published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

肌腱損傷發生率高，並且癒合結果很不理想，因為少量的肌腱細胞缺乏有效的修復能力，僅僅通過瘢痕形成來癒合, 肌腱瘢痕癒合難以恢復原本的肌腱組織結構及力學特性。目前，國內外臨床上治療肌腱損傷的方法很多，包括藥物、物理治療、手術等，這些並不能獲得滿意的療效。因此，如何採用肌腱組織工程技術迅速、安全、有效的修復肌腱損傷已成為運動醫學領域急需解決的重要問題。 / 有研究表明，骨髓間充質幹細胞、表皮成纖維細胞、肌腱細胞和胚胎幹細胞通過肌腱組織工程技術用於肌腱修復及再生取得了不錯的療效。但是，這些來源的細胞存在分化效率低，形成畸胎瘤和異位骨化等風險。近來，有研究報導可從人、小鼠、大鼠和兔的肌腱組織中分離培養出幹細胞，可作為肌腱組織工程種子細胞的一種新選擇，用於肌腱修復和再生。對於間充質幹細胞的成肌腱分化，有研究報導結締組織生長因子（CTGF）和抗壞血酸（維生素C的一種形式）在膠原及細胞外基質合成、調節細胞成肌腱分化方面扮演者重要的角色。 / 本研究的旨在：（1）在大鼠髕腱損傷模型中，證實肌腱幹細胞可作為一種新的幹細胞來源用於肌腱修復；（2）檢驗結締組織生長因子和抗壞血酸能在體外促進肌腱幹細胞的成肌腱分化；（3）嘗試通過肌腱幹細胞的成肌腱分化過程在體外構建不含外源性支架的肌腱樣組織；（4）探索該肌腱樣組織在大鼠髕腱損傷模型中是否可以促進肌腱癒合。 / 在大鼠急性髕腱損傷動物模型中，與對照組相比，肌腱幹細胞組具有更好的膠原排列，顯著增高的最大張力和楊氏模量，表明肌腱幹細胞可作為一種新的幹細胞來源用於肌腱損傷的修復。結締組織生長因子和抗壞血酸體外誘導肌腱幹細胞2周後，可顯著增加Tenomodulin, Scleraxis, Thbs4, I型膠原等肌腱相關基因的表達以及膠原蛋白的合成，說明結締組織生長因子和抗壞血酸可促進肌腱幹細胞的成肌腱分化。被結締組織生長因子和抗壞血酸誘導兩周後，肌腱幹細胞可形成了細胞膜樣結構，將這種細胞膜纏繞在迴紋針上，構建成肌腱樣組織，其具有相對疏鬆的細胞外基質和雜亂排列其中的肌腱幹細胞，以及表達Tenomodulin，I型膠原和III型膠原。將該肌腱樣組織移植到裸鼠體內8周和12周可形成新生肌腱組織，梭形細胞縱行分佈在平行的膠原纖維之間，並表達Tenomodulin，I型膠原和III型膠原蛋白。在大鼠髕腱損傷動物模型中，與對照組相比較，該肌腱樣組織可通過恢復肌腱組織結構及生物力學特性來促進肌腱癒合。 / 總的來說，本研究證實肌腱幹細胞可作為一種新的幹細胞來源用於肌腱組織工程促進肌腱再生。結締組織生長因子和抗壞血酸可調控肌腱幹細胞的成肌腱分化，並形成細胞膜結構。該細胞膜結構可在體外構建出不含外源性支架的肌腱樣組織，進而在裸鼠體內形成新生肌腱，並且在大鼠髕腱損傷模型中可有效的促進損傷肌腱的癒合。這種不含外源性支架的肌腱樣組織有希望成為肌腱組織工程技術的新手段，在肌腱再生和肌腱修復的臨床應用及基礎研究方面有廣泛的前景。 / Tendon injuries are common and tendon healing outcome is poor, because tendon contains few cells with limited capacities for self-repair/regeneration. The current treatments on tendon injuries including drugs, physiotherapy, and surgery are not ideal and there is a need for the development of novel tissue-engineering strategies for tendon repair. / Previous studies have shown positive effects of bone marrow-derived mesenchymal stem cells (BMSCs), dermal fibroblast, tenocytes, and embryonic stem cells-derived MSCs for tendon repair/regeneration. However, these cells have limitations including insufficient differentiation; risk of teratoma and ectopic bone formation etc. Recently, stem cells have been isolated from tendons of human, mouse, rat and rabbit and considered as a new alternative cell source for tendon tissue engineering (TDSCs). For tenogenic differention of MSCs, connective tissue growth factor (CTGF) and ascorbic acid (one form of vitamin C) are reported to play important roles in promoting collagen and other extracellular matrixes (ECM) production, and regulating the MSCs differentiation towards tenogenic pathway. / The aims of the current study are: (1) To investigate the use of TDSCs in tendon repair in a rat acute patellar tendon injury model; (2) To test the effects of CTGF and ascorbic acid on tenogenic differentiation of TDSCs in vitro; (3) To construct scaffold-free tendon-like tissues in vitro using tenogenically differentiated TDSCs; (4) To promote tendon healing by engineered tendon-like tissues in a rat acute patellar tendon injury model. / In the rat acute patellar tendon injury model, in contract to control group, TDSCs treated group showed better alignment of collagen fibers and the significantly higher ultimate stress and Young’s modulus, indicating TDSCs may be an alternative cell source for tendon repair. The effects of CTGF and ascorbic acid on tenogenic differentiation of TDSCs were also confirmed with higher expression of tendon related markers such as Tenomodulin, Scleraxis, Thbs4, Type I Collagen, etc; with higher production of collagenous proteins. After treatment with CTGF and ascorbic acid for 2 weeks, TDSCs can form cell sheets, which can be harvested, rolled up on a U-shaped spring to form tendon-like tissues in culture, which had loose extracellular matrices and randomly distributed TDSCs and also expressed Tenomodulin, Type I & III collagen. Following transplantation of the engineered tendon-like tissue in nude mice for 8 and 12 weeks, neo-tendon tissues were formed, with thin and parallel collagen fibrils and extracellular matrices of Tenomodulin, Type I & III collagen. Finally in the rat patellar tendon window injury model, data suggested that the engineered tendon-like tissue could