IN VITRO HAIR FOLLICLE ENGINEERING PAN JING NATIONAL UNIVERSITY OF SINGAPORE 2014 IN VITRO HAIR FOLLICLE ENGINEERING PAN JING (B. Sc., China Pharmaceutical University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledges all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Pan Jing 24 January 2014 ACKNOWLEDGEMENTS I would like to thank and acknowledge many people for their contributions to this thesis. First of all, I am very grateful to my supervisor Dr Kang Lifeng. Thank you for your encouragement, enthusiasm, positive attitude, staunch support and guidance for my project which otherwise would not have accomplished. I would also like to express my thanks to my co-supervisor A/P Chan Sui Yung for her valuable suggestions and being always there for me. She has impressed me with her ability to communicate optimism which has helped me grow both personally and professionally. I am grateful to the Department of Pharmacy at NUS, for providing me scholarship and this wonderful opportunity. I thank other faculty members who advised me and gave their insights at some point or the other. I thank present and past members of the lab, Li Hairui, Dr Jaspreet Singh Kochhar, Dr Li Fang, Yan Jun and Sara Dana, with whom I spent numerous fun-filled hours at the lab. I would like to thank the Final Year Project students Wong Xin Yi Cheryl, Liew Xin Yi Cindy, Kuek Qi Min and Undergraduate Research Opportunities Program student Hiew Tze Ning for the time spent together in research. I I also want to thank the lab-support and administrative staff of our Pharmily, my research wouldn’t progress if not for your timely assistance. Lastly, and most importantly, I am deeply thankful to my wonderful parents for their love, support, and sacrifices. Without them, this thesis would never have been written. II CONTENTS ACKNOWLEDGEMENTS .I CONTENTS . III SUMMARY VI LIST OF PUBLICATIONS IX LIST OF TABLES X LIST OF FIGURES . XI LIST OF ABBREVIATIONS XIX Chapter Background 1.1. Introduction 1.1.1. Hair follicle morphogenesis 1.1.2. Hair follicle generation from dissociated cells 1.1.3. Optimizing positional relationship and cell compartmentalization to enhance EMIs 1.2. Literature Review . 1.2.1. Human hair biology, pathophysiology and treatment 1.2.1.1. Hair size, shape and pigment . 1.2.1.2 Human hair cycle . 11 1.2.2. Androgenetic alopecia . 15 1.2.2.1 Pathophysiology . 15 1.2.2.2. Treatment 16 1.2.3. 3D microstructure fabrication in tissue engineering 18 1.3. Objectives and scope 21 Chapter Poly (ethylene glycol) diacrylate (PEGDA) based 3D microstructural hydrogel as potential substrate for hair follicle cells 25 2.1. Materials and Methods 26 2.1.1 Master fabrication 26 2.1.2. Polydimethylsiloxane (PDMS)-stamp fabrication 27 2.1.3. Microwell fabrication 28 2.1.4. Microwell stability . 28 2.1.6. Mechanical testing . 29 2.1.7. Cell culture . 29 2.1.8. Toxicity exclusion tests 29 2.1.9. HaCaT cell seeding into microwells . 31 2.1.10. Field emission scanning electron microscope (FE-SEM) study . 31 2.1.11. Encapsulation of HDF cells in PEGDA hydrogel 32 2.1.12. Statistics 32 2.2. Results . 33 2.2.1. Microwell Fabrication . 33 2.2.2 Microwell stability 36 2.2.4. Mechanical testing . 38 2.2.5. Toxicity exclusion tests 40 2.2.5.1. Investigating effects of PEGDA solution to HDF viability 40 III 2.2.5.2. Effects of UV light exposure to HDF viability 41 2.2.5.3. Effects of photoinitiator (HHEMP) to HDF viability 42 2.2.5.4. Effects of combination of UV exposure and photoinitiator (HHEMP) on HDF viability 43 2.2.6. Cell compatibility 45 2.2.7. HaCaT cell seeding into microwells . 48 2.2.8. Cell growth (HaCaT) in the microwells . 49 2.2.9. Cell growth (HDF) in the microstructured hydrogels 52 2.3. Discussion 54 2.3.1. Microwell fabrication 54 2.3.2. Microwell stability . 56 2.3.4. HaCaT cell seeding into microwells . 56 2.3.3. HDF cell encapsulation . 57 2.4. Summary . 58 Chapter Hyaluronic acid based 3D microstructural hydrogel as potential substrate for hair follicle cells . 59 3.1. Materials and Methods 60 3.1.1. MeHA synthesis . 60 3.1.2. Proton nuclear magnetic resonance (1H-NMR) spectroscopy . 60 3.1.3. Hydrogel preparation 60 3.1.4. Hydrogel morphology by scanning electron microscopy . 