| BackgroundIn tissue engineering,the most common,and simplest,culturing strategy is to cultivate a cell-seeded construct in static media.However,due to poor nutrient/waste exchange long-term static culture leads to limited cell viability and extracellular matrix formation in the centre of the construct.Recent research has shown that the problems associated with poor diffusion can be mitigated with a flow perfusion bioreactor;in which media is forced through the scaffold pore network.An additional,and critical,advantage of culture in a flow perfusion bioreactor is flow-induced mechanical stimulation of cells within the construct,which can enhance matrix production.Despite several flow perfusion bioreactor systems have been developed for bone and cartilage,cardiac muscle and vascular tissue engineering in vitro,however,the requirements for tissue engineering skin culture are different from those of other tissues,which require the gas-liquid interface and low shear stress,and the uniformity of the nutrient supply in the flow field.we designed and developed a new perfusion bioreactor for the construction of uniform tissue engineering skin in vitro called a quasi-static plane homogeneous flow field(QSPHFF),and through the tray design to improve the culture efficiency and scale of production.Developing such a tissue engineered skin and native skin biopsy model is to simulate the growth environment of cells in vivo,and to evaluate skin transplantation,skin diseases,drug application and wound healing.How to explore suitable culture conditions according to different tissues,to extend the culture periods of tissues and organs,and to establish a stable skin organ model? That is an urgent problem to be solved in the field of in vitro culture.In this study,we applied the quasi-static plane homogeneous flow field to culture tissue engineered skin,mouse skin and human foreskin in vitro,gradually improved the difficulty of culture,explored and optimized the operating conditions of the flow field.It is proved that the flow field can be used for the culture of different tissues,and has broad prospects for scientific research and clinical application.Objective1.To optimize the structure of the reactor,determine the optimal theory flow rate based on computational fluid dynamics(CFD)simulation analysis of flow field flow characteristics,and to investigate the uniformity of flow field.2.To expand the application of QSPFFB,comparing the survival state of rat skin grafts in vitro with that of conventional static culture.3.To compare the effects different perfusion velocity of QSPFFB on the survival of human derived skin specimens,and to develop larger free skin models.Materials and methods1.Optimize the structural design of QSPFFB by CFD simulation method;Separate fibroblasts of prepuce skin to fabricate of the composite chitosan dermal equivalent.Collect Samples every 48 h,use a colorimetric method of MTT to evaluate the cell viabilities of the dermal equivalents,measure the absorbance of glucose and lactate in fresh medium by a microplate reader;On day 8,use Hoechst 33342/PI double staining method to detect the proportion of dead cells,and measure the absorbance values by a hydroxyproline assay to assess the collagens synthesized in dermal equivalents,determine the ultimate tensile strength of the samples using our in-house high-sensitivity small tension membranous biomaterial mechanical test device,use haematoxylin and eosin(H&E)staining to evaluate the morphology and distribution of fibroblasts in tissue engineering skin.2.Culture 4mm rat skin biopsies in both QSPFFB and static culture plate,collect the samples at 3,6,9 and 12 days.Use H&E staining,periodic acid-Schiff(PAS)staining and picrosirius red(PSR)staining to observe the integrity of the morphological structure of skin,the changes of basement membrane,epidermal thickness and epidermal cell number,and dermal thickness and dermal cell number;Use immunohistochemical staining to detect the expression of CK10 and PCNA in skin specimens;Then use TUNEL method to detect the apoptosis of the cells in the free skin.3.Place 4mm prepuce grafts were in the QSPFFB,and cultured at two different perfusion velocities,collect the samples at 3,6,9 and 12 days.Observe the skin by the naked eye,use H&E staining,PAS staining and VG staining to observe the structure integrity,basement membrane,cell morphology and dermal collagen fibers;Use TUNEL method to detect the changes of apoptosis of skin graft in two groups.Measure the dissolved oxygen levels in the two groups by means of a dissolved oxygen meter.Then culture 1cm size foreskin in both QSPFFB with high perfusion frequency and