promote tendon healing with significantly improved histological features and biomechanical properties comparing to the control group. / In conclusion, our study has indicated that TDSCs can be an alternative cell source in tendon tissue engineering for tendon regeneration. The tenogenic differentiation of TDSCs, induced by CTGF and ascorbic acid in vitro, produces cell sheets, which can be constructed tendon-like tissues in vitro; to form neo-tendon and repair tendon injuries in vivo. The use of engineered scaffold-free tendon tissue for tendon tissue engineering has potentials in clinical application for tendon repair/regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ni, Ming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 107-126). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / DEDICATION --- p.I / ACKNOWLEDGEMENT --- p.II-III / TABLE OF CONTENTS --- p.IV-IX / PUBLICATIONS --- p.X-XII / ABBREVIATION --- p.XIII-XV / ABSTRACT (ENGLISH) --- p.XVI-XVIII / ABSTRACT (CHINESE) --- p.XIX-XX / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of tendon injury --- p.1 / Chapter 1.2 --- Healing process of tendon injury --- p.1 / Chapter 1.3 --- Tendon tissue engineering for tendon repair --- p.2 / Chapter 1.4 --- Stem cells in tendon repair --- p.2 / Chapter 1.5 --- Tenogenic differentiation of tendon derived stem cells --- p.7 / Chapter 1.6 --- Growth factors for tenogenic differentiation --- p.8 / Chapter 1.7 --- Vitamin C for tenogenic differentiation --- p.9 / Chapter 1.8 --- Summary --- p.10 / Chapter CHAPTER 2 --- Hypothesis, Objectives and Study Design --- p.11 / Chapter 2.1 --- Hypothesis --- p.11 / Chapter 2.1.1 --- Overall hypothesis --- p.11 / Chapter 2.1.2 --- Specific hypothesis --- p.11 / Chapter 2.2 --- Objectives --- p.12 / Chapter 2.3 --- Study design --- p.12 / Chapter 2.3.1 --- Study I --- p.12 / Chapter 2.3.2 --- Study II --- p.14 / Chapter 2.3.3 --- Study III --- p.14 / Chapter 2.3.4 --- Study IV --- p.17 / Chapter CHAPTER 3 --- Tendon-derived Stem Cells (TDSCs): A New Cell Source for Tendon Repair (Study I) --- p.19 / Chapter 3.1 --- Materials and Methods --- p.19 / Chapter 3.1.1 --- Isolation and characterization of rat GFP-TDSCs --- p.19 / Chapter 3.1.2 --- Animal surgery --- p.20 / Chapter 3.1.3 --- Ultrasound imaging --- p.25 / Chapter 3.1.4 --- Histology --- p.27 / Chapter 3.1.5 --- Biomechanical test --- p.27 / Chapter 3.1.6 --- Ex vivo fluorescence imaging --- p.28 / Chapter 3.1.7 --- Data analysis --- p.29 / Chapter 3.2 --- Results --- p.29 / Chapter 3.2.1 --- Gross observation of the injured knee and patellar tendon --- p.29 / Chapter 3.2.2 --- Histology of regenerated tendon tissue --- p.30 / Chapter 3.2.3 --- Biomechanical test of regenerated tendon tissue --- p.32 / Chapter 3.2.4 --- Ex vivo fluorescence imaging of GFP-TDSCs --- p.33 / Chapter 3.2.5 --- Ultrasound imaging of wound gap volume --- p.34 / Chapter 3.3 --- Discussion --- p.35 / Chapter 3.4 --- Conclusion --- p.50 / Chapter CHAPTER 4 --- Tenogenic Differentiation of Tendon-derived Stem Cells (TDSCs) (Study II) --- p.51 / Chapter 4.1 --- Materials and Methods --- p.51 / Chapter 4.1.1 --- Tenogenic differentiation of tendon-derived stem cells (TDSCs) --- p.51 / Chapter 4.1.2 --- Quantification of collagenous proteins --- p.51 / Chapter 4.1.3 --- Quantitative Real Time PCR (qRT-PCR) --- p.52 / Chapter 4.1.4 --- Data analysis --- p.54 / Chapter 4.2 --- Results --- p.55 / Chapter 4.2.1 --- Quantification of collagenous proteins --- p.55 / Chapter 4.2.2 --- Tenogenic, osteogenic and chondrogenic markers mRNA expression --- p.57 / Chapter 4.2.3 --- Tendon extracellular matrix markers mRNA expression --- p.57 / Chapter 4.3 --- Discussion --- p.59 / Chapter 4.4 --- Conclusion --- p.66 / Chapter CHAPTER 5 --- Engineered Scaffold-free Tendon Tissue Produced by Tendon-derived Stem Cells (TDSCs) Cell Sheet (Study III) --- p.67 / Chapter 5.1 --- Materials and Methods --- p.67 / Chapter 5.1.1 --- In vitro engineered scaffold-free tendon tissue by TDSCs cell sheet --- p.67 / Chapter 5.1.2 --- In vivo