| BackgroundHair follicle is a accessory organ of skin that controls hair growth. During the post-natal life process of mammalians, hair follicles undergo a repeatedly cycle that containing growth period (anagen), regression period (catagen) and resting phase (telogen). Based on such cycled re-growth, hair follicles exhibited a fully ability in self-renewal and regeneration. Many factors can affect the hair cycle, which can cause various hair-loss diseases. Baldness diseases that most commonly seen in clinical medicine includes androgen alopecia, alopecia areata and so on. Among these diseases, androgen alopecia is the most common form of hair-loss. Although they do not belong to a serious disease, but they will cause a great psychological pressure to most patients that showing a serious impact on patient’s life. At present, the treatment of alopecia mainly includes the followings:one is the drug therapy, namely using minoxidil and finasteride. But it can only slow down the process of hair-loss temporarily; Second, surgical treatment, namely autogenous hair transplantation. By transplanting hair-follicles from unbald area to bald area, however, it can only fulfill a redistribution of hair follicles on scalp region and dose not increase the actual amount of hairs. Especially to those who suffer from a large baldness area and lower hair follicle number on donar area, this method encounters many limits. In addition, for those who suffer from scar on donor area, plus a unstable survival rate after transplantation and a relatively lower efficiency for performing operation may also affect the therapeutic outcome of hair transplantation. In recent years, with the development of tissue engineering and regenerative medicine, bioengineering the hair follicle by tissue-engineering technologies has gradually become an ideal option for the treatment of alopecia with large area.The premise condition for reconstruction of bioengineered hair-follicles is to obtain sufficient and seed cells possessed with biological function. The composition of hair follicle contains two parts-epithelium and dermis. The interaction between the epithelial and mesenchymal determines the normal process for hair follicle morphogenesis, cycle and regeneration. The epidermal components include hair follicle stem cells, hair germinal matrix cells, the inner and outer root sheath, while the dermal components mainly contain dermal papilla (dermal papilla cell, DPC) and connective tissue sheath. Among these, the DPC hair follicle, induced periodic cycle and play a indispensible role in hair-follicle morphogenesis, cyclic activities and induction.In the normal body, DPCs exhibit an aggregative and closely connected pattern of cell growth, forming into a papilla-like three-dimensional structure (DP) that ranging between 150-250 microns. Under conventional two-dimensional (2D) in vitro culture, although DPCs could still retain some of their unique biological characteristics and functions, including self-aggregation pattern of cell behavior, expression of various and specific cell markers, and the ability to induce hair-follicle formation. However, with the increase of passaged number, cultured DPCs gradually lose such characteristics and biological functions. As far as now, this gradual loss of biological characteristics and function in cultured high-passaged DPCs has not yet been fully understood. Therefore, the key to the reconstruction of human hair follicle by tissue engineering is that how to maintain such intrinsic characteristics and biological function of DPCs during passaging in vitro. Studies have shown that three dimensional culture system helps to establish a biomimetic microenvironment for cell culture in vitro. Among these 3D systems, hanging-drop (HD) is relatively common and simple method. In recent years, a great advance that combines traditional hanging drop to high-throughput technology has prevailed the utilization of hanging drop culture plates (hanging drop plate, HDP). Such innovative three-dimensional culturing platform has demonstrated great advantage in reconstructing 3D cell culturing model and 3D microtissues.Therefore, in this study, we thought to establish an in vitro 3D model for both controllable and scaleable production of hair-inductive high-passaged human DPC microtissues by utilizing advanced hanging-drop micro-arrays. We believe that this model not will only provide a valuable tool for exploring the cell biology of human dermal papilla, but also hold a potential to produce sufficient hair-inductive high-passaged DPCs to be applied for future regenerative medicine.Objective1. To establish an in vitro 3D hanging-drop model for high-passaged human dermal papilla cells, to further produce bimimetic 3D microtissues of high-passaged human dermal papilla cells based on the in vitro 3D hanging-drop model2. To characterize and evaluate the bimimetic 3D microtissues of high-passaged human dermal papilla cells3. to apply bimimetic 3D microtissues of high-passaged human dermal papilla cells for in vivo hair-follicle reconstructionMethods1. The establishment of 3D hanging-drop culturing model and 3D microtissue for high-passaged human dermal papilla cellsA method that combined enzyme digestion with micro-dissection was applied for the isolation of DP. Isolated DP was observed for morphology, adherence and cell migration. P1-P10 DPCs were cultured and observed the changes in morphology and cell behavior; CCK 8 assays were conducted for cell proliferative activities in P1-P10 DPCs; Immunofluorescence staining were performed to evaluate the DPC specific markers including ALP and alpha SMA.For the optimization of seeding density, passaged 3 DPCs were divided into four groups according to the diameter of a normal dermal papilla that ranges between 150-250 microns, then cells