| Articular cartilage is an avascular, aneural, and alymphatic tissue that covers the endof it. Because of the limitation of its natural healing ability, articular cartilage damage caneasily lead to osteoarthritis. Therefore, the research into methods of restoring thearticulation surfaces, which included the method of construction of functional cartilage byregenerative medicine, is always the topics of clinicians. Recently, tissue engineering hasbeen introduced as a new method of restoring the articular cartilage damage. Someprogress of these concepts in orthopaedics and traumatology provides new perspective andopportunity for the cartilage repair. Most progress in cartilage tissue engineeringincorporate three main components: multipotent mesenchymal cells which is capable ofdeveloping into chondrocytes, growth factor proteins and a suitable biocompatiblescaffold. Previous studies have proved that adult MSCs such as bone marrow stromal cells(BMSCs) and adipose-derived stem cells (ADSCs), which have proliferative potential andmultidifferentiation capacity, could be applied extensively in the study of cartilage tissueengineering. However, with the same culture environment, the difference of chondrogenic ability of BMSCs and ADSCs is unclear. In the clinical application, we must supplyexogenous cytokines and growth factors with the limitation of high-cost and shorterhalf-life, to stimulate chondrocytes to synthesize matrix components. Platelet-rich fibrincan be considered as a second-generation platelet concentrate, prepared from autologousblood after immediately centrifuged. This open-access technique is the most simple andfree systems, which can serve as a biological scaffold material and can slow, release manykinds of growth factors. It was developed by clinicians for clinical and experimentresearch in recent years. We’ve learned that by putting the cells cultured in vitroinoculation in particular biological scaffold material, and then transplanted to tissueregeneration and repair of the traditional method, there is a loss of cell volume andtransplant area inflammation. And cell sheet technology research and development hasopened up a new way for the solution of these problems, it not only can retain a largenumber of cells, but also can store rich extracellular matrix and reduce the postoperativeinflammatory response of transplant area, so it will have broad prospect of application intissue engineering. However, many studies have found that formed by conventional staticculture system in vitro tissue engineering cartilage in the exercise of normal function isinsufficient, and the pressure as important factors in the occurrence, development andreconstruction of cartilage and roles in cartilage regeneration has gradually beenconcerned. However, more research of cartilage tissue engineering were about stem cellsproliferation and differentiation by the control of chemical and growth factorsenvironment, it is rare to explore the chondrogenesis effect of stem cells by mechanicalstimulation. Therefore, this study explore the effect of pressure on the capacity ofchondrogenic of compound membranes in vitro, and compare the chondrogenic effect ofBMSCs and ADSCs in the same vitro culture environment by constructing a new type oftissue engineering graft, which used adult stem cell sheet to composite self-PRF, throughadopting controllable load device to simulate microenvironment within the joints in thebody mechanics. It can provide a new idea for improving the quantity and mass of tissueengineering cartilage, and establish theoretical foundation and experimental evidence forcartilage tissue engineering screening superior seed cells. The study was divided into four parts. The first part was the construction ofBMSCs/PRF compound membranes and identified the biological characteristics. A totalof two healthy male New Zealand rabbits aged three or four months. High purity ofBMSCs was obtained from New Zealand rabbits by density gradient centrifugation,BMSCs cell sheet was obtained after flow cytometric analysis was performed to detectcell surface markers, and osteogenic and adipogenic induction were explored. Auriclearterial10ml whole blood was taken from the same rabbit and centrifuged with3000rper min for10minutes to acquire PRF clot. PRF membrane was obtained after squeezingout the serum from the clot and then cut up it into particles, and mixed with BMSCs cellsheet fragments for4days. It was observed by scan electron microscope. The resultshowed that a tightly attached between BMSCs and PRF, which form a whole.Determined by MTT method to detect the proliferation of BMSCs in compoundmembranes, the result showed that the proliferation rate of BMSCs in group withBMSCs/PRF was obviously superior to without PRF group. ELISA method to detect thereleasing of growth factors of PRF in compound membranes, the results showed that theconcentration of various growth factors all gradually decreased over time, but the speedis relatively slow.After the first part, we conducted the second experiment to explore the effects of thepressure on BMSCs the proliferation and chondrogenic differentiation in compoundmembranes. By using liquid static pressure loading device and set to different levelpressure. Afterwards, the BMSCs/PRF compound membranes were stimulated by0KPa(control)、90KPa、120KPa、150KPa of hydrostatic pressure for1or6h(s) per day for2,4and6days, and under the of respectively. Gene expression of PCNA，Sox-9，Aggrecan，Col-II was analyzed by real-time PCR after2,4and6days. The real-timePCR study results showed that the proliferation of BMSCs could be promoted obviouslyin the condition of pressure, especially120KPa/1h for4days, which characterized bythe gene expression of PCNA. After2consecutive days of pressure, Aggrecan wasstatistically up-regulated, while the expression of Sox-9and Col-II was almost the sameas baseline. However, all these three genes expressions were up-regulated by4 consecutive days of pressure, particularly in the group of120KPa/1h. In the groups of6consecutive days of pressure, only