61 3.1.5. Contact angle measurement . 61 3.1.6. Rheological study . 61 3.1.7. Cell culture . 62 3.1.8. HaCaT cell seeding 62 3.1.9. HDF cell encapsulation . 63 3.1.10. Statistics 63 3.2. Results . 64 3.2.1. 1H-NMR characterization of MeHA 64 3.2.2. FE-SEM study . 65 3.2.3. Contact angle measurement . 65 3.2.4. Rheological properties 66 3.2.5. HaCaT cell seeding 67 3.2.6. HDF cell encapsulation . 69 3.3. Discussion 71 3.3.1. Degree of methacrylation (DM) . 71 3.3.2. Contact angle measurement . 71 3.3.3. Rheology properties of MeHA hydrogels 72 3.3.4. HaCaT cell seeding 72 3.3.5. HDF cell encapsulation . 73 3.4. Summary . 74 Chapter Tissue culture . 76 4.1. Materials and Methods 76 4.1.1. HDF-HaCaT co-culture 76 IV 4.1.2. Cell monitoring in 3D microenvironment . 77 4.1.3. Immunofluorescence . 77 4.1.4. Histology study 78 4.1.5. Real-time polymerase chain reaction (PCR) . 79 4.1.6. Statistics 80 4.2. Results . 80 4.2.1. HDF-HaCaT co-culture 80 4.2.2. Cell distributions in the microstructures . 84 4.2.3. Cell proliferation and differentiation in the microenvironment . 86 4.2.4. Multiple hair follicle specific genes expressing in microwell system . 89 4.3. Discussion 93 4.3.1. HDF-HaCaT co-culture 93 4.3.2. Histology study 95 4.3.3. Gene expression . 95 4.4. Summary . 98 Chapter Conclusion 99 Chapter Future study . 101 REFERENCES 103 V SUMMARY Hair is a complex mini-organ that is important for the integrity of skin. While hair loss is usually not life threatening, it has a substantial psychosocial impact on the sufferers and can severely undermine the confidence of affected individuals and degrade their quality of life. As such, regenerating hair is of great clinical interest. Clinicians have resorted to transplanting hair follicles either from the patients’ own peripheral hair-bearing regions or from donor skin to bald regions. Because of the inability to generate hair follicles de novo, there is a shortage of human hair follicles for surgical transplantation. One potential solution is to use tissue engineering approaches to generate large quantities of human hair follicles in vitro to meet the clinical needs. From previously reported studies, hair follicle-like structures can be reconstituted by combining and transplanting mouse or rate epidermal and dermal papilla (DP) cells in non-hair bearing skin of animal models. However, hair follicle-like structures cannot be regenerated by using dissociated human cells, which may be due to the difficulties in re-establishing the cellular interactions during hair follicle development in vivo. In this thesis, we aim to design and explore a 3D microstructure resembling the architecture of the human hair follicles. Microwells with center islets were fabricated by using a patterned polydimethylsiloxane (PDMS) stamp on a glass substrate. Within the hydrogel microstructure, hair follicle inductive dermal cells can be immobilized to grow close to, but separated from epidermal cells. Poly (ethylene glycol) diacrylate (PEGDA) and hyaluronic acid (HA) were both considered as the candidate materials of the microstructure. VI PEGDA is a synthetic polymer, which has been commonly used in tissue engineering due to its high hydrophilicity, photocrosslinkability and low toxicity. Prior to encapsulating cells in PEGDA hydrogels, cytotoxicity of various factors contributing to the photocrosslinking process, including monomer solution, photoinitiator solution and ultraviolet (UV) intensity, were tested. PEGDA with higher molecular weight was shown to be less toxic to cells. Hydrogel stability was found to be inversely correlated with PEGDA concentrations, i.e., microstructures made of higher PEGDA concentrations were easier to detach from the underlying glass slide when immersed in PBS over time. It was also shown that epithelial and dermal cells were accurately compartmentalized within microstructures. Moreover, polymeric microstructures were shown to support the cell growth over 14 days. The natural polymer, hyaluronic acid (HA), was also studied as an alternative material of the microstructural hydrogel because of its biocompatible and biodegradable nature. HA