were seeding with the following low cell densities:1× 104/40 μl/well,0.5 X 104/40μl/well,0.25 X 104/40μl/well,0.1 X 104/40 μl/well, respectively. Each group was seeded with 30 wells, and the formed microtissues were observed and recorded under a microscope. Statistical analysis was conducted to summarize the size distributions and counting numbers of microtissues in each group. After that, the optimal seeding density for DPC microtissues were determine.According to the optimal seeding density (0.25 X 104/40 μl/well), DPCs were culture for up to 14 days. Morphology and diameter of DPC microtissues were recorded under a reverse phase-contrast microscope at different time points of 0,1,4, 7,10,14 days during culture, respectively. On 7,10,14 days of culture, DPC microtissues were collected for Live/Dead staining for evaluating the cell survival within microtissues, respectively. After that, the optimal culturing days for DPC microtissues was determined according to the aforementioned results.Passage 2 and passage 8 DPCs were prepared into cell suspension, cell concentration were adjusted to 6.25×104/ml, then cells were seeded with the optimal condition and cultured for 7 days. After microtissues were formed, DPC microtissues were observed and recorded under microscopic observation.2. The characterization and evaluation of the 3D hanging drop culturing model for high-passaged human dermal papilla cellsDPC microtissues at passage 8 were collected, fixed in 4% paraformaldehyde solution, then procceded with H&E staining, proliferation markers--Ki67 staining and apoptosis markers--TUNEL staining;In vitro re-inoculation and simulation culture:DPC microtissues were collected, then transferred and injected to another culture plates using 200μl pipette for another 7 days of incubation. Cell morphology and outgrowth from microtissues were observed under the reverse phase-contrast microscope. Primary DP was used as a positive control.Passage 2 DPC, Passage 8 DPC, and Passage 3 dermal fibroblast (DF) were all prepared for either conventional 2D culture or 3D hanging-drop culture according to experimental design. All 2D cultured cells and 3D microtissues were characterized with the biological properties as the followings:qRT-PCR was conducted to evaluate the gene expression of ALP, alpha SMA and NCAM; immunofluorescence staining was performed to qualitatively detect the protein expression of ALP, alpha SMA and NCAM; western blotting was carried out to quantitatively characterize the protein expression of ALP, alpha SMA and NCAM.3. The application of 3D microtissues of high-passaged human dermal papilla cells for in vivo hair-follicle reconstructionPassage 8 DPCs were labeled with the fluorescent dye CM-Dil for in vivo cell-tracking prior to microtissue fabrication. Either newborn mouse epidermal cells alone (1×106cells) or Passage 8 DPCs under 2D culture mixed with epidermal cells (1 ×106cells) were used as the control of experiment. After 3-4 weeks of implantation, all animals were sacrificed. For macroscopic observation, dissected samples were first characterized and imaged under a stereoscope. For further histological examination, the specimens were fixed in 10% paraformaldehyde buffered solution, paraffin-sectioned and processed for hematoxylin and eosin (H&E) staining. Frozen sections of grafts were also prepared. Induced HFs were observed and imaged under alight phase-contrast or fluorescent microscope.Results1. The establishment of 3D hanging-drop culturing model and 3D microtissue for high-passaged human dermal papilla cellsDP was isolated intactly and exhibited a pyriform or oval shape. After reinoculation, isolated DP can naturally adhere to culture plate and the cell migration was observed. Migrated cells were observed to exhibit a long-spindle or irregular polygon morphology, growing into a radiated pattern around the primary DP; when passage 1-6 human dermal papilla cells achieved confluent growth, they showed a distinct self-aggregation behavior, exhibiting a swirling cell pattern; After passage 7, such cell behavior and growth pattern became unconspicuous; cultured DPC exhibited a slightly larger size than that of fibroblasts. Cells less than passage 4 were even observed to be capable of forming spheroid self-aggregates; Such self-aggregation of DPCs were observed to be lost gradually with the increased number of passaging; Formed self-aggregates were found significantly reduced in passage 6 DPCs, while no aggregation growth was observed in passage 8-10 cells.To passage 1-10 DPCs, as culturing time extended, the cell viability of s gradually increased; while the proliferation ability gradually declined as the number of passaging increased in passage 1-10 DPCs.Cultured DPCs that originated from enzyme digestion combined with microdissection method exhibited positive expression of both ALP and a-SMA.DPCs were further seeded with low cell densities of 1.0× 104/40ul/well,0.5× 104/40ul/well,0.25×104/40ul/well and 0.1×104/40ul/well.Results demonstrated that the size of formed DPC microtissues were larger with increased density. Further analysis verified that most number of DPC microtissues in which the diameter ranged between 150 and 250 μm lies in the group of 0.25×104cells/well. The size of DPC microtissues within this range is the closest to that of a primary DP.0.25 X 104/40ul/well was determined to be the optimal seeding density for establishing DPC microtissue.Both low passaged (P2) and high passaged (P8) DPCs were seeded with the optimal density of 0.25×104/40ul/well and cultured for 7days, the numbers of established DPC microtissues that is the closest to a primary DP was found no obvious difference between two groups.2. The characterization and evaluation of the 3D hanging drop culturing model for human dermal papilla cellsThe appearance of