Experimental Study On Cultivation And Identification Of Nail Bed Cells And Organization Of The Tissue Engineering Nail Bed In Vitro

As the appendage tissue of the skin, nail can protect the finger end and assistpinch and grasp. Also they can strengthen the felling of the finger pulp. Inaddition, they can support the finger to do some special action in some daily life.Also the nail is also an important aesthetic part.According to the assess standard of the traditional hand function, the naildefection doesn't result in the hand function loss. But, with the advancement ofliving level and increase of social intercourse activity, more and more patientswith the nail defection demand the rehabilitation of the nail. So it seem to beimportant to repair and rehabilitate the nail bed defection and damage.At present, bed defection and damage on the clinic is cured by the skin graftor transplantation of flap and nail bed of nail or toes. A great number of the resultsof these opertation were not satisfactory. Or it was done at a cost of the loss ofother finger nail or toes bed. Therefore, how to repair the nail bed defection anddamage is a difficult question of the hand surgery in clinic that have not beensolved.Along with the technical development of tissue engineering, the means ofthe organization of the tissue engineering skin have inclined to maturity. Tissueengineering is defined as the application of principles and methods of engineeringand life sciences toward fundamental understanding of structure-functionrelationships in normal and pathological mammalian tissues, and the developmentof biological substitutes to restore, maintain, or improve tissue function.Sometimes also called reparative and regenerative medicine, tissue engineering isan emerging interdisciplinary area of research and technology development thathas the potential to revolutionize methods of health care treatment anddramatically improve the quality of life for millions of people throughout theworld. Some products have already applied to the clinic, and their number willassuredly increase rapidly in the future.As the appendage tissue of the skin, nail bed resembles skin in tissuestructure in many aspects. They all divide into the stratum of epidermis anddermis. The function cells of the epidermic stratum are epidermal cells. And thefunction cells of the dermis are fibroblasts. Drawing lessons from the method ofthe tissue engineering skin, we have organized the tissue engineering nail bed invitro.The experiment is divided into three parts:The first part is the cultivation and identification of nail bed cells in vitro.The epidermal cells and fibroblasts were dispersed with the method ofenzyme digestion in the experiment. The epidermal cells were dispersed withtrypsinase step by step and cultured by serum-free kerationcytes medium. Thefibroblasts were dispersed with collagenase and cultured by DMEM. The cellswere observe by upside-down microscope. The cells were counted and the thegrowth curve of cells were drawn. The cells were identified by immunochemistrystaining.The results show: The epidermal cells were flat round or ellipse and arrangedlike paving stone. The cells generated a passage every 10 days until 8 passages.The fibroblasts were long shuttle and arranged like radial, knittingor circinateform. The cells generated a passage every 7 days and been generated 8 passages inthe experiment..Anti-K17 antigen staining was positive in the cultured nail bed epedrmalcells. And the origin of the cultured epidermal cells was proved. Anti-vimentinantigen staining was positive in the cultured fibroblasts and the origin of thefibroblasts cultured was proved.From the first part experiment , we can make conclusions: Nail bed cellscould be successfully cultured. The purity of the obtained cells were high. Theycould proliferate naturally and could be generated in vitro. The character andgrowth regulation of the cultured cells was stable in vitro. They could used as theseed cell of tissue engineering.The second part is the preparatinon of acellular dermal matrix.Acellular dermal matrix (ADM) was prepared by treated with the low densitytrypsinase digestion and iterative freeze-melt. The ADM was observed withmorphological and histological examination. The ADM was detected withgermiculture examination and cell toxicity examination.The results show: The ADM was white, soft smooth. It was so elastic andtenacious that it could been processed different shapes. Under light microscope,there was no epidermis, no cells and epidermic appendages(hair, ducts and glands).The structure of ADM was like a three-dimensional net, colleagen fiber appearedorderly. The membrance was clear and closely intact. There was no bacterium inthe germiculture examination after it was cultured for 3 day. Cultured with thefragments of ADM, the epidermal cells could proliferate naturally.From the second part experiment, we can make conclusion: There was noepidermis, no cells and epidermic appendages in the ADM prepared by treatedwith the low density trypsinase digestion and iterative freeze-melt. The structureof ADM was like a three-dimensional net, colleagen fiber appeared orderly. Themembrance was clear and closely intact.The third part is the organization of tissue engineering nail bed.The experiment is divided into two team. Only epidermal cells were seeded onto theepidermal side of the ADM in the group A. Fibroblasts were seeded onto the dermal side andepidermal cells were seeded onto the epidermal side of an ADM in group B. Tissuestructure of the tissue engineering nail bed built in this experiment was observedby HE staining. The immunochemistry staining was been applied to identify theorigin of epidermal cells.The results show:1. After seeded onto the ADM, the epidermal cells could proliferate naturally.The stratum basale is intact. The epidermis is composed of a few stratum andsimilar to natural nail bed structure.2. The cells of the epidermis were positive by anti-K17 antigenimmunochemistry staining.3. The epidermises of the group A and B were not different by HE stainingand immunochemistry staining.From the third part experiment, we can make conclusions:1. After seeded onto the ADM, the epidermal cells could proliferate naturally.2. After seeded onto the ADM, fibroblasts have no influence on the proliferation anddifferentiation of epidermal cells.3. The tissue engineering bed was similar to natural nail bed structure. It isfeasible to organize the tissue engineering bed in vitro.The innovation of the research are:1. The cultural method that nail bed epithelia cells in vitro were cultured withthe method of trypsinase digestion step by sep in serum-free kerationcytesmedium, was experimented for the first time.2. The method combined the low density trypsinase digestion and iterativefreez-melt, simplify the process of preparation of acellular dermal matrix (ADM).3. For the first time, the possibility of the organization of tissue engineeringnail bed in vitro was explored. And the tissue engineering nail bed was elementarystudy.These results confirm the possibility of the organization of the tissueengineering nail bed in vitro, which will lays academic basis and experimentalevidence for the further the study and transplant of the bed substitute.