neo-tendon formation using engineered scaffold-free tendon tissue in nude mouse model --- p.67 / Chapter 5.1.3 --- Histology and immunohistochemistry staining --- p.68 / Chapter 5.1.4 --- In vivo fluorescence imaging --- p.69 / Chapter 5.1.5 --- Data analysis --- p.70 / Chapter 5.2 --- Results --- p.70 / Chapter 5.2.1 --- Gross observation of TDSCs cell sheet and engineered scaffold-free tendon tissue --- p.70 / Chapter 5.2.2 --- Histological and immunohistochemical characteristics in engineered scaffold-free tendon tissue --- p.71 / Chapter 5.2.3 --- Gross observation and in vivo fluorescence imaging of neo-tendon tissue --- p.74 / Chapter 5.2.4 --- Histology of neo-tendon tissue --- p.75 / Chapter 5.2.5 --- Immunohistochemistry staining in neo-tendon tissue --- p.76 / Chapter 5.3 --- Discussion --- p.78 / Chapter 5.4 --- Conclusion --- p.82 / Chapter CHAPTER 6 --- Use of Engineered Scaffold-free Tendon Tissue for Tendon Repair (Study IV) --- p.83 / Chapter 6.1 --- Materials and methods --- p.83 / Chapter 6.1.1 --- Animal surgery --- p.83 / Chapter 6.1.2 --- Ex vivo fluorescence imaging --- p.84 / Chapter 6.1.3 --- Histology and immunohistochemistry staining --- p.85 / Chapter 6.1.4 --- Biomechanical test --- p.86 / Chapter 6.1.5 --- Ultrasound imaging --- p.87 / Chapter 6.1.6 --- Data Analysis --- p.87 / Chapter 6.2 --- Results --- p.88 / Chapter 6.2.1 --- Gross observation of the injured knee and patellar tendon --- p.88 / Chapter 6.2.2 --- Histology of regenerated tendon tissue --- p.89 / Chapter 6.2.3 --- Tendon specific and ECM markers expression in regenerated tendon tissue --- p.91 / Chapter 6.2.4 --- Osteogenic and chondrogenic specific markers expression in neo-tendon tissue --- p.93 / Chapter 6.2.5 --- The fate of the transplanted engineered scaffold-free tendon tissue --- p.93 / Chapter 6.2.6 --- Biomechanical test of regenerated tendon tissues --- p.94 / Chapter 6.3 --- Discussion --- p.96 / Chapter 6.4 --- Conclusion --- p.102 / Chapter CHAPTER 7 --- General Conclusions --- p.103 / Chapter 7.1 --- General discussion --- p.103 / Chapter 7.2 --- General conclusions --- p.105 / FUNDING --- p.106 / REFERENCES --- p.107 / APPENDIX --- p.127

Opportunistic infection is always a potentially life threatening complication in pediatric oncology patients and hematopoietic stem cell transplant recipients. With the advances in various disease treatment protocols, the overall and event-free survivals of this high risk population improve significantly. In this thesis, the author reported a number of original studies to discuss different strategies in prevention of this serious complication. Firstly, the author demonstrates that pediatric oncology patients are still vulnerable to various vaccine-preventable infectious diseases up to 18 months after stopping chemotherapy. For those vaccine-preventable infectious diseases, pediatric oncology patients can mount a significant and persistent immune response to common inactivated vaccine (namely diphtheria-tetanus-pertussis vaccine). For non-vaccine preventable infectious diseases, regular monitoring of plasma viral load and strategic use of antiviral agents as pre-emptive or prophylactic agent is an effective approach to prevent infection. In hematopoietic stem cell transplant setting, adoptive transfer of acquired immunity from donor to recipient and incorporation of this parameter in donor selection process can be considered. The findings of the studies can be applied to clinical setting. The future direction of our studies includes the immune responses of other common vaccines namely pneumococcal vaccine and pandemic influenza vaccine in high risk population. The role of transfer of donor's varicella zoster immunity in prevention of herpes zoster infection in transplant recipient can be further explored. With the advances in supportive care of our vulnerable patients, the survival rate is expected to be further improved in the future. / by Frankie Wai Tsoi, Cheng. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 193-208). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Allogeneic haematopoietic stem cell transplantation (HSCT) has been used successfully to treat children and adults with high-risk or relapsed hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. When initially developed, allogeneic HSCT was conceived as a method of rescuing patients from the toxic side effects of dose-intensive chemoradiotherapy. Due to transplant-related toxicities, the application of myeloablative allogeneic HSCT has been limited to younger patients without organ dysfunctions. Since the early 1990s many groups of investigators have explored strategies using less intensive preparative regimens that would allow engraftment of hematopoietic progenitor cells from either identical or non-identical donors. These reduced-intensity conditioning (RIC) regimens result in less tissue damage, less inflammatory cytokine secretion, and possibly lower rates of graft-versus-host disease (GVHD) and non-relapse mortality (NRM). Such non-myeloablative approach, or "mini-transplant", has been suggested to benefit older patients as well as in conditions in which traditional myeloablative conditioning regimens are associated with high rates of non-relapse mortality. / Allogeneic HSCT is the only curative therapy for many patients with myeloid malignancies or myelodysplastic syndrome (MDS). The development of reduced-intensity preparative regimens may allow the extension of this form of treatment to older and patients with coexisting medical illness. On the other hand, relapse after transplantation remains the most important cause of treatment failure in patients with refractory acute myeloid leukemia (AML) or MDS, and is associated with poor survival. Evaluation of prognostic factors may help to improve the results of myeloablative and RIC allogeneic HSCT in this group of patients. Furthermore, the impact of comorbidities on outcomes of RIC allogeneic HSCT in this group of patients with refractory AML or MDS needs to be defined. / The application of embryonic and adult stem cells in regenerative and reparative therapies of non-hematopoietic diseases is emerging rapidly. Human umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and mesenchymal progenitor cells. Although clinical experience to date with UCB has focused on hematological application, early preclinical studies support the hypothesis that multipotential stem cells derived from UCB exhibit functional characteristics similar to that observed in adult marrow-derived stem cells in mediating vascular and organ regenerative capabilities. However, the application of these preclinical findings in clinical setting needs to be further studied. Mini-transplant of human UCB may be an effective approach to repair organ damage in patients with non-hematological diseases. / Wong Siu Ming Raymond. / Adviser: Joseph J.Y. Sung. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 187-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Kwok, Po Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-130). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 中文摘要 --- p.v / Acknowledgements --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Table of Abbreviations --- p.xii / Contents --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- The Biology of Human Umbilical Cord Perivascular Cells (HUCPVs) and Their Potential Applications in Tissue Regeneration / Chapter 2.1 --- INTRODUCTION --- p.5 / Chapter 2.1.1 --- Stem cells --- p.5 / Chapter 2.1.2.1 --- Embryonic stem cells --- p.6 / Chapter 2.1.2.2 --- iPS cells --- p.7 / Chapter 2.1.2.3 --- Somatic stem cells --- p.8 / Chapter 2.1.3 --- Mesenchymal stem cells --- p.9 / Chapter 2.1.4 --- Pericytes --- p.11 / Chapter 2.1.5 --- CD146 positive MSCs --- p.12 / Chapter 2.1.6 --- Human umbilical cord perivascular cells (HUCPVs) --- p.13 / Chapter 2.1.7 --- The biology of stem cell microenvironment (niche) --- p.14 / Chapter 2.1.8 --- Current applications of HUCPVs --- p.17 / Chapter 2.1.9 --- Regenerative medicine --- p.17 / Chapter 2.1.10 --- Applications of stem cells in bone