| Tissue engineering skin has obtained the good curative effect both in refractory ulcer and small wound. However, it is a pity that due to lack of skin appendages, there are much difference from normal skin in many aspects such as morphology, function and texture. Hair follicle is an important skin appendage, which not only have hairdressing effect, but also participates in the physiological metabolism, defense, update of the skin. Dermal papilla is located at the base of the hair follicle and plays a vital role on the formation of hair follicle and the regulation of hair cycle; and many papers have shown that the hair inductive ability of DPCs(dermal papilla cells) is closely related with its aggregative growth pattern.Hair follicles can be more easily induced in vivo, while there is no breakthro ugh in vitro. The most important reason is that hair follicle formation needs not only the interaction between epithelial and mesenchymal cells, but also a suitable microenvironment. In vivo, the flowing blood can provide an environment which signal molecu les, hormones and extracellular matrix coordinate with each other, but these factors are hardly to imitate the complexity in vitro. With the use of perfusion, the nutrition supply, mechanical force and molecular penetration from circulation system in vivo can be simulated in a certain extent.In this study, the method to isolate DPCs of rat whisker is explored. 3D collagen gel culture is developed to construct DPCs to gel spheroids. And in perfusion device, the tissue engineering skin which contains the DPCs gel spheroids were cultured, which laid a foundation for further research of the follicle induction in vitro.Objective:1. The combination method of microdissection and collagenase and the two-step enzymatic digestion method are uesd to seperate and culture DPCs of the hair follicle from rat vibrissae. To compare the results of the two methods to set up an efficient, fast, and easy method for isolation and culture of DPCs.2. Using collagen gel culture to construct DPCs into 3D gel spheroids which are rich in extracellular matrix and explore the suitable reconstruction manner in vitro. In this way, the aggregative growth property of high passage DPCs can be partly restored.3. The tissue engineering skin including DPCs gel spheroids is made to be dynamic cultured in perfusion bioreactor in order to observe whether the hair follicle can be induced.Methods:1.Using the two-step enzyme digestion method and microdissection collagenase combined method, DPs from SD rat vibrissae were respectively isolated and cultured t o compare with the harvest rate, shape, type, adherence time and operation efficiency. These results were statistically analyzed;2.Collagen derived from the rat tail was chosen as scaffolds. In order to find the suitable manner for the reconstruction of DP in vitro, the combination of different amount of gel with different number of cells were used to construct the three-dimensional DP gel spheroids and the pattern, final shape and size of the different combination were observed.3.Passage1 and 6 of DPCs were used respectively to build the dermal papilla gel spheroids, their vitality is compared. The alpha SMA and versican immunofluorescence staining were used to test the aggressive property and inducing ability.4.Using the two-step enzymatic digestion method to separate and cultivate human KC and Fb. In perfusion bioreactor, the tissue engineering skin including DPCs gel spheroids was made and dynamic cultured, comparing with static culture. After 10 days, HE staining was used to observe whether there is a hair follicle structure formation.Results:1. Two-step enzymatic digestion and microdissection collagenase combined method both could obtain complete dermal papillae. The operation time of two-step enzymatic method is shorter with more DPCs including coat and vibrissae type compared with microdissection collagenase combined method which the number of harvested DPs was smaller but high purity. There was no significant difference in the adherent time and cell outgrowth time.2. The newly created collagen gel 3D culture method could successfully construct DPCs gel spheroids.3.The shape of gel spheroids was round or oval with diameter in 200-1250μm;Cell number and the amount of gel both could affect the diameter, and there was interaction effect in exploring experimental conditions, the case of collage 100 μl with 105 cells cause the predicted minimum diameter: 352μm. As the amount of cells to reduce, while the increasing of the amount of gel, the diameter had a tendency to gradually increase.4. After taking the spheroids to a new plate, complete aggregative growth of dermal papilla cells could been found around the passage 1 DPCs gel spheroids, whi le there were single dermal papilla cells radiated out from passage 6 DPCs gel spheroids.5. Alpha SMA, versican immunofluorescence test were all positive in passage 1 and 6 DPCs gel spheroids. However, the test of alpha SMA, versican immunofluorescence te st of passage 6 DPCs in 2D culture were negative.6. After 10 days culture, the tissue engineering skin in dynamic perfusion device on the HE staining showed that more clearly structure could been seen in tissue engineering skin cultured than that with static culture, and epidermal layer did not show hyperkeratosis or fall off;7. Dermal papilla cell gel spheroids fit better with human tissue engineering skin in perfusion culture, and above the DPCs gel spheroids, a down growth structure of epidermis like “hair germ” can be found.Conclusion:Two-step enzymatic digestion method is a convenient, fast and efficient method to isolate DP of rat vibrissae. The collagen gel 3D culture is an effective way to reconstruct 3D dermal papilla spheroids and the aggregative growth property of high passage DPCs can be restored partly; Comparing with static culture, perfusin culture is more suitable for the cultivation of the tissue engineering skin. Under the condition of perfusion, the chimeric tissue engineering skin has the potential to induce the formation of hair follicle in vitro. |
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