was grafted with methacrylate groups to be photocrosslinkable. It was found that the hydrophilicity of methacrylated HA (MeHA) hydrogels decreased with increasing macromer concentration while the stiffness of MeHA hydrogels increased with increasing the macromer concentrations. These results are consistent with other reports. Also, the results of field emission scan electron microscopy (FE-SEM) study showed that high macromer concentration hydrogels possess smaller and more compact porous structure. Similar to PEGDA hydrogels, MeHA hydrogels were also shown to sustain cell survival and growth over time. However, gel swelling and weak stability hindered the use of MeHA hydrogels in long-term study. Thus, PEGDA was used as the material of microstructures for the study of cell-cell co-culture, cell proliferation and differentiation, and gene expression. Epithelial and dermal cells were co-cultured within PEGDA based VII performed in the future to test the function of the genes in the microwell-cultured cells. For example, it has been demonstrated that sonic hedgehog (Shh) is one of hair follicle inductive signals generated in the hair follicle placode and it is required for regulating epithelial proliferation during hair follicle development 9. In Shh-null hair follicles, epithelial proliferation is decreased 176 . Thus, we can transfect HaCaT cells with Shh-siRNA to knockdown Shh and determined whether Shh plays a role in the epithelial proliferation. 4.4. Summary HDF and HaCaT cells were co-cultured within microstructures upon 21 days. Most of cells remained viable at day 21. However, increasing hydrogel thickness resulted in a dramatic decrease in cell viability and very few cells were alive at day 14. From the confocal images, we have demonstrated that HDF cell distribution was relatively uniform in the 3D microstructures meanwhile HaCaT cells grew into a multi-layered cap-shaped cell aggregates. The results from immunostaining assays have shown that the microstructures sustained the cell proliferation and cell differentiation. In addition, cell aggregates on non-patterned hydrogels and 3D microstructures is AE-13 positive, indicating self-aggregation may be important for the differentiation of HaCaT cells into cortical keratinocytes. Furthermore, compared to 2D cultured cell mixture, the expression of gene Wnt10a, Wnt10b, Shh, KGF and BNDF was higher in 3D cultured cells, exhibiting the potential of 3D microstructures in guiding cell development for hair follicle regeneration. 98 Chapter Conclusion This thesis presents a novel system for hair follicle engineering in vitro by combining microtechnologies and tissue engineering. A 3D microstructure, containing a microwell and a center islet, was fabricated to mimic the architecture of the human hair bulb. In Chapter 2, the synthetic polymer, PEGDA, was employed as the base material of the microstructure. 10% and 20% (w/w) PEGDA microwells proved to be the most stable after 10-day incubation. Cytotoxicity of variable factors involved in polymerization process was evaluated. After microstructure fabrication, HaCaT cells were seeded in the microwells and the number of HaCaT cells in the microwell increased with cell seeding density. It was demonstrated that HaCaT cells grew into cell aggregates and remained viable over days. From the results of HDF cell encapsulation study, PEGDA MW 3500 showed the least cytotoxicity compared to PEGDA MW 575 and PEGDA MW 700. HDF cells remained alive and cell spreading was observed in the PEGDA (MW 3500) hydrogels. In Chapter 3, MeHA was used as an alternative material of the microstructural hydrogel. The hydrophilicity and stiffness of MeHA hydrogels were tested. HaCaT and HDF cells were also incorporated into MeHA hydrogels, separately to investigate cell compatibility. Cell viability on or in the MeHA hydrogels was comparable to that within PEGDA hydrogels. Similarly, HaCaT cells aggregates formed and grew in the MeHA microwells. Nonetheless, gel swelling and weak stability of MeHA hydrogels may restrain themselves in the long term development. Hence, PEGDA was considered to be more suitable for following studies. In Chapter 4, epithelial