regeneration --- p.19 / Chapter 2.2 --- MATERIALS AND METHODS --- p.22 / Chapter 2.2.1 --- Cell culture --- p.22 / Chapter 2.2.2 --- Preparation of Human Umbilical Cord Perivascular (HUCPV) cells --- p.22 / Chapter 2.2.2.1 --- Isolation of Human Umbilical Cord Perivascular (HUCPV) cells from human umbilical cord --- p.22 / Chapter 2.2.2.2 --- Purification of HUCPV cells --- p.23 / Chapter 2.2.3 --- Immunocytochemsitry --- p.24 / Chapter 2.2.4 --- Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) --- p.25 / Chapter 2.2.4.1 --- Isolation of total cellular RNA --- p.25 / Chapter 2.2.4.2 --- Complementary DNA (cDNA) synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase chain reaction (PCR) --- p.26 / Chapter 2.2.5 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.30 / Chapter 2.2.6 --- In vitro differentiation assays --- p.33 / Chapter 2.2.6.1 --- Osteogenic differentiation --- p.33 / Chapter 2.2.6.2 --- Adipogenic differentiation --- p.33 / Chapter 2.2.6.3 --- Chondrogenic differentiation --- p.34 / Chapter 2.2.6.4 --- In vitro chondrogenic differentiation on gelfoam® --- p.34 / Chapter 2.2.7 --- Cytochemistry staining --- p.35 / Chapter 2.2.7.1 --- Alkaline Phosphatase staining --- p.35 / Chapter 2.2.7.2 --- Alizarin Red S staining --- p.35 / Chapter 2.2.7.3 --- Oil Red O staining --- p.36 / Chapter 2.2.7.4 --- Alcian Blue staining --- p.36 / Chapter 2.2.8 --- Scanning electron microscopy (SEM) --- p.37 / Chapter 2.2.9 --- Transmission electron microscopy (TEM) --- p.37 / Chapter 2.2.10 --- Paraffin tissue embedding --- p.38 / Chapter 2.2.10 --- Haematoxylin and Eosin staining --- p.38 / Chapter 2.3 --- RESULTS --- p.40 / Chapter 2.3.1 --- Isolation and purification of HUCPVs --- p.40 / Chapter 2.3.2 --- Osteogenic differentiation of HUCPVs under normoxia --- p.41 / Chapter 2.3.3 --- Osteogenic differentiation of HUCPVs under hypoxia --- p.42 / Chapter 2.3.4 --- Adipogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.5 --- Chondrogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.6 --- Chondrogenic differentiation of HUCPVs on gelfoam® --- p.44 / Chapter 2.4 --- DISCUSSION --- p.59 / Chapter Chapter 3 --- Calcium and Calcium-sensing Receptor (CaSR) in osteogenesis / Chapter 3.1 --- INTRODUCTION --- p.62 / Chapter 3.1.1 --- Metabolism of calcium --- p.62 / Chapter 3.1.2 --- Calcium-sensing receptor --- p.64 / Chapter 3.1.2.1 --- The molecular structure of calcium-sensing Receptor (CaSR) --- p.64 / Chapter 3.1.2.2 --- The expression pattern of calciumsensing receptor (CaSR) --- p.67 / Chapter 3.1.2.3 --- The physiological function of calcium-sensing receptor in different tissues or organs --- p.68 / Chapter 3.1.2.4 --- Regulatory role of calcium-sensing receptor in calcium sensing and homeostasis --- p.71 / Chapter 3.1.2.5 --- The role of calcium-sensing receptor in diseases --- p.72 / Chapter 3.1.2.6 --- Genetic animal models targeting calciumsensing receptor --- p.73 / Chapter 3.1.2.7 --- Calcium-sensing receptor in mesenchymal lineage Differentiation --- p.76 / Chapter 3.1.2.8 --- The role of calcium-sensing receptor in the skeleton --- p.76 / Chapter 3.1.3 --- Calcium-sensing receptor related pathway --- p.78 / Chapter 3.1.3.1 --- Cyclic AMP pathway --- p.78 / Chapter 3.1.3.2 --- Cyclic AMP response element-binding protein (CREB) --- p.80 / Chapter 3.2 --- MATERIALS AND METHODS --- p.83 / Chapter 3.2.1 --- Preparation of primary mouse osteoblasts (MOB) from long bone --- p.83 / Chapter 3.2.2 --- Preparation of primary mouse osteoblasts (CMOB) from calvaria --- p.84 / Chapter 3.2.3 --- Immunocytochemistry --- p.84 / Chapter 3.2.4 --- Osteogenic differentiation --- p.85 / Chapter 3.2.3 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.85 / Chapter 3.2.4 --- Cell proliferation measurement by BrdU ELISA (colorimetric) assay --- p.85 / Chapter 3.2.5 --- Western blotting analysis --- p.86 / Chapter 3.2.5.1 --- Preparation of the protein lysate --- p.86 / Chapter 3.2.5.2 --- Protein quantitation --- p.86 / Chapter 3.2.5.3 --- SDS-PAGE --- p.87 / Chapter 3.2.5.4 --- Protein transfer --- p.87 / Chapter 3.2.5.5 --- Immunodetection --- p.88 / Chapter 3.2.6 --- cAMP EIA assay --- p.89 / Chapter 3.3 --- RESULTS --- p.91 / Chapter 3.3.1 --- "Expression of CD 146 and CaSR in HUCPVs, primary mouse long bone osteoblasts and MC3T3-E1 cell line" --- p.91 / Chapter 3.3.2 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under normoxia --- p.91 / Chapter 3.3.3 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under hypoxia --- p.92 / Chapter 3.3.4 --- The effect of calcium treatment on cell proliferation in primary mouse long bone osteoblasts --- p.93 / Chapter 3.3.5 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse long bone osteoblasts --- p.94 / Chapter 3.3.6 --- The effect of calcium treatment on calcium-sensing receptor expression in HUCPVs --- p.95 / Chapter 3.3.7 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse calvarian osteoblasts --- p.96 / Chapter 3.3.8 --- The effect of calcium treatment on cyclic AMP levels in primary mouse long bone osteoblasts --- p.97 / Chapter 3.4 --- DISCUSSION --- p.117 / Chapter Chapter 4 --- General Discussions --- p.121 / References --- p.124 / Appendices --- p.131

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

Made available in DSpace on 2014-08-13T14:50:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-11Bitstream added on 2014-08-13T18:00:42Z : No. of bitstreams: 1 000755550_20151031.pdf: 220836 bytes, checksum: 87b354d7a24b15fa538662e0ef64bad7 (MD5) Bitstreams deleted on 2015-11-03T14:39:17Z: 000755550_20151031.pdf,. Added 1 bitstream(s) on 2015-11-03T14:40:19Z : No. of bitstreams: 1 000755550.pdf: 553044 bytes, checksum: 424382ed9bb35c0860f257d171e5ed77 (MD5) / O trabalho teve por objetivo avaliar clínica e laboratorialmente a viabilidade e a segurança do transplante intratecal de células-tronco mesenquimais autólogas e alogênicas provenientes da medula óssea (CTM-MO) de cães. Foram utilizados 15 cães, sem raça definida, adultos, de ambos os sexos, clinicamente saudáveis, excetuando dois que apresentavam trauma medular crônico. Foram divididos em três grupos, contendo cinco animais cada. As CTM-MO foram obtidas pela punção da medula óssea, isoladas e cultivadas. O grupo A foi transplantado com CTM-MO autólogas por via intratecal através da cisterna magna, enquanto que o grupo B recebeu as CTM-MO alogênicas e o grupo C (controle) foi aplicado solução tampão fosfato-salina (PBS). O líquido cefalorraquidiano (LCR) foi obtido imediatamente antes do transplante das CTM-MO (momento 1) e no quinto dia após (momento 2), para avaliação físico-química, citológica e da concentração de metaloproteinases (MMP-2 e MMP-9). Todos os animais foram acompanhados por exame físico neurológico pré e pós-transplante. Pelas avaliações clínicas e laboratoriais, não se observaram alterações significativas após o transplante das CTM-MO autólogas e alogênicas por via intratecal, demonstrando ser uma via segura e viável para a utilização de terapia celular em cães / The study aimed to evaluate clinical and laboratory viability and safety of intrathecal transplantation of bone marrow mesenchymal stem cells (BM-MSC) autologous and allogeneic of dogs. A total of 15 dogs, mixed breed, adults of both sexes, clinically healthy, except two who had chronic spinal trauma. They were divided into three groups with five animals each. BM-MSC were obtained by puncture of the bone marrow, isolated and cultured. Group A was transplanted with autologous BM-MSC intrathecally via the cisterna magna, while group B received allogenic BM-MSC and group C (control) was administered phosphate buffered saline (PBS). Cerebrospinal fluid (CSF) was obtained immediately before transplantation of BM-MSC (moment 1) and the fifth day after (moment 2) for physico-chemical, cytological and the concentration of matrix metalloproteinases (MMP-2 and MMP-9 .) All Animals were monitored by neurological examination before and after transplantation. By clinical and laboratory evaluations, no significant changes were observed after transplantation of BM-MSC autologous and allogeneic intrathecal, proving to be a safe and viable for the use of cell therapy in dogs

