques of bone tissue engineering training room. Detection of the bio-reactor mechanical performance, flow, temperature effects, aseptic performance, gas / liquid and liquid-liquid exchange performance , thereby evaluated the feasibility of the system as a platform to culture engineered tissue.2,The density grade centrifugal technique combined with the adhering wall cultural technique to separate the human mesenchymal stem cells (hMSCs) from the red medulla ossium of healthy volunteer and cultivate them, and identified their surface marker and osteogenic differentiation.Tissue engineered bone was fabricated with the implanting technique of collagen gel , using the hMSCs as the seed-cells and the Demineralized Bone Matrix (DBM) as scaffold.The fabricated tissue engineered bone constructs were distributed into experimental group and control group, the control group was cultured statically in incubator, and the experimental group was subjected to press load of displacement of 2%, 5%, 10%, 15%, and20%at frequency of 0.5Hz, 1.0Hz and 2.0Hz for 21days, 2h/d. The constructs were detected by microscope, scan electronic microscope and H&E staining; the proliferated levels and cycles of the implanted cells were monitored by MTT assay and DNA content analysis, thereby study the proliferated effect of press load on the MSCs at different frequency and amplitude; the osteogenic effects of different frequency and amplitude of the press load on the MSC were monitored by the Calcium content assay, the alkaline phosphatase (ALP) activities analysis and the technique of immunohistochemial staining of osteocalcin .Results:1,Successfully produced a bioreactor Intelligence. Achieve the automation of tissue-engineered bone to cultivate. Nurturing environment for intelligent feedback to the machine performance is stable, high accuracy.2,The displacement scope of the bellows pump, a circulatory dynamic component of trail reactor, ranged from 0 to 15mm, adjustable augment value:0.1mm; the bellows pump could operate in sequent and intermittent models, the flux of the pump ranged from 0 to 143.2ml/min. 3,The path of the press load of trail reactor ranged from 0 to 48mm, adjustable augment value:0.1mm; the displacement adjustable in range of 0 to 48mm, run models: sequent and intermittent.4,The temperature in cultural cabin could be set in the scope of 22℃to 40℃, and temperature was set at 37℃,it would change in the range of 36.9℃to 37.1℃.5,The cultural medium circulated in the circulatory system for 3 weeks without contamination though serially monitoring.6,By changing the gas flux, the gas-liquid and liquid-liquid incorporate exchanger of the trail reactor could change the PH value of the culture medium in the range of 6.55 to 8.37, the PCO2of which do in the range of 5mmHg to 150mmHg, the PO2 of which do in the range of 112mmHg to 385mmHg. The fabricated engineered bone cultivated in the trail reactor for 14 days, the PH value of cultural medium ranged between 7.51 and 7.633, the PCO2 of which did between 177mmHg to 199.75mmHg, the PO2 of which did between 183.5mmHg and 199.75mmHg, these variational scopes were distinctly less than that of the engineered bone cultured in the Petri dish(PH value: 7.152 to 7.633, PCO2: 13.325 to 183.5mmHg, PO2: 177mmHg to 199.75mmHg).7,The scan electron microscope image revealed the engineered bones subjected to the press load of 10% compress amplitude and 0.5%Hz, covering with a mass of cells on the hollow wall, the cells tended to align in lines, distributed more homogeneous and its density is distinctly lower than that control groups. The histological slice and H&E staining showed engineered bones that subjected to a load of 10% displacement at 0.5Hz had significantly greater cells amount than the control ones, cells distributed more homogeneous, excreted more extracellular matrix. While in the group subjected to a load of displacement of 20% and 2.0Hz, only small quantity of cells and extracellular matrix could be seen in the scaffold and the cell density was obvious lower than the control ones.8,The compress load could promote the implanted hMSCs proliferation with frequencies of 0.5Hz and 1.0Hz and amplitudes of 2%, 5%, 10% respective, all load regime therein, the cells in the samples cultured under 10% amplitude at 0.5Hz exhibited a most significant effect of proliferation, its growth curve shifted ahead distinctly, and its percentage(27.45±0.25)% of cells among S phrase heightened by 43.43% relative to that of control group(19.15±0.26 )%, the DNA content at the 21st day was 291.95% that of time 0 , which heightened by 43.72%,4.25%relative to the control group. While the compress load inhibited cells'proliferation with the amplitude increasing to 15% and 20%. At 2.0Hz compress frequency, the compress load inhibit the cells implanted from proliferation regardless of the compressive amplitude, therein, the group under 20% compressive amplitude was the most distinct. The percentage of cells among S phrase (4.25±0.89)% decreased by 77.81%relative to the control group(4.25±0.89)%, the DNA content at 21st day point heightened by 183.07%relative to time 0, which reduced relative to the control group.9,The data from the activity of ALP exhibited the compressive load of 5% amplitude and 0.5Hz could rise the activity of ALP distinctly, just the verse when the load , 20%compress amplitude and 0.5Hz,was applied. The load ,at 0.5Hz combined with compress amplitude of2%, 5%, 10%and 15% rose Calcium content obviously when compared with that of the control group(P<0.05), and the risen degree of the groups subjected to 0.5Hz 10%and 15% compressive amplitude were the most distinct, the groups cultured under 20%compressive amplitude and at 2Hz combined with 10%and 15%compressive amplitude, obviously reduced their calcium content relative to the control group. The immunohistochemical staining of osteocalcin showed the group subjected to the load, 0.5Hz combined with 20%compressive amplitude resulted in a higher positive rate, on the contrary, which subjected to the load 2.0Hz and 20%compress amplitude got a lower positive rate.Conclusion:1. The intelligentized engineered tissue bionical cultural system and all its functional components operate well, Can be cultivated tissue-engineered bone by biomimetic to impose various forms of stress stimulate and nurture a stable micro-environment, Four organizations can be at the same time the cultivation of tissue engineering bone, Achieve the requirements of intelligence, automation, the industrialization of bone tissue engineering . 2. Keep good air-tightness and sterility, so as to they offer a suitable condition to the engineered bone to grow in a long period, its components, the gas-liquid and liquid-liquid incorporate exchanger supplied the long-term cultured engineered bone with a stable microenvironment.3. The compressive stress plays a significant role in hMSCs proliferation, the response of hMSCs to the compressive stress has a threshold scope. A suitable intensity promotes the hMSCs proliferation, the load at the frequency of 0.5Hz and 5%～10%compressive amplitude is the feasible intensity to stimulate the hMSCs proliferation , while the proliferated effect on the hMSCs might be inhibited under 20% compressive amplitude or at the frequency of 0.5Hz.4. The compressive stress has a significant effect on the osteogenic differentiation of hMSCs, likewise, it has a definite threshold scope. A compressive stress with suitable intensity can promote the hMSCs to osteogenic differentiation, the compressive stress at 0.5Hz combined with compressive amplitude of 5%～10%is the moderate intensity to boost hMSCs'osteogenic differentiation, on the contrary , the effect of osteogenic differentiation on hMSCs might be inhibited when the compressive stress of the frequency reaches 2.0Hz or the compressive amplitude increases to 20%.5. The bioreactor culture can promote the maturation of bone tissue engineering, because it can provide appropriate and stable compressive stress to stimulate the cultivation of micro-environment.