| Cartilage tissue has simple structure without vascular or nerves and is therefore limited in its ability to repair itself. Traditional cartilage defect treatment methods have limitations which can not completely recover their functionalities, using the method of tissue engineering to build large osteochondral constructs would be one of the best ways to solve large scale of osteochondral defects in the future. In this research, we take porcine bone which has strong biocompatibility, similar structure to human bone tissue as the bone biomimetic scaffolds after the procedures of derosination, decalcification and deproteinization. Take chitosan/gelatin hydrogel scaffold which was made through mixing, freeze-drying and cross-linking as the cartilage biomimetic scaffolds. The feasibility of the osteochondral constructs for osteochondral defection treatment was evaluated after inoculation of osteogenic and chondrogenic differentiated Adipose-derived stem cells (ADSCs).First, prepare cancellous bone and detect the porosity and distribution of pore size. Then, observe surface morphology with the scanning electron microscope (SEM) and detect its composition using infrared instrument. A lot of nanoscale protuberance was found on the surface of cancellous bone through SEM observation, and holes were connected inside the scaffold with the average pore size of410±59μm and porosity of70.64±1.71%. Through infrared detection, we could find that organic substances like hydroxyapatite and protein were contained in the scaffold. Prepare chitosan/gelatin hydrogel with different concentrations and detect their porosity, degradation ratio and swelling ratio. Then choose the group with best comprehensive performance for the later experiment and observe its pore size, micro surface morphology and analyze the chemical changes after cross-linked through infrared detection. The mass ratio of the gelatin and chitosan in the hydrogel was3:1and we set the total concentration of chitosan gelatin at3%,4%,5%. After the detection of relevant physical properties, the group of4%was shown to have the optimal comprehensive performance, whose porosity, swelling rate and degradation rate were suitable for cartilage biomimetic scaffolds and holes inside the hydrogel scaffold were connected to each other. The hydrogel scaffold has an average pore size of117±21μm. Its porosity and swelling rate were83.40±0.79%and362.00±2.38%respectively. The hydrogel remained80.76%±1.6%of the primal dry weight after six weeks of soaking in the PBS. The infrared detection showed that there were chemical reactions between the functional groups of gelatin and chitosan after crosslinking. Cancellous bone scaffold and hydrogel scaffold have similar physical performances with in situ cartilage and bone. Besides, the bone scaffold and hydrogel scaffold also have strong biocompatibilities.Human ADSCs were used as resource cells after separation, proliferation and induction towards adipose cells, osteoblast and cartilage cells. Then, growth conditions and morphologic changes were observed and ability to differentiate into osteoblast, cartilage and adipose cells was tested by conventional staining. The separated ADSCs grew with adherence, shuttle shaped. They grew speedily with5to6days per generation. After two weeks of induction in adipocyte medium, cells began to have morphological changes and the results of oil red staining indicated that there were red small droplets of oil in cells. After three weeks of induction in cartilage medium, polysaccharides extracellular matrix secreted by induced stem cells were stained lavender by toluidine blue staining. After bone induction, the experimental group was tested positive while the control group was not stained or was not apparently stained by ALP staining, von Kossa staining and alizarin red staining. Overall, ADSCs stayed stable in morphology and grew speedily and had the ability to differentiate into adipose cells. osteoblast and cartilage cells, which were suitable resource cells for osteochondral constructs.Bi-layered scaffold were prepared using the following procedures. First, differentiated ADSCs were inoculated to cancellous bone and hydrogel scaffold with the density of1×107cells/mL followed by a two weeks induction in24-well plate. Then we combined these two parts to construct osteochondral constructs. The construction of osteochondral constructs were divided into three groups:bi-layered osteochondral constructs as the experimental group, separation culture scaffold as the control group and blank group without cells seeded. After two weeks of co-culture, use inverted microscope to observe cell growth conditions and Dead/Live fluorescent staining to observe cell proliferation. Besides, analyze staining results and cell viability with IpWin5software and observe the bone differentiation by alizarin red staining. The cell growth state and extracellular secretions on hydrogel scaffold and cancellous bone in experimental group were observed using SEM. The results of inverted microscope examination showed that adhesion of the ADSCs on the holder was very well and there were cell colonies in various sizes. IpWin5software was used to analyze the optical density and the staining area of fluorescence staining and t-test was used to test significance. The accumulated optical density of the experimental group and the control group on the cartilage scaffold were350059dpi.163129dpi respectively, while that on the bone scaffold were306222dpi.31595dpi respectively. P<0.005. The fluorescence staining area of experimental group and control group on the bone scaffold were14815.10μm2,1412.13μm2respectively, while that on the cartilage scaffold were16836.96μm2,7829.27μm2 respectively, P<0.005. The result showed that there were significant differences on the optical density and the staining area between experimental group and control group, which indicated that cells on the osteochondral constructs (experimental group) had stronger proliferation ability and activity than those on the monolayer biomimetic scaffold during isolated culture. Alizarin red staining showed that staining of the experimental group on the cancellous bone scaffold was deeper than the control group. The cell clusters growing in the holes of hydrogel scaffold and cancellous bone could be seen under the SEM. Extracellular secretions like mineral salt and hydroxyapatite crystals on cancellous bone and white extracellular secretions on the surface of hydrogel scaffold could also be observed under SEM. The above detections indicated that the osteochondral constructs (experimental group) had apparently achieved better construction results. Therefore, osteochondral constructs prepared in this experiment can be used in the constructs of osteochondral tissue engineering. |
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