Orientador: Fernanda da Cruz Landim / Coorientador: Leandro Maia / Banca: Betânia Souza Monteiro / Banca: Bruna de Vita / Resumo: Células tronco mesenquimais (CTMs) possuem potencial terapêutico regulando a inflamação, prevenindo a formação de feridas e regenerando tecidos. Por sua propriedade imunomoduladora, tem sido estudadas no transplante alogênico. As CTMs derivadas de anexos fetais são destaque por serem de fácil obtenção, de tecidos geralmente descartados e com grande possibilidade de formação de bancos. O presente trabalho objetiva avaliara resposta inflamatória in vivo do músculo glúteo médio à inoculação alogênica de CTMs derivadas de cordão umbilical equino. As CTMs foram cultivadas e caracterizadas por meio de imunofenotipagem e diferenciação em linhagens mesodermais e então, criopreservadas. Após descongelação e expansão as CTMs foram transplantadas no dia 0 no músculo glúteo médio direito e esquerdo, em duas regiões diferentes, em seis éguas hígidas. Como controle foram realizadas injeções de Solução Balanceada de Hank's caudalmente às regiões onde foram transplantadas as CTMs. As biópsias musculares forma realizadas 30 dias antes do transplante de CTMs, bem como dois dias depois do transplante e 7 dias após o transplante. Todos os transplantes e biópsias foram precedidos pelo exame ultrassonográfico além de colheita de sangue para os parâmetros hematológicos. As CTMs foram bem caracterizadas, apresentaram alta clonicidade e não provocaram alteração nos parâmetros hematológicos. O exame ultrassonográfico revelou aspecto sugestivo de reação inflamatória 48 horas após os transplantes em ambos os grupos, controle e tratadoNa avaliação histológica não foram encontradas diferenças entre os grupos tratado e controle em nenhum dos momentos estudados. Uma discreta diferença no parâmetro de infiltrado neutrofílico foi encontrada entre D30 e D2 apenas no grupo tratado (P<0,05), entretanto não houveram diferenças em D7. Desta forma, podemos concluir a terapia celular não incitou resposta... / Abstract: Mesenchymal stem cells (MSCs) have therapeutic potential regulating inflammation, preventing the formation of wounds and regenerating tissues. MSCs derived from fetal membranes are highlighted because they are easy to obtain, from tissues usually discarded and with great possibility of bank formation. This study evaluated the in vivo inflammatory response of the gluteus medius muscle to the inoculation of allogeneic MSCs derived from equine umbilical cord. MSCs were cultured and characterized by immunophenotyping and differentiation into mesodermal lineages, and next cryopreserved. After thawing and expansion of MSCs transplantation was performed on day 0 in the right and left middle gluteal muscle at two different regions in six healthy mares. Likewise, for the control group, an injection of Hank's-Balanced Solution was performed caudally to the regions where MSCs were transplanted. Muscle biopsies were performed 30 days before the transplantation of MSCs, 2 days and 7 days after transplantation. All biopsies and transplants were preceded by ultrasonographic examination, and blood samples retrieval for hematological parameters. The biopsies were be analyzed by histological techniques. The MSCs were well characterized, and possessed high clonogenicity causing no changes in the hematological parameters evaluated. Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups, control (C) and treated (T). Histological evaluation found no differences between C and T groups in any of the time points studied. A discrete temporal inflammation were found between D30 and D2 (P <0.05) only in the T group, however no differences were found in D7. Thus, we conclude that the cellular therapy did not incited systemic inflammatory response to recipients, demonstrating, for the first time, that the allogeneic use of MSCs from equine umbilical cord are safety for use in horse ... / Mestre

