IN VIVO AND EX VIVO OSTEOGENESIS OF HUMAN EMBRYONIC STEM CELLS DR. SUBAKUMAR LAKSHMI (B.D.S., Dr. MGR Medical University, India) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 i ACKNOWLEDGEMENTS I am extremely lucky to have met a number of wonderful people during the course of my masters’ program at Faculty of Dentistry, NUS. My utmost gratitude goes to my supervisor, Associate Professor Yeo Jin Fei, for giving me the opportunity to pursue this research program. Without his warm support and encouragement, I would not be where I am today. I am deeply grateful to my co-supervisor Associate Professor Cao Tong for his generous support and unfailing guidance throughout this work. It is no exaggeration to say that without his support this thesis would not have happened. I would like to specially thank Dr.Yang Zheng for spreading her enthusiasm and extending her guidance during the many challenging days. I am indebted to her for having critically reviewed my thesis and helped me improve my skills in scientific writing. I would like to thank Dr. Ge Zigang and Dr. Alexis Heng Boon Chin for their support. I would like to extend my gratitude to Ms. Han Tok Lin for patiently training me in histological work. My heartfelt thanks go to my colleagues Dr. Vinoth Kumar, Dr. Liu Hua, Mr. Toh Wei Seong, Mr. Lu Kai, Ms. Fu Xin, Ms. Sui Lin, and Mr. Li MingMing for their valuable help and making my stay in NUS memorable. ii TABLE OF CONTENTS 1 LITERATURE REVIEW ....................................................................................................................... 2 1.1 2 Bone ........................................................................................................................................ 2 1.1.1 Bone composition 1 ......................................................................................................... 2 1.1.2 Bone structure 2............................................................................................................... 4 1.1.3 Bone types ...................................................................................................................... 5 1.1.4 Bone formation and remodelling 1 .................................................................................. 5 1.2 Clinical repair of bone defects ................................................................................................ 7 1.3 Cell based tissue engineering................................................................................................ 10 1.3.1 Scaffolds ........................................................................................................................ 10 1.3.2 Materials used as scaffolds ........................................................................................... 10 1.3.3 Seeding of cells into scaffolds ....................................................................................... 12 1.3.4 Cell source for cell-based tissue engineering for bone regeneration ........................... 12 1.4 Stem cells in bone repair....................................................................................................... 13 1.5 Human Embryonic stem cells ................................................................................................ 15 1.5.1 Derivation of HES cells .................................................................................................. 15 1.5.2 Expansion and maintenance of pluripotency of HES cells ............................................ 16 1.5.3 Characterization of hES cells ......................................................................................... 18 1.6 Differentiation of hES cells into osteogenic lineage ............................................................. 18 1.7 In vivo bone formation using Stem Cells .............................................................................. 20 MATERIAL & METHODS ................................................................................................................ 23 2.1 Culture and maintenance of Human Embryonic Stem Cells ................................................. 23 2.2 Initiation of osteogenic differentiation in hESCs .................................................................. 24 2.3 Human fetal osteoblasts and their culture conditions ......................................................... 24 2.4 Methods for assessment of in vitro osteogenic differentiation ........................................... 25 2.4.1 RNA extraction and c-DNA synthesis ............................................................................ 25 2.4.2 Conventional Polymerase Chain Reaction .................................................................... 25 2.4.3 Detection of calcium deposition by alizarin red staining .............................................. 26 2.4.4 Hoechst DNA quantification assay ................................................................................ 26 2.5 Embedment of H1 & hFOB cell in ExtracelTM ........................................................................ 27 iii Assessment of cell viability in ExtracelTM .......................................................................... 27 2.5.1 2.6 2.6.1 Culture of H1 cells in Matrigel ...................................................................................... 28 2.6.2 Pre-differentiation of cells in scaffolds prior to in vivo implantation ........................... 29 2.6.3 Labeling of differentiating cell with CFDA-SE................................................................ 29 2.6.4 In vivo grouping............................................................................................................. 30 2.6.5 Surgical procedure ........................................................................................................ 30 2.6.6 Tissue Processing .......................................................................................................... 31 2.7 3 In vivo experiments ............................................................................................................... 28 Histology Staining.................................................................................................................. 31 2.7.1 H & E staining ................................................................................................................ 31 2.7.2 von Kossa staining ......................................................................................................... 31 2.7.3 Immuno-Fluoresence Staining ...................................................................................... 32 RESULTS ........................................................................................................................................ 34 3.1 Characterisation of hESCs ..................................................................................................... 34 3.2 In vitro results - Confirmation of osteodifferentiation in hESCs and hFOB - 2D culture system .............................................................................................................................................. 35 3.2.1 Alizarin red staining....................................................................................................... 35 3.2.2 Hoechst DNA Quantification Assay ............................................................................... 37 3.2.3 PCR Results .................................................................................................................... 37 3.2.4 Cell viability in 3D culture system - Laser Scanning Confocal Microscopy ................... 38 3.3 In vivo results – Histology ..................................................................................................... 38 3.3.1 Samples removed after 2 weeks in vivo ....................................................................... 40 3.3.2 Samples removed after 4 weeks in vivo ....................................................................... 42 3.3.3 Samples removed after 7 weeks in vivo ....................................................................... 44 4 DISCUSSION................................................................................................................................... 53 5 CONCLUSION ................................................................................................................................. 59 5.1 Limitations and future direction ........................................................................................... 60 6 APPENDIX ...................................................................................................................................... 62 7 REFERENCES .................................................................................................................................. 