| BackgroundIt is possible to use engineered bone for treatment of clinical bone defects, while avoiding the adverse factors of autologous and allograft bone, such as pain, infection, rejection and so on. In recent years, it has been gradually accepted that construct the tissue engineered bone in vitro to treat the critical bone defects. However, construct a critical size tissue engineered bone in vitro has been facing a problem that the seeding cells can not be evenly distribution, proliferation and differentiation in the large scale scaffolds. Bioreactor is expected to solve this problem. Many scholars have simulated a three-dimensional dynamic culture environment by using of bioreactors in vitro. For bone tissue engineering, we had better use perfusion bioreactor. However, how to minimize the path of non-perfusion and increase perfusion efficiency has been a problem to overcome.Objective1. Develop a three-dimensional perfusion bioreactor and use to construct a critical size tissue engineered bone in vitro.2. Culture the BMSCs by whole bone marrow in vitro, research its biological characteristics and pluripotency.3. Compare the effect of BMSCs’ distribution, proliferation and differentiation in critical size scaffolds between two perfusion culture ways. Methods1. Perfusion bioreactor construction and performance testBioreactor design principles:①Culture in a controlled environment for long time;②Ensure that the vertical fluid flow through the scaffolds and minimize fluid flow through the non-perfusion path;③The module can be combined to culture the number of engineered tissues;④Ensure that the fluid can be repeated, controlled, and continuously through the system;⑤It is easy to sterilize the system and remain sterile during the culturing process;⑥The system is simple and reasonable in order to avoid the interference caused by unpredictable factors.Bioreactor performance test:(1)Mechanical properties and safety assessment:the bioreactor system was test by airtight, bacteriological, cytotoxicity and determination of fluid shear stress. (2)Evaluation of tissue culture:Used eGFP-BMSCs as seeding cells, composited withβ-TCP, and then put into the bioreactor. Experimental group: bioreactor culture group; control group:static culture group. At 3,7,14 d was observed the distribution and proliferation in the scaffold for cell viability, scanning electron microscopy and fluorescence microscopy on cells (n=3). 2. SD rat BMSCs isolation, culture and identificationSD rat BMSCs isolation and culture:sacrificed SD rat, soaked in 75% alcohol for 15 min, then removed to sterile workbench. Removed soft tissue attached to femur and tibia bone completely, washed the marrow cavity with sterile normal saline 1-2 times. Centrifuged it with 1000 rpm 10 min, used the standard SD rat BMSCs culture medium, inoculated in 25 cm2 flasks, placed in 37℃,5% CO2 incubator for culture. Changed the medium after 24 h, then every 2-3 days did 1 time. Digested and subcultured it after 80%～90% confluences.SD rat BMSCs identification:Selected the P4 cells, detected the surface of the flow cytometry antibody. P3 cells were respectively inducted into the bone, cartilage and fat, then taken of ALP staining, alizarin red staining, toluidine blue, alcian blue staining, oil red O staining to testify the potential ability of multi-directional differentiation. 3. Influence of distribution, proliferation and differentiation of BMSCs in scaffolds by two perfusion culture methodsThere were three groups divided by different culture methods. Control group was the static culture, which do not open the pump. Experiment group A was the persistent perfusion culture, which open the pump to 10 rpm continuously perfusion culture to the end. Experiment group B was the intermittent perfusion culture, which open the pump to 10 rpm, every 3 hours perfusion culture 1 hour during the day and at night continuously perfusion culture 8 hours. Cell-scaffold complexes were harvested at time points and observed under SEM, assayed cells in various parts of the scaffolds, glucose consumption in the bioreactor, the cells ALP activity and osteogenic gene expression.Results1. Perfusion bioreactor construction and performance testBioreactor system had been running 14 days. The mechanical performance, tightness and flow rate were all good, without any abnormal conditions. When the pump rate was 10 rpm, the fluid shear stress was 0.0895dyne/cm2 (8.95×10-3Pa). Cytotoxicity and bacteriological testing results showed that using this bioreactor is reliable.Observed under the SEM and FM showed that experimental group cell’s distribution and number in the scaffolds were superior to control groups. The cells of experimental groups could be seen well-distributed, while