and mesenchymal cells were compartmentalized within microstructures in a similar way as in vivo and co-cultured over 21 days. Based on the results, it appears that the methods of cell-encapsulation and 99 cell-seeding are able to place different types of cells at designed locations and in turn to facilitate cell-cell communication. Cell proliferation and differentiation were evaluated by immunofluorescence staining. The results implied that cell proliferation was maintained within microstructures and the microstructure may help to guide the differentiation of HaCaT cells to cortical keratinocytes. On the other hand, gene expression within microstructures was examined using real-time PCR. Gene Wnt10a, Wnt10b, Shh, KGF and BDNF were all positively expressed in the mRNA isolated from cell mixtures extracted from microwell arrays. In conclusion, this microstructure provided a 3D microenvironment for co-culturing epithelial and mesenchymal cells in vitro and optimized cell compartmentalization according to their anatomical relationship in vivo. Multiple genes associated with hair follicle development were positively expressed in the microwell-cultured cells. Nevertheless, the potential of this microwell system in the application of hair follicle regeneration should be further tested in vivo. 100 Chapter Future study One limitation of this study was that no histology result was provided for exploring the internal structures of cell mixtures within microwells. The main challenge is to section the microstructural hydrogel because the hydrogel contained 90% water and it may be too soft to cut or it may melt fast due to the generated heat during cutting. It would be interesting to develop other methods for discovering the internal structures of cell-laden microwells, i.e., cutting the hydrogel in the middle and observing lateral face of microstructures using confocal laser scanning microscopy. Another disadvantage of this study was that the viability of HDF cells in the microstructures was only above 50% and the number of cell spreading was limited until day 14. It may be due to the residual free radicals remaining in the microstructure after polymerization. To address this problem, biomolecules (i.e., Arg-Gly-Asp-Ser (RGDS) peptide and fibrinogen) should be used in future to modify PEGDA monomers and form biosynthetic microstructural hydrogels for improving cell adhesion and cell proliferation 177,178. Moreover, although 3D microstructure system exhibited some advantages over 2D culture in gene expression, the importance of such specific microstructure (a microwell with a center islet) was little verified. It would be useful to include another appropriate microwell control (a microwell without the center islet) and examine whether the up-regulation of gene expression was attributed to our specific 3D microstructure or the microwell control is sufficient to induce the gene expression changes. In addition, this study was restricted to in vitro work, since the focus of this thesis was to create biomimetic microstructures using microfabrication techniques and to facilitate cell distributions within the microstructures in a controlled way. However, the potential of cell-incorporated microstructures in hair follicle engineering should be further confirmed in vivo. In the future study, human dermal papilla cells will replace human dermal fibroblasts and primary keratinocytes will replace HaCaT cells 101 in the co-culture system. Intact dermal papillae are isolated from occipital scalp follicle and then papilla cells migrate out from papilla and undergo cell expansion in 2D culture. 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Tissue Eng Part A 2009;15(3):579-85. 113 [...]