64 iv Summary Aim This study was aimed at comparing the in vivo osteogenic differentiation potential of human embryonic stem cells (hESC) and human somatic osteoblast cell line. Method HESCs were propagated on mouse embryonic feeder cells. They were shown to be pluripotent by expression oct4, sox2 and nanog molecular markers. Human fetal osteoblasts (hFOB) cell lines were cultured in DMEM media without phenol red. Osteogenic differentiation was initiated by supplementing the culture medium with βglycerophosphate, ascorbic acid, dexamethasone and vitamin D3. After 21 days of in vitro culture, osteogenesis in both cell types was confirmed by expression of osteocalcin and bone sialprotein molecular markers and calcium deposition by alizarin red staining. For in vivo assessment of osteogenesis, hESCs were propagated on feeder free culture system for 2 weeks. Both hESCs and hFOB cells were then seeded into PLGA scaffolds with cell carrier, Extracel, and left in culture media containing the same osteogenic supplements used for in vitro osteogenic differentiation. Before going in vivo the cells were stained with CFDA, a cell tracing reagent. After 14 days of in vitro differentiation, the cell-scaffold constructs were placed subcutaneous into the dorsum of the mice. Sacrifices were made at the end of 2nd, 4thand 7th weeks after implantation. The removed samples were processed and stained to assess the capacity of both hESCs and hFOB to form mineralized tissue. The tissue sections v were stained with H&E, von Kossa followed by immunostaining for osteonectin and alkaline phosphatase to confirm osteogenic differentiation. Results In hESC differentiated samples harvested after 7 weeks of implantation, discrete areas of mineralized tissue could be observed within the scaffolds. This was confirmed by von Kossa staining, H&E staining and immunostaining with osteonectin and alkaline phosphatase (ALP). There was no evidence of teratoma formation. In hFOB samples harvested 7 weeks after implantation, immunostaining with osteonectin and ALP confirmed the progression to osteogenic differentiation within the scaffolds. However, the samples did not stain positive for von Kossa staining. A possible explanation for this could be that the hFOB cells were at an early stage in differentiation. In samples harvested after 4 weeks of implantation, the scaffolds were found to be empty and devoid of the implanted cells. This was observed in samples of both cell types. Mineralization could not be detected in samples of both cell types harvested after 2 weeks of implantation. Conclusion This study demonstrates the possibility of generating osteoblasts from direct differentiation of hESCs under our osteogenic conditions. These osteoblasts when implanted subcutaneous into SCID mice, where able to form mineralized tissue. A comparison of in vivo osteogenesis in hFOB and hESCs was done. This comparison helped us to confirm the efficacy of in vivo osteogenesis in hESC. vi LIST OF FIGURES Figure 1 (a) human ES colonies on embryonic fibroblast feeder layer (b) RT-PCR analysis of Oct4, Nanog and Sox2 in undifferentiated hESCs ................................................................... 34 Figure 2 Alizarin red staining of mineralized nodules in H1 derived osteoblasts and hFOB on day 7 and day 21 of in vitro osteogenic culture........................................................................ 36 Figure 3 RT-PCR analysis of osteocalcin expressions in H1 derived osteoblasts and hFOB on day 7 and day21 of osteogenic culture...................................................................................... 39 Figure 4 FDA staining shows viable H1 cells (green), PI staining shows dead H1 cells (red) .............................................................................................................................................................. 39 Figure 5 Cell-scaffold construct extraction after 7-weeks in vivo ............................................... 39 Figure 6 At 2-week time point: H&E staining and von Kossa staining of sections showing scaffold and cells (e) hFOB - higher magnification showing scaffold (arrow) ........................... 41 Figure 7 At 4-week time point: H&E staining and von Kossa staining of sections showing scaffolds devoid of cells. ................................................................................................................... 43 Figure 8 At 7 week time-point: (a) H&E staining showing newly formed tissue within the scaffold (arrow showing scaffold remains) (b) von Kossa staining and counterstaining with nuclear fast red showing deposition of mineralized tissue in the area marked and shown in higher magnification: x20. Sites of mineralization indicated by arrow in fig (d ), (c) H&E staining showing newly formed tissue (arrow showing blood vessel infiltration into newly formed tissue) ..................................................................................................................................... 45 Figure 9 7 week time-point (b) Immunostaining for osteonectin (arrow) (d) Immunostaining for bone specific ALP (arrow) (a) (c) nuclear counterstaining with DAPI (blue) ....................... 46 Figure 10 Combination picture showing (a) H1generated osteoblasts taking up dapi, osteonection & CFDA stains (arrow) (b) H1 generated osteoblasts taking up dapi, ALP & CFDA stains (arrow) (c) & (d) CFDA-SE staining showing the implanted cells in tissue sections. ............................................................................................................................................... 47 Figure 11 At 7 week time-point: (a) H&E staining (c) H & E showing newly formed tissue (arrow showing blood vessel), (b) von Kossa staining did not show the presence of mineralised tissue............................................................................................................................... 49 vii Figure 12 At 7 week time-point: (a) (c) nuclear staining with DAPI (b) Immunostaining with bone specific ALP (arrow showing the cells positive for ALP stain) (d) Immunostaining with osteonectin (arrow showing cells positive for osteonectin staining) ........................................... 50 Figure 13 Combination pictures (a) hFOB taking up dapi, osteonection & CFDA stains (arrow) (b) hFOB taking up dapi, ALP & CFDA stains (arrow) (c) & (d) CFDA-SE staining showing implanted cells .................................................................................................................... 51 viii CHAPTER 1 LITERATURE REVIEW 1 1 LITERATURE REVIEW 1.1 Bone Bone is a mineralized connective tissue that acts as an internal scaffold for the body. Arranged both inside and around the bone are softer structures such as the vital organs, muscles, cartilages, nerves, blood vessels, adipose tissue and skin. Bone also serves as a reservoir for minerals such as calcium, phosphate and magnesium. Its unique cellular content and structural organization helps it to meet its biochemical and biomechanical demands in vivo. 1.1.1 Bone composition 1 Bone consists by weight of about 67% inorganic and 33% organic substances. The organic substances include both collagen and noncollagenous proteins such as proteoglycans, bone sialoprotein, osteocalcin, osteopontin and osteonectin. This organic matrix is permeated by hydroxyapatite crystals which constitute the inorganic portion of the bone. These crystals are responsible for a physiological mechanism called mineralization that happens in bone. The cellular components responsible for maintenance of the bone architecture include the osteogenic cells and osteoclasts. Osteogenic cells which form and maintain bone are represented by osteoprogenitors, preosteoblast, osteoblasts, osteocytes and bone lining cells constituting the different maturation stages of osteogenic cells. Osteoclasts are responsible for physiological bone resorption and remodeling of bone tissue. 2 Osteoblasts are cells derived from the mesoderm. Under the microscope, these mononucleated cells are distinct with their presence of extensive endoplasmic reticulum and numerous free ribosomes in the cytoplasm suggestive of their strong role in protein synthesis. Osteoblasts are cells specialized to perform two important functions, extracellular matrix formation and mineralization. Osteoblasts synthesize both the collagenous and non collagenous bone matrix proteins. This uncalcified bone matrix called the osteoid acts as a scaffold for deposition of the apatite crystals. Some osteoblasts, during their process of bone formation get trapped within their own matrix forming osteocytes. Generally, woven bone has more osteocytes than the lamellar bone. These osteocytes after their formation become reduced in size. The space in the bone matrix occupied by an osteocyte is referred to as osteocytic lacuna. Extensions of these lacunae form cannaliculi and house the osteocytic processes. Through these channels and processes, the osteocytes maintain contact with adjacent osteocytes, osteoblasts and the bone lining cells. This places the osteocytes in a very ideal position to sense the biochemical and mechanical environment around them and transduce signals to other cells involved in maintenance of bone integrity and vitality. When bone is no longer forming, osteoblasts flatten and extend along the bone surfaces. These cells are called bone lining cells. Osteoclasts are bone resorbing cells derived from the hematopoietic lineage. Typically, these cells are found in hollowed out depressions called the Howships lacunae. Under a scanning electron microscope these lacunae appear as shallow troughs with irregular shapes, reflecting the activity and mobility of osteoclasts during active bone resorption. 3 Osteoclasts are multinucleated cells with cell membrane thrown into numerous folds to form ruffled borders. At the periphery of the border, the plasma membrane is apposed closely to the bone surface. This clear zone not only attaches the osteoclasts to the mineralized surface but also isolates a microenvironment between them and the bone surface. Another feature of osteoclasts is a proton pump associated with the ruffled border that pumps hydrogen ions into the sealed compartment. The sequence of resorption is as follows: 1. Osteoclasts attach to the mineralized surface of the bone. 2. A sealed microenvironment is created through the action of proton pump which demineralizes bone and exposes the organic matrix. 3. The exposed bone matrix is degraded by enzymes such as acid phosphatase and cathepsin B. 4. Endocytosis of the degraded organic matrix happens at the ruffled borders. 5. These degraded products are released along the membrane opposite the ruffled border. 