the Col-II gene expression under90KPa or120KPafor1hour increased. At the same time, all pressure groups have high genes expressingfor4days and decreased after6days, there have no significant difference between1hand6h groups with the same pressure. Thus,120KPa/1h for4days is the optimumpressure condition. In this connection, we further observed the effect of the suitablepressure on morphology of BMSCs in compound membranes. BMSCs cell sheets wererandomized to4groups：BMSCs cell sheet was as control group, BMSCs/PRF group,BMSCs cell sheet+pressure group, BMSCs/PRF+pressure group.120KPa/1h per dayas the pressure stimulation, all groups were sacrificed at4days. BMSCs ultrastructurechanges in the compound membranes were observed by transmission electronmicroscope（TEM） and BMSCs sheet surface changes were observed by scanningelectron microscope (SEM). TEM and SEM observed results showed that compared withcontrol group, BMSCs endoplasmic reticulum showed height expansion in BMSCs/PRF+pressure group. At the same time; a mass of collagen fibrillar network and granularsecretions distributed on the sheet surface in BMSCs+pressure and BMSCs/PRF+pressure group, especially the BMSCs/PRF+pressure group was the most significant.In the third part of the experiment, we use the easier obtained stem cell(ADSCs) toconstruct the ADSCs/PRF compound membranes, and observed the effect of suitablepressure on ADSCs the capability of chondrogenesis and compared with BMSCs,explored ADSCs whether can truly replace BMSCs for construction of tissue engineeringcartilage. Cultivating rabbit ADSCs by tissue block method, ADSCs cell sheet wasobtained after flow cytometric analysis was performed to detect cell surface markers, andosteogenic and adipogenic induction were explored. Auricle arterial10ml whole bloodwas taken from the same rabbit and centrifuged with3000r per min for10minutes toacquire PRF clot. PRF membrane was obtained after squeezing out the serum from theclot and then cut up it into particles, by using the same method of BMSCs/PRF toconstruct ADSCs/PRF compound membranes. ADSCs cell sheets were randomized to4groups: ADSCs cell sheet were as control groups, ADSCs/PRF group, ADSCs cell sheet +pressure group, ADSCs/PRF+pressure group. ADSCs groups were sacrificed at4days,120KPa/1h per day as the pressure stimulation, all groups were sacrificed at4days.ADSCs ultrastructure changes in all groups were observed by TEM. The results showedthat pressure stimulated has no obvious effect on ADSCs. Gene expression of PCNA，Sox-9，Aggrecan，Col-II was analyzed by real-time PCR for all groups after2,4and6days.Real-time PCR results show that the expression of PCNA., Aggrecan and Col-IImRNA both reached the highest after2days, compared all the treatment groups, theexpression of PCNA and Aggrecan and Col-II had no significant difference, only on theexpression of the Sox-9, all experimental groups with more higher expression than pureADSCs sheet group. Compared all the level of genes expression above of ADSCs to thecorresponding BMSCs group, the results showed that BMSCs whether or not in thecondition of stress or PRF, PCNA, Sox-9, Aggrecan, Col-II gene mRNA expressionwere significantly higher than the corresponding amount of ADSCs group. The groups ofADSCs have lower genes expressions of proliferation and chondrogenic differentiationthan BMSCs.On the basis of the first three parts, in the fourth part of the experiment based onBMSCs/PRF compound membranes, to construct tissue engineering cartilage and toobserve the effects of optimal pressure on the construction of cartilage. We constructedtissue blocks by using three layers BMSCs cell sheets combined one layer PRF. Thetissue blocks were randomized to4groups：tissue block was as control group, appliedthree different treatments respectively to the tissue block per day were single optimalpressure stimulated, single chondrogenesis, and both of them concurrence. Tissuemorphology characteristic was observed by Hematein Eosin and Toluidine Blue stainingafter2and4weeks. The results showed that lots of chondrocytes were both observed inpressure stimulated group and pressure combined with chondrogenesis group after4weeks, and cartilage matrix were distributed both in the two groups too, especiallyobviously observed in pressure combined with chondrogenesis group, but after onlycultured2weeks, there have no chondrocyte and cartilage matrix in all groups. Conclusion:1. Using BMSCs cell membrane fragments according to the suitable proportion tocomposite autologous PRF membrane particles can build a new type of tissueengineering graft. This graft has good biological characteristics, and can providedouble support matrix and growth factors microenvironment for BMSCs.2. Stress stimulation can obviously promote the capacity of proliferation andchondrogenic of BMSCs in the BMSCs/PRF compound membranes,120KPastatic pressure1h per day for four days is the optimal pressure condition forBMSCs/PRF chondrogenic differentiation in vitro. Under the pressure, it canpromote the synthesis of protein and polysaccharide macromolecules substancesby stimulating the expansion of endoplasmic reticulum of BMSCs, and activatingthe organelles; while in the group without pressure,, there have no correspondingphenomenon.3. Under the same pressure condition, the ability of proliferation and chondrogenicdifferentiation of ADSCs/PRF compound membranes is weaken than thecorresponding BMSCs; in the same culture environment, BMSCs has strongerchondrogenesis capability than ADSCs in vitro.4. By using three layers of BMSCs cell sheets to composite one self-PRF sheet, itcan form cartilage after4weeks under120KPa/1h pressure per day orchondrogenesis induced. We found that pure stress stimulus can replacechondrogenesis induced, but if these two factors combined together, the quality ofthe tissue engineering cartilage will be improved. If neither pressure norchondrogenesis induced environment, BMSCs/PRF compound membranes wasnot formed cartilage mass after4weeks in vitro. |
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