cultured DPCs displayed a relatively long spindle or irregular polygon shape, self-aggregation behavior was visible, presenting a distinct swirl pattern; while DF exhibited a long and thin spindle-shape, smaller than DPCs in size, no self-aggregation was observed, presenting a weaving cell pattern.Histological features:microtissues possessed a complete structure;uniformity of cell arrangement was observed within microtissue; large amount of extracellular matrix components were found to be filled with intercellular space, similar to the normal DP organization.Immunohistochemical properties:Ki67 staining and apoptostic TUNEL assay revealed that cell characteristics within DPC microtissues exhibited both relatively low percentages of proliferation and apoptosis, where only few (5.2%) Ki67-positive cells mainly located at the edge and the apoptostic cells (5.93%) distributed sparsely within the DPC microtissue.To validate the integrity and functionality of DPC microtissues after in vitro simulating injection, DPC microtissue were re-transferred and injected into new dishes for another 7 days of incubation. As expected, the re-inoculated DPC microtissue still maintained an intact morphology after injection. Being similar to a primary DP, grafted DPC microtissue became attached on the culture dish after 3 days of re-seeding and then cell migration was observed within 7 days of culture, which was also confirmed by SEM observation.qRT-PCR, immunofluorescence and western-blotting test showed that:signature genes and proteins of DPCs including ALP, a-SMA and NCAM were all restored and well-preserved in high-passaged DPC microtissues; expression of these distinct DPC markers associated with HF-inductivity were all found significantly up-regulated in P8-DPC microtissues, when normalized against the P8 cells under 2D culture. However, no significant expression was found up-regulated in DFs-formed microtissues.3. The application of 3D microtissues of high-passaged human dermal papilla cells for in vivo hair-follicle reconstructionTo induce de novo HF in vivo, P8-DPC microtissues mixed with newborn mice epidermal cells (EPCs) were subcutaneously implanted into nude mice. Aabundant newly-formed HFs and regenerated hair shafts were observed within the hypodermis of nude mice after 5 weeks of implantation. However, when P82D DPCs combined with epidermal cells or just epidermal cells alone (1×106 cells)were employed for subcutaneous implantation, no hair induction was presented in the recipient sites. H&E section showed that a great amount of mature HFs distributed within the hypodermis layer, while the fluorescence image further confirmed the human origin of these grafted DPC microtissues in the reconstituted HF.Conclusion1. The Dermal papilla from human scalp can be isolated and obtained by the enzyme digestion combined with micro-dissection method, which possesses a great advantages including high productivity, high efficiency, reduced labor intensity, and high adherence and cell emigration rate; this method does not significantly affect the cellular morphology, growth pattern, and specific markers expression including ALP and α-SMA of cultured DPCs; such method was used in our following experiment.2. Under in vitro culture condition, the distinct self-aggregation behavior of DPCs is gradually lost with increasing passages; no self-aggregation was observed in high-passaged cells (above P6) basically; compared with low passaged cells (less than P6), high-passaged cells (above P6) displayed a relatively lower proliferative capacity and doubling rate.3.3D hanging-drop culture is beneficial to assembly human DPCs to form into multicellular spheroid microtissues; the optimal conditions for culturing:DPCs are seeded with density of 0.25 X 104/40 μl/well, and kept under culture in 37℃ contains 5% CO2 incubator for 7 days, in which the established microtissues are the most stable and possessed an appropriate diameter that resembling a normal human dermal papilla; in addition, the survival rate of cells within microtissue was the best at 7days of culture; thus, such optimal condition was used to establish high-passaged human DP microtissues in our following experiments.4. Human high-passaged DPC microtissues that developed by 3D hanging drop model resembled a primary DP; cell characteristics within microtissues presented a relatively low level of both proliferation and apoptosis activities; microtissues were proved to be both injectable and transplantable, microtissues were verified to be capable of adhering and possessed the functionality of cell migration, similar to the normal dermal papilla in the human body.5. Results from qRT-PCR, immunofluorescence staining and immunoblotting demonstrated that:develop model constructed by 3D hanging drop, high-passaged DPC within microtissues can express DPC’s markers including ALP, alpha SMA and NCAM, suggests that the formation of 3D microtissues can restore and maintain the unique biological characteristics of high-passaged DPC.6. Developed by the 3D hanging-drop culturing model, our high-passaged microtissues were proved to resemble a primary DP in human body that referring to the similarity including cell biology, microtissue diameter, and biological characteristics and specific markers; hence,it is a biomimetic microtissue.7. DPC microtissues that established by the 3D hanging-drop culturing model, not only can restore and maintain both the intrinsic properties and biological function of high-passaged DPCs, but also can fulfill both a controllable and scaleable production of hair-inductive high-passaged human DPC microtissues that can be used for injection to reconstruct hair-follicles after transplantation in vivo, possessing a great potential for clinical application. |
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