1.1.2 Bone structure 2 A section of a bone would reveal a dense sheet of outer compact bone and a central, medullary cavity filled with red or yellow bone marrow that is interrupted by a network of bone trabeculae. This network of bone trabaculae is called the cancellous or spongy bone. Adult bone whether compact or spongy consists of microscopic layers or lamellae. Three distant types of layering can be seen under the microscope – 4 circumferential lamellae, concentric lamellae with the Haversian canal and the interstitial lamellae. Surrounding the outer aspect of the compact bone is a connective tissue membrane called the periosteum. The outer layer of periosteum consists of fibrous connective tissue. The inner layer of periosteum consists of bone cells, their precursors and a rich vascular supply. Calcification prevents diffusion of nutrients into all the bone cells. Periosteum thus provides these cells with direct vascular supply. 1.1.3 Bone types Many types of classifications exist for bone. Based on their shape, bone can be classified as long bones (eg: femur, tibia, humerus, fibula), short bones (eg: carpals, metacarpals, tarsals, metatarsals), flat bones (eg: frontal, parietal, scapula) and irregular bones (eg: maxilla, sphenoid). Bone can also be broadly classified as woven bone and lamellar bone. Woven bone is highly cellular, formed in response to growth or injury and rich in bone sialoprotein. Lamellar bone is the mature bone which has collagen fibers arranged in lamellae and contains large quantities of osteocalcin. Bones can also classified into dense outer cortical bone and inner cancellous or spongy bone. 1.1.4 Bone formation and remodelling 1 Throughout embryogenesis bone development happens by two distinct processes. Intramembraneous ossification occurs in the bones of cranial vault, maxilla, and body of the mandible and the midshaft of long bones. In this type of ossification, bone develops directly within the soft connective tissue. First, 5 mesenchymal cells proliferate and condense as a membrane in the area of future bone. Vascularity at these sites increases. Osteoblasts differentiate from the mesenchymal cells and begin to produce bone matrix. The newly formed bone is termed woven bone. From early fetal development to full expression of the adult skeleton a continual slow transition occurs from woven bone to lamellar bone. Endochondral ossification occurs at extremities of long bones, vertebrae, ribs and at the articular extremities of mandible and the base of the skull. First there is condensation of mesenchymal cells. Chondrocytes differentiate from these condensed mesenchymal cells to form cartilage. The cartilage then undergoes a degenerative process with chondrocyte hypertrophy, programmed cell death and matrix calcification. Blood vessels penetrate bringing osteoprogenitor cells to the region which after differentiating to osteoblast, produced bone matrix overlaying the cartilaginous matrix remnants. Establishment of the overall size and shape of the bone extends from intrauterine stage to preadult period of human growth. During this phase bone is rapidly formed in the periosteal surface and destroyed along the endosteal surface within the compact bone. The adult skeleton also undergoes remodeling. In a healthy individual the amount of bone lost is balanced by the amount of bone formed. However, in case of certain diseases and with age, bone resorption exceeds formation resulting in overall loss of bone. Also defects that arise due to trauma, tumors or abnormal skeletal development cannot be addressed by the normal bone remodeling processes. 1 Ten Cate’s oral histology: Development, structure and function 2 The anatomical basis of dentistry, Bernard Liebgott 6 1.2 Clinical repair of bone defects Orthopedists, oral surgeons and scientists have always been looking for ways to stimulate fracture repair, heal non-union or restore lost segments of bone. Currently, skeletal defects are largely addressed in clinics by autogenous bone grafts, allogeneic bone grafts or by bone graft substitutes which include demineralized bone matrix, ceramics, graft composites and bone morphogenetic protein. The biology of these grafts varies and may provide one or more of these several essential components: (1) Osteogenic cells (2) Osteoconductive matrix (3) Osteoinductive proteins (Finkemeier, 2002). Autografts are derived from the patient’s own bone. These grafts have osteogenic, osteoinductive and osteoconductive properties. Common harvest sites for these grafts include iliac crest, tibia, fibula and radial bone. Advantages of autografts include their excellent success rate, low risk of transmission of diseases and histo-compatibility. However the disadvantages include the limitation in availability of graft material and the potential donor site morbidity. Tang et al reported that of the thirty nine patients who had a free fibular graft harvested for treatment of avascular necrosis of femoral head, 42% had a subjective sense of instability and 37% had a subjective sense of weakness in the lower extremities potentially due to muscle stripping during fibula harvest. Another source of autogenous graft material is the autogenous bone marrow. The injection of bone marrow provides a graft that is osteogenic (bone marrow stem cells) and osteoinductive (cytokines and growth factors secreted by the marrow cells). Bone marrow can be aspirated from the posterior iliac crest and injected into 7 the defect. The tendency of the injected materials to be washed away from the defect site is a potential problem in this technique. Demineralised bone matrix which is both an osteoconductive and osteoinductive material is used as carrier for autogenous bone marrow. Injection of autogeneous bone marrow with or without carriers has been used to treat several bone defects in clinics. With the development of immune suppressant, allogenic bone grafts are an alternate option and are available in various preparations. Demineralized bone matrix, cancellous chips, corticocancellous and cortical grafts, osteochondral and whole bone segments are some of them. Cortical, cancellous, osteochondral and the whole bone segments that are available are osteoconductive and provide immediate mechanical support. These are typically non vital bones harvested from cadavers and processed for use in clinics. However, most allografts possess the risk of disease transmission. Demineralised bone matrix (DBM) is prepared by a standardized process in which allogeneic bone is crushed into small particles and demineralized in HCL. DBM preparations are available as gels, puttys or strips for clinical use. These grafts offer both as osteoconductive and osteoinductive properties. However they do not offer structural support. Another disadvantage of demineralized bone matrix is that different batches may have different potencies because of the wide variety of donors used (Finkemeier, 2002). Bone graft substitutes are commercially available alternatives to auto and allogeneic bone grafts which possess many of the bone forming properties as human bone. Ceramics, ceramics phosphates and bioactive glasses are a few examples of these substitutes available for clinical use. Calcium phosphate ceramics are 8 osteoconductive and are used to fill bone defects. But these crystals by themselves are brittle and have poor tensile strength. Hence they should be placed in intact bone or rigid stabilized bone in order to protect the ceramic from shear stress and they should be tightly packed into adjacent host bone to maximize in-growth (Bucholz et al, 1987). Another bone graft substitute currently in clinical use is calcium collagen graft material. This graft also does not provide structural support but is used as a bone graft expander to augment fracture repair. Bone morphogenetic proteins (BMP) are proteins naturally produced in the body to regulate bone formation and healing. Recombinant human bone morphogenetic protein (rhBMP) is an osteoinductive material currently available and FDA approved for treatment in certain kinds of bone defects. Combinations BMP with gelatin foam, collagen, or calcium phosphate pastes is said to increase its retention in the defect site. Clinical studies have shown higher BMP doses are needed for osteoinductivity in humans (Termaat et al, 2005). Also the production and purification of BMP makes this an expensive treatment. To summarize, graft rejection, shortage in graft material, insufficient biocompatibility, lack of strength of the graft material and absence of cells with reparative potential to fill in large defects are the major disadvantages faced by most of these treatment methods. Thus, alternative approaches such as regenerative medicine is needed to address this growing problem. 9 1.3 Cell based tissue engineering Cell based tissue engineering deals with the use of biodegradable materials seeded with living cells to regenerate the form and function of a damaged or diseased tissue in a human body. Tissue engineering could be seen as a tool for regenerative medicine. The concept of tissue engineering involves cells on biodegradable scaffolds to generate functionally active tissues for in vivo applications. Investigations into the cell types available for this purpose, the scaffolds used and their combined role in bone formation are the need of the hour. 1.3.1 Scaffolds The ideal scaffolds are 3-dimensional, porous, osteoconductive biomaterials that have the following functions to perform: (1) promote cell adhesion and extracellular matrix deposition (2) permit the transport of nutrients, gases (3) biodegrade at a controlled rate (4) provoke minimal inflammation or toxicity in vivo. While considering scaffolds for bone regeneration, appropriate mechanical properties and design have to be taken into consideration (Principles of Tissue Engineering, Lanza, Langer and Vacanti). 