Made available in DSpace on 2015-12-10T14:22:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-05-21. Added 1 bitstream(s) on 2015-12-10T14:29:11Z : No. of bitstreams: 1 000851315.pdf: 1476331 bytes, checksum: eba120cc7ab9336605a5035f69abf08e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Células tronco mesenquimais (CTMs) possuem potencial terapêutico regulando a inflamação, prevenindo a formação de feridas e regenerando tecidos. Por sua propriedade imunomoduladora, tem sido estudadas no transplante alogênico. As CTMs derivadas de anexos fetais são destaque por serem de fácil obtenção, de tecidos geralmente descartados e com grande possibilidade de formação de bancos. O presente trabalho objetiva avaliara resposta inflamatória in vivo do músculo glúteo médio à inoculação alogênica de CTMs derivadas de cordão umbilical equino. As CTMs foram cultivadas e caracterizadas por meio de imunofenotipagem e diferenciação em linhagens mesodermais e então, criopreservadas. Após descongelação e expansão as CTMs foram transplantadas no dia 0 no músculo glúteo médio direito e esquerdo, em duas regiões diferentes, em seis éguas hígidas. Como controle foram realizadas injeções de Solução Balanceada de Hank's caudalmente às regiões onde foram transplantadas as CTMs. As biópsias musculares forma realizadas 30 dias antes do transplante de CTMs, bem como dois dias depois do transplante e 7 dias após o transplante. Todos os transplantes e biópsias foram precedidos pelo exame ultrassonográfico além de colheita de sangue para os parâmetros hematológicos. As CTMs foram bem caracterizadas, apresentaram alta clonicidade e não provocaram alteração nos parâmetros hematológicos. O exame ultrassonográfico revelou aspecto sugestivo de reação inflamatória 48 horas após os transplantes em ambos os grupos, controle e tratadoNa avaliação histológica não foram encontradas diferenças entre os grupos tratado e controle em nenhum dos momentos estudados. Uma discreta diferença no parâmetro de infiltrado neutrofílico foi encontrada entre D30 e D2 apenas no grupo tratado (P<0,05), entretanto não houveram diferenças em D7. Desta forma, podemos concluir a terapia celular não incitou resposta... / Mesenchymal stem cells (MSCs) have therapeutic potential regulating inflammation, preventing the formation of wounds and regenerating tissues. MSCs derived from fetal membranes are highlighted because they are easy to obtain, from tissues usually discarded and with great possibility of bank formation. This study evaluated the in vivo inflammatory response of the gluteus medius muscle to the inoculation of allogeneic MSCs derived from equine umbilical cord. MSCs were cultured and characterized by immunophenotyping and differentiation into mesodermal lineages, and next cryopreserved. After thawing and expansion of MSCs transplantation was performed on day 0 in the right and left middle gluteal muscle at two different regions in six healthy mares. Likewise, for the control group, an injection of Hank's-Balanced Solution was performed caudally to the regions where MSCs were transplanted. Muscle biopsies were performed 30 days before the transplantation of MSCs, 2 days and 7 days after transplantation. All biopsies and transplants were preceded by ultrasonographic examination, and blood samples retrieval for hematological parameters. The biopsies were be analyzed by histological techniques. The MSCs were well characterized, and possessed high clonogenicity causing no changes in the hematological parameters evaluated. Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups, control (C) and treated (T). Histological evaluation found no differences between C and T groups in any of the time points studied. A discrete temporal inflammation were found between D30 and D2 (P <0.05) only in the T group, however no differences were found in D7. Thus, we conclude that the cellular therapy did not incited systemic inflammatory response to recipients, demonstrating, for the first time, that the allogeneic use of MSCs from equine umbilical cord are safety for use in horse ... / FAPESP: 2013/01383-0