... interactions in the native hair follicle Thus, the 3D hydrogel microstructure may serve as a suitable model for cell compartmentalization in studying hair follicle interactions in vitro, with the possibility to be further explored for human hair follicle engineering VIII LIST OF PUBLICATIONS Journal 1 Pan J, Chan SY, Common JE, Amini S, Miserez A, Lane EB and Kang L Fabrication of a 3D hair follicle- like... remaining Me display signs of degeneration, and the hair fiber 10 only contains melanin debris 58 It was also reported that the antioxidant systems within the hair follicle Me become impaired with age, likely resulting in hair graying/canities 62 1.2.1.2 Human hair cycle The human hair cycle consists of 3 main phases – anagen (growth phase), catagen (involutional/regression phase) and telogen (resting... weeks of gestation, when hair numbers are specified and fixed throughout life The size and shape of the follicle determine the size and shape of the hair fiber 38 Large hair follicles produce ‘terminal’ hairs such as those on the scalp Curved follicles produce curly hair fibers 39 Small follicles produce short, fine, light-colored hairs that are characteristic of body hair The hair follicle bulb consists... in the MC1R gene In the adult hair follicle, follicular Me lie in the matrix zone and their activity is hair cycle dependent (details of human hair cycle will be discussed in Chapter 1.2.1.2) During the anagen phase, activated Me transfer the melanin granules to the surrounding keratinocytes and thereby direct the pigmentation of the hair fiber 58 Melanin formation is switched-off in catagen remaining... alternatives of harvesting hair follicles other than from human donors 6 Therefore, it is important to explore a new method to generate hair follicles in large quantity to meet the clinic needs Recently, scientists attempt to emphasize on the understanding of hair follicle morphogenesis, to investigate the mechanism of hair follicle initiation and development, and to form hair follicles from dissociated... tissue engineering scaffold with the vasculature directly embedded into the scaffold (Reproduced from Biomedical Microdevices, Vol 4, 2002, pp 167-175, Microfabrication Technology for Vascularized Tissue Engineering, Jeffrey T Borenstein, H Terai, Kevin R King, E.J Weinberg, M.R Kaazempur-Mofrad, J.P Vacanti, Figure 6, with kind permission from Springer Science and Business Media) 98 C: Cells were printed... curly hair follicle morphology In the world, over 90% human population has deep brown-black hair and the other 5% – 10% people who mostly originate in northern Europe have different hair colors, ranging from white blonde, yellow blonde, auburn to red and all shades in between 52 As we know, eumelanin and pheomelanin are two types of melanin and they regulate the pigmentation of hair follicles Eumelanin... dissociated cells This chapter will provide a brief account of hair follicle morphogenesis, research progress of hair follicle generation and the importance of cell compartmentalization in hair follicle bioengineering 1 1.1.1 Hair follicle morphogenesis Hair follicle formation is dependent on reciprocal, sequential, epithelial-mesenchymal interactions (EMIs) The communication between epithelial and mesenchymal... indicate that hair follicles could be well reconstituted in vivo using homospecific mouse cells and even heterospecific mouse–rat combinations However, it was found that homospecific human cells could not grow into normal hair follicles while human hair follicle like structures could be obtained by co-grafting human foreskin-derived epidermal cells with rodent DP-enriched cells 27 These hair follicle- like... years 4 Currently, treatments of AGA include using anti hair loss drugs and/or surgical implantation Minoxidil and finasteride are two drugs for AGA 5 However, some side effects appear during the treatment and hair fall resumes upon withdrawal of the drugs On the other hand, using surgical procedure is effective in hair regeneration by which grafts containing hair follicles were transplanted onto the . IN VITRO HAIR FOLLICLE ENGINEERING PAN JING NATIONAL UNIVERSITY OF SINGAPORE 2014 IN VITRO HAIR FOLLICLE ENGINEERING PAN JING (B. Sc., China. epithelial-mesenchymal interactions in the native hair follicle. Thus, the 3D hydrogel microstructure may serve as a suitable model for cell compartmentalization in studying hair follicle interactions in vitro, . is to use tissue engineering approaches to generate large quantities of human hair follicles in vitro to meet the clinical needs. From previously reported studies, hair follicle- like structures