1.3.2 Materials used as scaffolds Natural Polymers Collagen scaffolds have been reported to heal tibial bone defects in rats (Rocha et al, 2002). Collagen being the most commonly available protein in the body has the following advantages - excellent biocompatibility, biodegradability and promoting cell adhesion. But the disadvantages have been its low mechanical strength. Scaffolds made from hyaluronic acid have been used for osteogenic 10 differentiation of murine pluripotent cells (Kim and Valentini, 2002). But, like collagen they also fail to meet the mechanical demands. Ceramics Bioceramics such as hydroxyapatite and tricalcium phosphate, because of their similarities in composition with the inorganic portion of bone, have been used as scaffolding materials for bone regeneration. However, the major limitations in their use include their brittleness and difficulty in processing. Synthetic Polymers Biodegradable homopolymers and copolymers of lactic and glycolic acid have also been frequently used as scaffold materials. Many types of cells have been shown to attach and grow on these materials. Neonatal rat osteoblasts have been shown to attach to PLA (poly lactic acid), PGA (poly glycolic acid), PLGA (poly lactic co glycolic acid) substrates and synthesize collagen in culture (Ishaug et al, 1994). PCL (polycaprolactone) -HA (hydroxyapatite) scaffolds were used to tissue engineer bone in a study conducted by Yu et al in 2008. These synthetic polymers have for long been used in clinics as degradable suture materials. PLGA scaffolds are biocompatible, undergo controlled biodegradability and have good mechanical properties. In addition, reports have demonstrated that PLGA scaffolds support proliferation and differentiation of osteoprogenitor cells (Yang. et al, 2001). Lactic acid and glycolic acid are end products of their biodegradation that are eventually metabolized by the body. In addition, they have a high three dimensional design flexibility and can be fabricated into scaffolds with different size and structure. 11 The PLGA scaffolds used in this study are porous with a pore size of about 50 µm and channel size of about 2.4 mm. 1.3.3 Seeding of cells into scaffolds Various gels have been used to carry cells into scaffolds. An ideal cell carrier would be one which supports homogeneous and high cell-seeding efficiency as well as proliferation/differentiation. While considering gels for this purpose the following requirements have to be met: (1) close to neutral pH (2) quick setting time (3) stable in the culture media during the in vitro culture (4) biodegradation approximates that of scaffolds (5) minimal toxic residue. Extracel TM used as a cell carrier in this study is a hydrogel that is made up of three biocompatible components: thiol-modified hyaluronon, thiol-modified gelatin and a thiol-reactive crosslinker; polyethylene glycol diacrylate. Collagen and hyaluronic acid are of bovine origin. 1.3.4 Cell source for cell-based tissue engineering for bone regeneration Cellular components used in tissue engineering encompass viable cells of autologous, allogeneic or animal origin. While using viable cells, care is needed to control introduction of infectious diseases, cross-contamination from donors or introduction of infectious agents from materials used to process cells. Also, consequences of immune and inflammatory responses after the implantation of the cell- scaffold construct need to be considered. Different types of cells have been used for the reconstruction of bone tissue. Periosteal cells, skeletal muscle cells (Deasy et al, 2001), cells derived directly from 12 bone, as well as cells transduced with bone morphogenetic protein genes (Lee JY et al, 2001) have been used. Xiao et al in 2003 concluded that cells from human alveolar bone can retain their osteogenic properties in a three-dimensional collagen scaffold and subsequently synthesize a bone matrix, which after implantation in SCID mice can induce new bone formation in critical-size calvarial bone defects. Muschler and Midura, (Hutmacher and Sittinger, 2003) based on their mathematical model, calculated that 70 million osteoblasts are needed to regenerate 1cm3 of bone. But, there are limitations in expanding differentiated osteoblasts to such high numbers. Vacanti et al in the year 2001 (Hutmacher and Sittinger, 2003) reported a clinical case in which periosteal cells were used in bone tissue engineering. A biopsy taken after 10 months revealed that the cell-scaffold construct was vascularized and well integrated into the tissues. However, only 5% of the constructs showed newly formed lamellar bone and endochondral tissue. A clinical trial conducted by the University of Freiburg in 2003 (Hutmacher and Sittinger, 2003) resulted in commercial availability of bone grafts using autologous periosteal cells for clinical applications (BioSeed B; BioTissue Technologies, Freiburg, Germany). However, autologous procedures are time consuming due to the prolonged ex vivo cell culture. 1.4 Stem cells in bone repair Stem cells are the fundamental source of tissue for any organism. They provide the body with cells that are needed both for growth as well as repair. Stem cells are found both in a developing embryo as well as in an adult body. 13 Stem cells can be broadly classified based on the variability of derivatives they give rise to. Unipotent stem cells can generate only one mature cell type, multipotent stem cells give rise to two or more differentiated cell types and pluripotent stem cells that can give rise to representative of all three germ layers. Two important sources of stem cells available for regenerative therapy include (1) the adult stem cells from the adult tissue (autologous or allogenic) and from the bone marrow (mesenchymal stem cells, hematopoietic stem cells) and (2) embryonic stem and embryonic germ cells derived from discarded human embryos and germ line stem cells. Adult stem cells can be derived from various parts of the body including bone marrow, blood, deciduous teeth, pulp tissue and muscle. These lineage-restricted stem cells have been isolated from both fetal and adult tissues for application in regenerative medicine. Although, these cells have a high capacity to self renew in culture, their ability to expand is less than that for embryonic stem cells. The self renewal and the proliferative capacity of these stem cells also decrease with age (Pittinger et al, 1999; Murphy et al, 2002; Heng et al 2004). Recent studies have shown that MSCs derived from different tissue origins have different potential in lineage differentiation (Im et al, 2005; Chen et al 2006; Kern et al, 2006). In general MSCs are a heterogeneous collection of progenitor cells with varying degrees of replicative potential. Thus purification is required for its use in lineage specific differentiation. In comparison, embryonic stem cells (ESC) have the capacity to self renew infinitely. It is also relatively easy to generate sufficient cells for clinical use. However, immune rejection (Heng et al, 2004) of the ESC derived tissue poses a 14 major hurdle in the use of these cells. Although lifelong immunosuppression can be possible, it would be preferable to design bioengineered products that would be tolerated by the recipients without the use of immunosuppressive drugs. With reports of reprogramming of adult somatic cells to ESC like cells (Yu et al, 2007) concerns over immune rejection could be eliminated very soon. Other challenges in the use of ESC include controlling lineage specific differentiation and elimination of residual stem cells. Thus to obtain a purified population of osteoblasts from ESCs a good understanding of the mechanism underlying the lineage specific differentiation of hES cells is mandatory. 1.5 Human Embryonic stem cells As the name suggests, the human embryonic stem cells are derived from the early embryo. These cells have the capacity to self renew and can be directed to differentiate into derivates of all the three germ layers. However, in an intact embryo pluripotent undifferentiated cells are short lived. Thus stable maintenance of undifferentiated ESCs is only possible in vitro cultures. 1.5.1 Derivation of HES cells Human embryonic stem cells are derived from the inner cell mass of the developing blastocyst- stage embryo. The blastocyst is structurally a hollow sphere composed of the outer trophoblast, which develops into placenta and its supporting structures, the blastocoel, a fluid filled cavity inside the blastocyst and the inner cell mass (ICM) which gives rise to all cell types within the embryo. In vitro culture of isolated ICMs from human blastocysts was first reported in 1994 (Bongso et al 1994); however these cells were kept in culture for only a few 15 passages. The first derivation of a hES cell line from ICM of a blastocyst was published in 1998 by Thomson et al. To date hES cells have been derived from a variety of sources, including morula stage embryo, single human blastomeres and later blastocyst stage embryos. 