static culture plate at 7 days.Use Immunofluorescence staining to detect the expression of undifferentiated keratinocyte marker CK15 in the basal layer of skin samples,use immunohistochemical technique to detect the expression of cell proliferation marker PCNA,detect the apoptosis of samples by TUNEL method.Results1.CFD simulation study indicated that when the vertical distance between the bottom of the tissue culture plates and the tray was 2mm,the nutrient solution volume fraction distribution below the culture plates was uniform.When the diversion trenches were set,the liquid flow resistance was weakened,which was beneficial to the absorption of nutrient solution at downstream.2.CFD simulation results showed that the flow field was uniform and quasi static at the perfusion velocity of 80ml/min,which was the optimum perfusion velocity,and the uniformity of flow field is destroyed at 800ml/min.3.When the tissue engineered skin was cultured at 80ml/min perfusion velocity,MTT colorimetric showed an increase in absorbance values significantly higher than in other flow velocity and static culture groups,and the increase of glucose content and the decrease of lactate content were significantly higher than those of other flow rates and static culture group.The perfusion at 80 ml/min was most suitable for dermal equivalent maturation in vitro.4.After 8 days of culture in vitro,MTT showed no significant difference in cell viability at the same tray.H&E staining and Hoechst 33342/PI double staining showed that the cells and dead cells were evenly distributed in the dermal equivalent.The tensile strength and hydroxyproline content of dermal equivalent in different positions of 12-well plate were not statistically different.These results suggested that the tissue engineered skin in the QSPFFB is homogeneous.5.After Maintenance of ex vivo rat skin 12 days in dynamic culture compared to static culture conditions,H&E staining,PAS staining and PSR staining in dynamic culture showed that the preservation of epidermal and dermal structural integrity,subcutaneous connective tissue was relatively intact,the basement membrane was continuous,and the density of collagen fibers increased significantly,furthermore,the arrangement of the collagen fibers was more regular than that of the static group.6.Immunohistochemical staining for CK10 showed a disintegrated and weak staining in the static culture compared to dynamic culture,whereas those in dynamic culture increased in their staining intensity after 12 days of in vitro culture.7.The PCNA positive cells were still observed in the skin samples in perfusion culture group at 12 days,while the expression level of PCNA in the static culture group reached the peak at 6 days,and then the proliferation activity of the cells decreased significantly.The results of TUNEL showed that a large number of TUNEL positive cells appeared in the static culture group over time,which was significantly more than that in the perfusion group.8.Two kinds of perfusion frequency were carried out to culture 4mm prepuce skin for 12 days.Naked eye observation,H&E staining,PAS staining and PSR staining in Perfusion group 2(perfusion,1h,inter stop 5h)showedthat skin necrosis,Nuclear pyknosis,a decrease in number of cells,severe separation of dermis,disappearance of basement membrane,and collagen fiber accumulation,parallel to the skin surface.The results of TUNEL showed that the level of apoptosis was significantly higher than that in the perfusion group 1(perfusion,4min,inter stop 2min).9.Measurements of the oxygen dissolved into the culture medium showed that the levels in the high frequency perfusion group were better than those under low frequency perfusion.10.After Maintenance of ex vivo prepuce skin 7 days in dynamic culture compared to static culture conditions,H&E staining showed that the perfusion group preserved the skin structure better and existed a large number of cells.The expression of CK15 in the perfusion group markers was more active.The results of PCNA and TUNEL showed that the perfusion group had a higher cell proliferation activity and a lower apoptosis level,which was better than that of static culture.Conclusions1.The QSPFFB can provide a uniform,stable and quasi static environment for the large-scale production of tissue engineered skin products.2.The QSPFFB is superior to the traditional method of static culture,has fluent penetration of nutrition,suitable for culture and long-term preservation of skin tissues and organs from different sources.And the flow field provides a new and more scientific method for scientific research. |
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