cells in control groups were gathered in the scaffold’s edges. With the culture time, the cells of experimental group began to proliferate overall the scaffold while the control group proliferated in the edge of the pore, it could be seen the dead cells in the inter pore.Cell viability assay showed that it decreased with the incubation time at two groups (P<0.05). Cell activities of Experimental group at 3,7,14 d were 1.87±0.38, 5.11±0.75 and 7.64±1.01; control group were 1.42±0.13,2.55±0.88 and 3.86±0.73. After culture 3 days, the viability of the two groups was no significant difference (t=1.917, P>0.05). After cultured 7 days (t=3.838, P<0.05) and 14 days (t=5.259, P<0.01), cell activities of experimental group were higher than the control group. 2. SD rat BMSCs isolation, culture and identificationUse of bone marrow adherent culture method successfully isolated SD rat BMSCs, identified by flow cytometry cell surface marker-positive cells were seized by the proportion of cells were:CD29 99.86%, CD44 98.99%, CD90 99.49%; BMSCs surface marker negative cells were seized by the proportion of cells were: CD451.26%, CD34 2.16%, CD 106 1.75%.After osteogenic induction 7 days and 28 days, ALP staining and alizarin red staining were active. After chondrocytes induction 10 days and 21 days, the toluidine blue staining and Alcian blue cartilage staining were active. After adipogenic induction 21 days, oil red O staining was active.3. Influence of distribution, proliferation and differentiation of BMSCs in scaffolds by two perfusion culture methodsSEM showed that after culturing 14 days, it was better that the density of cells of experimental B gourp inside the material than the experimental A group. After culturing 28 days, the visible form of persistent perfusion culture cells changed with increasing the cell body, more polygonal and was similar with osteoblasts cell.Glucose consumption showed that each group in the first 3 d was minimum and significantly increasing with time. Control group and the experiment B group went into the platform at 15 d and experimental A group went into about 20 d. Cultured for 28 days, the two experimental groups at the glucose consumption are 3.7 and 4 times to the control group. During all the time, two experimental groups were higher than control groups (P<0.01). After culturing for 20 days, there was no significant difference between two experimental groups (P>0.05).Determination of cells number in different scaffold parts showed that there were significant differences of control group, of which the bottom (part D) gathered the most of cells (P<0.01). There were no significant differences in the 7,28 d about experimental A group (P>0.05), but at 14 d there was a significant difference (P<0.01). LSD comparison showed the cell’s number at the bottorn>top> lower> upper part (D>A>C>B). Experimental B group in different parts of the cell number was no significant difference all the time (P>0.05). ALP activity assay showed that three groups continued to rise over time. Compared with 14 d, ALP activity in control group decreased slightly at 21 d. Two experimental groups were higher than the control group all the time (P<0.05). At 7d experimental A group’s ALP activity was higher than B group (P<0.05). At 14,21 d, there were no significant differences between two experimental groups (P>0.05).The three genes increased the expression of different degrees in all groups with the culture time. The two experimental groups regulated gene expression multiples was higher than control group at each time point. At 3 d, excluded OCN(P>0.05), the experimental A group was higher than B group in OPN and Col-I mRNA expression(P<0.05). At 7d, Experimental A group of OCN mRNA expression was higher than B group, while there were no significant differences of the OPN and Col-I (P<0.05). After culturing 14 days, there were no statistically significant differences of 3 mRNA’s expression between the two experimental groups (P>0.05).Conclusion1. The perfusion bioreactor was easily to operate, stable performance; compared with static culture, it was more suitable for constructing critical size tissue engineered bone in vitro.2. Primary cultured SD rat BMSCs with whole marrow adherent method was efficiency and high purity.3. This bioreactor could improve the distribution of cells in the critical size materials, increase the amplification efficiency and promote BMSCs’ osteogenic differentiation; compared with the persistent perfusion, intermittent perfusion had higher amplification efficiency. After culturing 14 days, there were no significant differences in ALP activity and Col-I, OCN, OPN mRNA expression between two groups. |
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