1.5.2 Expansion and maintenance of pluripotency of HES cells Under the microscope, undifferentiated hES cells have a distinct morphology. They appear in tightly packed colonies with sharp borders. Most hES cells are heterogeneous as they contain both undifferentiated stem cells and some differentiated derivatives. Long term stability of hES cells is a requirement that has to be met for prolonged expansion of these cells in culture. Specialized culture conditions and culture medium are required to maintain its undifferentiated phenotype. In vitro, hES cells are cultured on feeder cells which produce various factors essential for the growth and maintenance of hES cells in their pluripotent state. Mouse embryonic fibroblasts (MEF) have been successfully used for this purpose. The propagation medium consists of DMEM/F-12 supplemented with Knockout serum replacement, supplemented with basic fibroblast growth factor to sustain the undifferentiated stage of the hES cells. In addition to conventional feeder based cultures, feeder-free culture systems are also widely in use. Matrigel is a soluble basement membrane extract of the Engelbreth-Holm-Swarm tumor that gels at room temperature to form a basement membrane (Kleinman et al, 1986). The major components of Matrigel matrix are laminin, collagen IV, entactin and heparan sulfate proteoglycan (Kleinman et al, 16 1982). Growth factors, collagenases, plasminogen activators and other undefined components have also been reported in Matrigel matrix (Vukicevic et al, 1992). Xu et al reported a culture system that utilized matrigel and conditioned media of mouse embryonic fibroblasts containing animal component-containing serum replacement and basic fibroblast growth factor, bFGF, for hESC culture. Studies on molecular signaling of hESCs have revealed various pathways that are crucial for the maintenance of pluripotency. These include FGF, TGFβ/activin/ nodal, BMP, Wnt pathways (Sato et al, 2003). Fibroblast growth factor2 signaling is crucial for the self renewal of hES cells. The mechanism of FGF2 signaling include induction of supportive factors secretion in MEF, up-regulation of major genes expressed in TGFβ/activin/nodal signaling and inhibition of BMP signaling (Gerber et al, 2007). The activation of the TGFβ/activin/nodal signaling through SMAD2/3 is associated with pluripotency and is required for the maintenance of the undifferentiated state in hES cells (James D et al, 2005). The Wnt pathway is also reported to enhance cell survival and proliferation transiently in hES cells (Dravid et al, 2005; Cai et al, 2007). With the identification of factors responsible for the maintenance of ESC pluripotency, defined feeder free culture media, TeSR1, has been developed by Ludwig and colleagues in which defined growth factors ( bFGF, TGFβ, aminobutyric acid, pipecolic acid and lithium chloride) have been added to replace the dependence on mouse embryonic fibroblasts-derived media. The modified TeSR-1 (mTeSR1) used for propagation of hESCs also includes some animal sourced proteins yet retains the advantage of being serum free. 17 1.5.3 Characterization of hES cells Several markers are used to identify the pluripotency in hES cells. These include surface markers such as glycolipids, glycoproteins, stage-specific embryonic antigens (SSEA) 3/4, tumor rejection antigens (TRA)-1-60, TRA-1-81 (Amit et al, 2000; Reubnoff et al, 2000; Rosler et al 2004) surface antigens: AC133, CA117, CD135, CD9 and transcription factors oct3/4, Sox-2 and Nanog (Hoffman et al, 2005). About 16 novel genes have been identified that are unique to hES cells. These include zinc finger proteins, GSH1 homeodomain protein containing a HOX domain and ESC-specific transcription factor Nanog (Bhattacharya et al, 2004). Telomerase activity, mitochondrial metabolism, genomic stability and epigenetic stability are some of the other standards in characterizing hES cells (Loring et al 2006). Human embryonic stem cells have shown to retain a stable diploid karyotype and continuously express high levels of telomerase during long term cultures (Zeng and Rao, 2006). HESCs capability to differentiate into all the three germ layers can be demonstrated by their ability to form embryoid bodies in vitro (Wobus et al, 2001) and teratoma in vivo (Thomson et al, 1998). 1.6 Differentiation of hES cells into osteogenic lineage The first report on osteogenic differentiation of ESCs was based on mouse ESCs and was published in 2001. The mouse ESCs were induced to form embryoid bodies following which they were directed to osteogenic differentiation (Buttery et al, 2001). 18 In vitro differentiation of ESCs into osteoblasts needs the addition of some supplements into the culture media. These include β-glycerolphosphate, ascorbic acid and vitamin D3 which are needed for matrix formation and mineralization. Stimulation of the culture medium with dexamethasone resulted in increased bone nodule formation (Beilby et al, 2004). Similar methods have been followed for osteogenic differentiation of human ES cells (Sottile et al 2003, Bielby et al 2004). Karp et al demonstrated the formation of bone nodules in 10 to 12 days without the EB step. Ahn et al cocultured hES cells with primary bone derived cells and showed that primary bone derived cells are able to induce differentiation of hES cells into osteoblasts without the addition of exogenous factors. Zur Nieden et al studied the expression pattern of osteoblastic markers during the differentiation of ES cells. The pattern of expression of marker genes can be related to phases of osteogenesis. Three phases include (1) proliferative phase (2) matrix deposition phase (3) mineralization phase. Type1 collagen mRNA is expressed at the end of proliferative phase and during matrix deposition phase. Osteopontin mRNA is expressed at the end of matrix deposition phase and at the beginning of the mineralization phase. BSP, Cbfa1/Runx2 are expressed during the mineralization phase indicating the presence of mature osteoblasts. Osteocalcin mRNA is considered an essential marker of the mineralization state. It was suggested that the length of time that differentiating hES cells are maintained in cultures (about 21 days) is in accordance with the time scale for osteogenic differentiation pathway of human primary osteoblasts and MSC cultures (Buttery et al, 2001). The capacity of the hES cells to differentiate into osteoblasts 19 and their ability to mineralize can be detected through staining procedures such as alizarin red or von Kossa. 1.7 In vivo bone formation using Stem Cells Bone formation through in vivo implantation of the stem cells that are directed to osteogenic differentiation are being studied and reported. First clinical reports of human MSC loaded onto a scaffold for trial on patients was by Quarto et al in 2001. The implants showed good osteointegration of newly formed bone. However the results were solely based on radiographic examinations and biopsies were not taken. Meijer et al (2007) have mentioned about their pilot study in which 10 patients were treated with cultured MSCs loaded on coralline hydroxyapatite (HA) scaffolds, for intra-oral defects. Only in one patient were they able to establish that the newly formed bone was due to the implanted cells. There are also reports on in vivo osteogenic potential of hES cells, when implanted into animal models. Bielby et al (2004) showed that culture conditions that resulted in vitro osteogenic differentiation of hES cells also yielded mineralized tissue when implanted in vivo into the dorsum of SCID mice. Bone formation by endochrondal ossification was achieved in vivo in immunodeficient mice, by using mouse ES cell derived cartilage constructs as base material (Jukes et al, 2008). A cartilage matrix was formed in vitro by using mouse ES cells seeded on to scaffolds. When these tissue engineered constructs were implanted subcutaneously, the cartilage matured became hypertrophic, calcified and was ultimately replaced by bone in 21 days. When the authors followed the same 20 approach with hESCs, they found that the chondrogenic potential of hESCs was insufficient to form cartilage in vitro and bone in vivo. Tremoleda et al in 2008 compared the in vivo osteogenic potential of hES cells with that of hMSC. They demonstrated that bone formed by in vivo directed differentiation of hESCs in diffusion chamber model had no obvious qualitative difference to bone formed by hMSC. A comparison of osteogenesis of hES cells within 2D and 3D culture systems demonstrated that the osteogenic differentiation of hES cells is enhanced in a 3D culture system compared to a 2D culture environment in vitro. When these cellscaffold constructs were seeded onto cortical defects created on rabbit bone, they yielded bone after about 4 weeks. (Tian et al 2007). In our study we have compared in vivo formation of bone from osteoblasts differentiated from hES cells with a human somatic osteoblasts cell line. HES cells differentiated osteoblasts are genetically young bone forming cells which can ultimately be used for bone reconstruction. This study would help us establish the efficacy of applying hESCs-dervied osteogenic cells in bone formation. 21 CHAPTER 2 MATERIAL & METHODS 22 2 2.1 MATERIAL & METHODS Culture and maintenance of Human Embryonic Stem Cells Human embryonic stem cells (hESCs) used in this study were the H1 cell lines (isolated and established at the University of Wisconsin) with normal karyotype. These H1cells were cultured on a layer of murine embryonic fibroblasts (MEFs) as described in chapter 1. Culture plates (6 well plates) were first coated with 0.1% (v/v) gelatin (Sigma) and left overnight. The next day the plates were seeded with mitomycin-C inactivated MEFs at a density of 1.4 million cells per 6-well plate. These MEFs were allowed to attach for a day prior to use as feeder layers. Then H1 cells were propagated on this layer of MEF cells. H1 cells were maintained in hESC culture media comprised of Dulbeccos modified eagles medium/ F12 (DMEM/F12, Gibco BRL, Grand Islands, NY, USA) supplemented with 20% (v/v) KnockoutTM Serum Replacement (Gibco BRL), 1% (v/v) nonessential amino-acids (Gibco BRL), 1mM L-glutamine (Gibco BRL), 0.1nM β-mercaptoethanol (Sigma) and 4 ng/ml basic fibroblast growth factor (bFGF; Gibco, BRL). Cultures were kept in an undifferentiated state at 37 C and in a 5% CO2 atmosphere and checked visually under a light microscope. Culture media was changed every day. H1 cells usually reached confluence by day five. These cells were then removed from the culture dish by incubation with 1 mg/ml collagenase type-IV (Gibco BRL) for about 3 to 5 minutes at 37 C followed by mechanical scraping of the culture plates. The H1 cells were then re-plated at a splitting ratio of 1:6 to new 6-well culture plates seeded with MEFs. 23 The MEFs were prepared by expanding them in basic media which consisted of DMEM (Sigma, St.Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (FBS; Hyclone, USA). MEFs were passaged at 1:5 splitting ratio up to five passages after which they were inactivated with 10 µg/ml mitomycin-C (Kyowa,Tokyo, Japan) for 2 hrs at 37 C before use as feeder cells. 2.2 Initiation of osteogenic differentiation in hESCs To initiate differentiation, H1 cells were obtained after treatment with collagenase. Approximately 500,000 cells were seeded onto each well of a 24-well culture plate. The 24-well culture plate was coated with gelatin the previous day and left overnight. The seeded cells were first left in basic media (DMEM with 10% FBS) for a day and then changed to culture media treated with osteogenic supplements. The osteogenic media consisted of basic media supplemented with 50 µg/ml ascorbic acid (sigma), 10 mM β-glycerolphosphate (sigma), 10-8 M vitamin D3 and 10-6M dexamethasone (Sigma). Culture media was changed once every 2 days until day 21. The cells were maintained in incubators at 37 C and in a humid 5% CO2 atmosphere. 2.3 Human fetal osteoblasts and their culture conditions Human fetal osteoblasts (hFOB; CRL- 11372, ATCC, VA, USA) were used as control cells in this study. Human fetal osteoblasts were cultured in DMEM/F-12 (Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, UT, USA) and 0.3 mg/ml Geneticin (G418 sulfate, Gibco). All cultures were incubated at 34 C in 5% CO2 according to recommendation of ATCC. A temperature of 34 C was considered more optimal for the proliferation of hFOB instead of the 24 usual 37 C (Ge.Z et al, 2008). Culture media was changed once every three days. Osteoblasts were trypsinized when confluent and replated at a splitting ratio of 1:3. For differentiation about 500,000 cells were plated onto each well of a 24-well plate. The cells were maintained for a day in hFOB culture media and then changed to osteogenic culture media. The osteogenic culture media consists of DMEM/F-12 supplemented with 10% FBS, 50 µg/ml ascorbic acid, 10 mM β- glycerolphosphate 10-8 M vitamin D3 and 10-6 M dexamethasone. Cultures were maintained in osteogenic differentiation medium for 21 days with the media changed once every three days. 2.4 Methods for assessment of in vitro osteogenic differentiation 2.4.1 RNA extraction and c-DNA synthesis Total RNA was collected from the osteogenic cultures after 7 and 21 days using RNeasy Mini Kit (Qiagen, Chatsworth, USA) following the manufacturer’s instructions. Samples were taken from undifferentiated H1 cells and osteodifferentiated H1 & hFOB cultures. Samples were treated with RNase-free DNase ŀ (Qiagen) to remove any genomic DNA contamination. The extracted RNA was quantified using Nanodrop (Nanodrop Technologies, Wilmington, DE). About 500 ng of mRNA was used to synthesize the required cDNA. cDNA synthesis kit (Bio-Rad, Hercules, CA) was used for this purpose. 2.4.2 Conventional Polymerase Chain Reaction Conventional PCR was performed using PCR thermal cycler, Mycycler (BioRad). Samples were amplified for 35 cycles at 95 C for 5 minutes, 35 cycles at 95 C for 30 seconds, 55-65 C for 45 seconds, 72 C for 1 minute, followed by 72 C for 5 25 minutes. β-actin was the house keeping gene used to normalize the PCR reactions. Electrophoresis of the amplified products was run on 2% agarose gel. The products were stained with ethidium bromide and viewed using Light Imaging System (BioRad). PCR primers, annealing temperature, primer sequence and their product size are listed in Table 1 2.4.3 Detection of calcium deposition by alizarin red staining As an indicator of mineralization within both the H1 and hFOB osteogenic differentiation cultures, calcium deposition was analyzed by alizarin red staining at 7, 14 and 21 day time points. 2 g of alizarin red S (sigma) was dissolved in 100 ml of de-ionized water. The pH of the solution was adjusted to 4.2 using 0.5% ammonium hydroxide. The solution was filtered before use. After washing with PBS the osteogenic cell cultures were fixed in 4% paraformaldehyde for 30 minutes at room temperature. Then the cultures were again washed with PBS and stained with alizarin red for about 5 minutes. The dye was removed and the cell cultures washed with deionized water. The mineralization was observed under the microscope. The calcium salts within the bone nodules stained red while the collagenous extra cellular matrix turned yellow. 2.4.4 Hoechst DNA quantification assay Samples were taken on day 7, 14 and 21 of osteogenesis in both hESC and hFOB osteogenic differentiation. DNA concentration was assessed to compare the cell density during the mentioned time points. 10 mg/ml calf thymus DNA was diluted in 1xTNE buffer composed of 10 mM Tris Base, 10 mM EDTA and 100 nM NaCl with pH adjusted to 7. Twelve different 26 concentrations of calf thymus DNA was used to establish standard curve for DNA content. 10 µl of each sample was diluted with PBS to a final volume of 100 µl followed by addition of 100 µl of Hoechst33258 Dye for fluorescence measurement by fluorescence plate reader (Tecan Safire, Austria) at 355 nm excitation and 460 nm emission. DNA concentration in each sample was extrapolated from the DNA standard curve. 2.5 Embedment of H1 & hFOB cell in ExtracelTM About 500,000 cells of both H1 and hFOB were grown in Extracel (Glycosan Biosystems, Salt Lake City, Utah). TM Extracel is made up of three biocompatible components: Gelin-S, Glycosil and Extralink. Hydrogel was formed when the crosslinking agent, Extralink™ was added to a mixture of Glycosil™ and Gelin-S™ which also contained the cells. This mixture was then placed in culture plates and allowed to gel at 37 C. Gelation occurred in about twenty minutes. Then the respective culture media were added to each of the wells holding the cell-gel construct. Media was changed every day for the H1 cultures and once in two days for hFOB cultures. The cultures were maintained in gel for a week and then assessed for cell viability. Extracel is intended to be used as a cell carrier for seeding cells into the scaffolds for in vivo experiments. 2.5.1 Assessment of cell viability in ExtracelTM 2.5.1.1 FDA/PI staining Cell viability in Extracel was assessed by Fluorescein diacetate/ Propidium iodide (FDA/PI) staining. 12 µM stock solution was prepared by dissolving of 5 mg FDA (Sigma) in 1 ml of acetone. Then FDA working solution was prepared by adding 27 0.04 ml of stock to 10 ml of dulbeccos phosphate buffered saline (DPBS). 1.5 µM working solution was prepared by dissolving 1 mg of PI (Sigma) in 50 ml of DPBS for use. After a week in Extracel culture, the culture media was removed and the cells (both H1 and hFOB) were stained with 0.1 ml of FDA (2 µg) and 0.03 ml (0.6 µg) of PI for about 3 mins and then placed in ice. 2.5.1.2 Confocal microscopy Confocal microscopic analysis of the FDA/PI stained cells embedded in Extracel was carried out using a Ziess Fluoview laser confocal microscope. The wavelength of the excitation and emission for FDA were 488 nm and 505-503 nm and for PI were 543 nm and 585 nm. Image processing was completed using software supplied by the confocal microscope’s manufacturer. 2.6 2.6.1 In vivo experiments Culture of H1 cells in Matrigel For in vivo experiments H1 cells cultured in feeder-free culture system were used. Confluent H1 cells grown on feeder layer were removed from the culture plastics after incubation with collagenase. The H1 cells were replated into mTeSR1TM (Stemcell Technologies Inc). The new culture plates were coated with BD MatrigelTM (BD Biosciences) an hour prior to replating of H1 cells. Media was changed once every day. Cells were removed from the culture plastics when reaching confluence after incubation with dispase (Stemcell Technologies) for 7 minutes at 37 C and mechanical scraping. The cells were then replated onto new Matrigel-coated culture 28 plates. The H1 cells were cultured in feeder-free culture for 2 weeks prior to implantation in vivo. 2.6.2 Pre-differentiation of cells in scaffolds prior to in vivo implantation About 500,000 cells of either H1 or hFOB were seeded into each of the PLGA scaffold (Bio-scaffold International, Singapore) using ExtracelTM as the cell carrier. The cell-scaffold constructs were left in their respective culture media for a day. After 24 hrs, osteogenic differentiation was initiated in these cells by replacing the culture media with their respective osteo-differentiation media. The cell scaffold constructs were maintained in osteogenic culture media for two weeks before implantation in vivo. Media was changed once every two days. 2.6.3 Labeling of differentiating cell with CFDA-SE Vybrant R CFDA SE kit was used for in vivo tracing of the cells in this study. 10 mM stock solution was prepared by dissolving 500 µg Carboxy-flurorescein diacetate, succinimidyl ester (CFDA SE) powder into 90 µl of dimethylsulfoxide (DMSO). This stock solution was then diluted in PBS to the required working concentration of about 15 µM. The cell-scaffold constructs were maintained in osteogenic differentiation media for about 2 weeks before labeling. Culture media was removed from the dishes. The cells were incubated for 15 minutes in prewarmed (37 C) PBS containing CFDA SE. This loading solution was then replaced with fresh prewarmed (37 C) osteogenic differentiation media. The cell scaffold constructs were incubated for about 30 minutes in 37 C before observation under the fluorescent microscope. 29 Then the cell scaffold constructs were left in the incubator for a day before implantation in vivo. 2.6.4 In vivo grouping A total of 6 SCID mice were used for in vivo experiments, with each mouse carrying 5 - 6 samples of scaffold-cell constructs. Three of the mice carried test samples which were scaffold-hESC constructs, while the other three carried positive control samples which were scaffold-hFOB constructs. One mouse each from the test and control groups were sacrificed at 2nd, 4th and 7th weeks. Group Sample Test PLGA scaffold + cell carrier +d-hESC PLGA scaffold + cell carrier + hFOB Positive control 2nd week sacrifice 5 samples x 1 mice 4th week sacrifice 5 samples x 1 mice 7th week sacrifice 6 samples x 1 mice Total 5 samples x 1 mice 5 samples x 1 mice 6 samples x 1 mice 16 samples x 3 mice 16 samples x 3 mice 2.6.5 Surgical procedure Ethical approval was obtained from the animal welfare committee, National University of Singapore, licensed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Seven weeks old male and female SCID mice were used in this study. The animals were acclimatized to the animal holding unit one week prior to the surgery. The SCID mice were anasthetized with an intraperitoneal administration of AHU CRU mixture (Ketamine + medetomidine). A one centimeter incision was made on the dorsum of the mice. Then cell-scaffold constructs (H1 & hFOB osteodifferentiated cultures) were transplanted subcutaneously into both the sides on the dorsal surface. The grouping was done as mentioned in the above table. The 30 skin was closed using 5-0 Prolene suture material. After 2, 4 and 7 weeks, the mice were sacrificed and the specimens were harvested. 2.6.6 Tissue Processing The harvested explants which included the scaffold and the surrounding tissue from the SCID mice were fixed in 4% para-formaldehyde for 7 days. Routine tissue processing using an automatic process machine (Leica) was carried out. Following this the tissue-scaffold explants were embedded in paraffin blocks. Then, each block was sectioned at 5 µm thickness and collected on Silane coated slides. Sections were de-paraffined and re-hydrated in Xylene for 3 times followed by two time serial incubation in absolute and 95% ethanol before staining. 2.7 Histology Staining 2.7.1 H & E staining Sections were incubated in hematoxylin (Sigma) for 5 minutes. They were then washed in tap-water followed by a dip in Scott’s tap-water. Then the sections were again washed in tap-water. Then the sections were stained in eosin (Sigma) for 5 minutes. After staining the sections were dehydrated in alcohol and xylene. Sections were then sealed with cover slips using permanent mounting media. 2.7.2 von Kossa staining This technique helps to visually establish the presence of calcium or calcium salt deposits in tissue sections. Sections were incubated with 1% silver nitrate solution (Sigma) in a clear glass coplin jar and placed under ultraviolet light for 30 minutes. Sections were then rinsed in several changes of distilled water. Un-reacted silver was removed with 5% sodium thiosulfate (Sigma) for 5 minutes. Sections were 31 again rinsed in distilled water and counterstained with nuclear fast red for 5 minutes. Sections were once again rinsed in distilled water. Further sections were dehydrated through graded alcohol and cleared in xylene and coverslipped using permanent mounting medium. 2.7.3 Immuno-Fluoresence Staining After de-parafinizing, antigen retrieval was achieved through microwave antigen retrieval method. The sections were then blocked for 20 minutes in PBS-BSA followed by incubation overnight with primary antibodies. Monoclonal antibodies to bone- specific-ALP (B4-78, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City) and osteonectin (AON-1, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City) were diluted in PBS and 2% BSA at a dilution factor of 1:40 for use in this study. Sections were then washed in PBS and incubated with 10 nM Qdot655 antimouse IgG secondary antibody (Invitrogen, Oregon, USA) at room temperature for 60 minutes. Sections were then washed twice with 0.05% v/v Tween20 (Bio-Rad) and one time PBS and mounted with slow Fade Gold antifade reagent containing DAPI (Invitrogen). 32 CHAPTER 3 RESULTS 33 3 3.1 RESULTS Characterisation of hESCs Undifferentiated hESCs were characterized by their distinct morphology and by their ability to express molecular markers that are typical of mammalian pluripotency cells. The expression of pluripotency markers in undifferentiated hESCs allows us to distinguish them from their differentiated progenies. hESCs expanded on the feeder MEF layer maintained the typical undifferentiated tightly packed colonies with sharp borders (Fig 1a). The pluripotency status of H1 cells was confirmed by their expression of Oct 4, Nanog and Sox 2 mRNA (Fig 1b). β-actin oct4 nanog sox2 (a) (b) Figure 1 (a) human ES colonies on embryonic fibroblast feeder layer (b) RT-PCR analysis of Oct4, Nanog and Sox2 in undifferentiated hESCs 34 3.2 In vitro results - Confirmation of osteodifferentiation in hESCs and hFOB - 2D culture system 3.2.1 Alizarin red staining hESCs and hFOB were seeded as monolayer and induced to differentiate in osteogenic media containing ascorbic acid, dexamethasone, and β-glycerophosphate and vitamin D3. Calcium deposition in hESCs and hFOB on days 7 and 21 were assessed by alizarin red staining. The calcium salts in the bone nodules picked up the red stain which was indicative of hESCs and hFOB differentiating down the osteoblastic lineage (Fig 2). The day 7 samples showed initial stage of mineralization with fewer nodules when compared to the day 21 for both the hESCs and hFOB. 35 (a) H1 osteodifferentiation – day7 (c) H1 osteodifferentiation – day 21 (b) hFOB osteodifferentiation – day 7 (d) hFOB osteodifferentiation – day 21 Figure 2 Alizarin red staining of mineralized nodules in H1 derived osteoblasts and hFOB on day 7 and day 21 of in vitro osteogenic culture 36 9 3.2.2 Hoechst DNA Quantification Assay 10 µl Sample Lysate Used No. 1 2 3 4 5 6 Samples H1 differentiation 7days H1 differentiation 14days H1 differentiation 21 days hFOB differentiation 7days hFOB differentiation14days hFOB differentiation 21days Concentration (µg/ml) 10.6 16.8 16.9 16.2 17.8 18.5 Samples were taken on day 7, 14 and 21 of osteogenesis in hESC and hFOB cultures. Samples taken on the 7th day of differentiation showed higher rate of cell proliferation in hFOB than in hESC. However, as the differentiation proceeded, cell proliferation rate was comparable in both the cell types, as seen from the samples taken on day 14 & 21. 3.2.3 PCR Results The osteogenic differentiation potential of H1 and hFOB under our osteogenic conditions was further assessed by their ability to express some molecular markers specific for osteogenic differentiation. Osteocalcin and bone sialoprotein molecular markers were used in this study. Both day 7 and day 21 samples expressed the osteocalcin with the expression being more evident on the day 21 sample. Expression level of osteocalcin in hFOB was much higher than that of H1 cells suggesting that hFOB were more differentiated than H1 cells in vitro (Fig 3). 37 3.2.4 Cell viability in 3D culture system - Laser Scanning Confocal Microscopy About 500,000 cells of H1 and hFOB were seeded on to Extracel and allowed to grow in culture plates. After a week in gel, the cells were stained with FDA and PI and viewed under a confocal microscope. The results showed that about 80% of the cells remained viable in the gel. The viable cells picked up FDA and appeared green under confocal microscope while the dead cells picked up PI and appeared red under the microscope (Fig 4). 3.3 In vivo results – Histology H1 and hFOB cells were seeded into PLGA scaffolds and allowed to differentiate into osteogenic lineage for 14 days in vitro. The cell-scaffold constructs were then transplanted subcutaneously into both sides on the dorsal surface of seven-week old SCID mice. After 2, 4 and 7 weeks, the mice were sacrificed (Fig 5) and the specimens were harvested. 38 H1 H1 d7 d21 hFOB hFOB d7 d21 Bone sialoprotein Osteocalcin β-actin Figure 3 RT-PCR analysis of osteocalcin expressions in H1 derived osteoblasts and hFOB on day 7 and day21 of osteogenic culture Figure 4 FDA staining shows viable H1 cells (green), PI staining shows dead H1 cells (red) scaffold Figure 5 Cell-scaffold construct extraction after 7-weeks in vivo 39 3.3.1 Samples removed after 2 weeks in vivo After two weeks in vivo, histological examination of the implanted samples, both H1 and hFOB, showed viable cells and vascular infiltration within the scaffolds (Fig 6a). However, von Kossa staining did not show sites of calcium deposition (Fig 6d). A possible explanation could be that even in an ideal environment, formation of new bone tissue to fill in defects could take about 2 months to complete. Hence, mineralised tissue could not be expected in samples after 2 weeks of implantation in an ectopic site. Presence of PLGA scaffolds was detected in the tissue sections confirming their controlled biodegradability in vivo. 40 (a) H1 H & E staining (b) H1 von Kossa staining (c) hFOB H & E staining (d) hFOB von Kossa staining Scaffold (e) hFOB H & E staining Figure 6 At 2-week time point: H&E staining and von Kossa staining of sections showing scaffold and cells (e) hFOB - higher magnification showing scaffold (arrow) 41 3.3.2 Samples removed after 4 weeks in vivo After four weeks in vivo, histological examination did not show the implanted cells within the scaffolds (Fig 7). The empty scaffolds contained few blood vessels within the pores of the scaffolds. A possible explanation for the loss of the implanted cells within the scaffolds could be due to the procedures involved in processing of these explanted tissue samples. 42 (a) H1 H & E staining (b) H1 von Kossa staining (c) hFOB H & E staining (d) hFOB von Kossa staining Figure 7 At 4-week time point: H&E staining and von Kossa staining of sections showing scaffolds devoid of cells. 43 3.3.3 Samples removed after 7 weeks in vivo 3.3.3.1 H1 - Histology After seven weeks, histological examination of the implanted scaffolds showed evidence of vascular infiltration (Fig 8) and new tissue formation within the scaffolds. von Kossa staining showed some dark areas suggestive of calcium deposition in these areas (Fig 8). This new tissue stained positive with antibodies specific for ALP and osteonectin (Fig 9). CFDA staining confirmed presence of implanted cells within the scaffold. CFDA staining also showed that the cells that stained positive for ALP and osteonectin were from the implanted cells and not from the host cells (Fig 10). However implanted cells did not completely infiltrate the scaffolds thus leaving some areas devoid of cells. H&E staining showed no evidence of teratoma formation around the site of the implanted scaffold. Portions of the scaffolds were still seen in the tissue sections (Fig 8). 44 A (a) H & E (b) von Kossa staining C (c) H & E magnified (d) von Kossa staining - magnified Figure 8 At 7 week time-point: (a) H&E staining showing newly formed tissue within the scaffold (arrow showing scaffold remains) (b) von Kossa staining and counterstaining with nuclear fast red showing deposition of mineralized tissue in the area marked and shown in higher magnification: x20. Sites of mineralization indicated by arrow in fig (d ), (c) H&E staining showing newly formed tissue (arrow showing blood vessel infiltration into newly formed tissue) 45 3.3.3.2 H1 - Immuno staining – Week 7 (a) Cell nuclei stained for dapi (b) Osteonectin + dapi (c) Cell nuclei stained for dapi (d) ALP + dapi Figure 9 7 week time-point (b) Immunostaining for osteonectin (arrow) (d) Immunostaining for bone specific ALP (arrow) (a) (c) nuclear counterstaining with DAPI (blue) 46 (a) Combination dapi + ON+ CFDA (b) Combination dapi + ALP + CFDA (c) CFDA staining for ON (d) CFDA staining for ALP Figure 10 Combination picture showing (a) H1generated osteoblasts taking up dapi, osteonection & CFDA stains (arrow) (b) H1 generated osteoblasts taking up dapi, ALP & CFDA stains (arrow) (c) & (d) CFDA-SE staining showing the implanted cells in tissue sections (arrow showing implanted cells). 47 3.3.3.3 hFOB – Histology – Week 7 After seven weeks, histological examination showed vascular infiltration and new tissue formation. Though this tissue stained positive for antibody specific for ALP and osteonectin (Fig 12), von Kossa staining did not demonstrate deposition of calcium salts in these tissue sections (Fig 11). H&E staining did not show any evidence of teratoma formation around the implanted site. CFDA staining confirmed the presence of the implanted cells within the scaffolds (Fig 13). 48 (a) H & E staining (b) vonKossa staining (c) H & E staining magnified Figure 11 At 7 week time-point: (a) H&E staining (c) H & E showing newly formed tissue (arrow showing blood vessel), (b) von Kossa staining did not show the presence of mineralised tissue 49 3.3.3.4 hFOB - Immunostaining – Week 7 (a) cell nuclei with dapi (c) cell nuclei with dapi (b) Osteonectin + dapi (d) ALP + dapi Figure 12 At 7 week time-point: (a) (c) nuclear staining with DAPI (b) Immunostaining with bone specific ALP (arrow showing the cells positive for ALP stain) (d) Immunostaining with osteonectin (arrow showing cells positive for osteonectin staining) 50 (a) dapi + ON+ CFDA (b) dapi + ALP + CFDA (c) CFDA staining for ON (d) CFDA staining for ALP Figure 13 Combination pictures (a) hFOB taking up dapi, osteonection & CFDA stains (arrow) (b) hFOB taking up dapi, ALP & CFDA stains (arrow) (c) & (d) CFDA-SE staining showing implanted cells 51 CHAPTER 4 DISCUSSION 52 4 DISCUSSION The derivation of human embryonic stem cells and establishment of their culture systems has provided us with a unique tool for bone regeneration. The plasticity shown by hESCs makes them ideal candidates in the field of tissue engineering. Several studies have been reported on the in vivo osteogenic potential of hESCs in animal models. Bielby et al in the year 2004 seeded osteoblasts generated from hESCs onto PDLLA scaffolds, implanted them subcutaneously into SCID mice and reported the formation mineralized tissue. However in this study other types of bone cells such as bone lining cells and osteocytes were not identified. Further to this, in a study by Jojanneke M. Jukes et al in 2008 mouse ESCs were first made to deposit cartilage. This cartilage template was implanted subcutaneously onto immunodeficient mice. In contrast to the samples which were differentiated into osteogenic lineage in vitro, bone like tissue formed in samples were derived from the above mentioned cartilage templates. However, hESCs were not used in this study. In our study we compared the in vivo mineralization of hESC derived osteoblasts with that of their somatic counterparts. The main focus of our study was to examine the capacity of hESCs to differentiate into osteogenic lineage within an in vitro environment and there after assess their ability to mineralize in an ectopic environment in vivo. Pluripotent H1 cells were used in this study. The H1 cells were grown on gelatin coated culture plates. The cells were allowed to differentiate in the presence of dexamethasone, ascorbic acid and βglycerolphosphate. Novel to our study was the use of human somatic 53 osteoblasts as control cells. This comparison of osteogenesis between H1 derived osteogenic cells and human somatic osteoblasts allowed us to test the efficacy of these H1 differentiated cells. The H1 cells were plated in monolayer and allowed to differentiate into osteoblastic phenotypes. The optimal seeding density used for this study was about 500,000 cells per well which provided enough space for cells to proliferate and provide cell to cell contact, followed by extracellular matrix secretion and mineralization. Seeding density was kept the same for hFOB osteogenic differentiation as well. Both the cell types were allowed to differentiate for about 21 days. Mineralization was assessed by Alizarin Red staining. Positive staining was detected on both the cell lines tested. A comparison of cell proliferation in both cell types on day 7, 14 and 21 of osteogenic differentiation was done by Hoechst DNA quantification assay. DNA concentration showed that in the initial stages of differentiation cell proliferation rate was higher in hFOB, but by 14th and 21st days of osteogenic differentiation, there were almost equal DNA concentration in hFOB and hESC cultures indicating similar cell density at these time points. Conventional PCR analysis done to screen the osteoblast specific genes demonstrated the expression of osteocalcin and bone sialoprotein on both these differentiated cell types at both time points (day 7 & 21). It was observed in our study that highest level of osteocalcin expression was seen on day 21 for both the cell type. The H1 cells that were differentiated into osteogenic lineage for the in vivo study were grown on feeder free culture system for two weeks. This excluded the possibility of a carryover of the mouse feeder cells during 54 implantation. PLGA scaffolds were used to carry the differentiated H1 cells and hFOB in vivo. The scaffolds had a pore size of about 50 µm and a channel size of about 2.4 mm. This pore size was believed to enhance cell growth and neovascularisation and might thus promote bone formation in vivo. The H1-derived osteoblasts - scaffold constructs when implanted in vivo were able to generate discrete areas of mineralized tissue which had close association with blood vessels. However this result was only observed in samples that were harvested after seven weeks in vivo. Differentiation into osteogenic lineage happens in three phases, proliferative phase, matrix deposition phase and mineralisation phase. The mechanism of mineralization by osteoblasts involves the deposition of calcium phosphate apatite within the osteoid. Mineralization was confirmed by the positive dark brown staining from von Kossa. Silver nitrate used in von Kossa staining helps detect the phosphate in the calcified matrix. Further to this, the presence of osteoblasts in the newly formed tissue was confirmed by immunostaining specific for bone specific ALP and osteonectin. As the H&E staining did not show any teratoma formation, it could be said that the presence of undifferentiated cells in the H1 differentiated cultures was minimised. The hFOB -scaffold constructs which were removed from the mice at the same time point did not stain positive for von Kossa. However, immunostaining with bone specific ALP and osteonectin gave positive results. As mentioned earlier mineralization is the last stage in osteogenesis. Each stage in osteogenesis is characterized by expression of distinct set of genes. During proliferative stage, Collagen type1 genes associated with formation of extra 55 cellular matrix are expressed. Osteopontin and ALP are the genes expressed at the end of matrix deposition. The last stage which is the mineralization stage is characterised by the expression of osteocalcin. Thus a possible explanation for the above mentioned results could be that the hFOB differentiation was at an early stage and had not reached the last phase of differentiation. Samples that were retrieved after four weeks of implantation had much of the scaffolds devoid of cells. This was the case for both H1 and hFOB derived osteoblasts. As the presence of the cells in the scaffold before going in vivo was not confirmed through scanning electron microscopy, it was difficult to say where the absence of cells in the tissue sections was because of improper seeding or due to tissue processing procedures. In the samples harvested after 14 days of implantation, tissue sections showed the presence of cells both H1 and hFOB within the scaffolds. These cells however did not stain positive for von Kossa. A possible explanation could be that, 14 days was not enough to achieve osteogenic differentiation in an ectopic site. Normal bone development follows a typical path for differentiation from early mesodermal progenitors to the fully differentiated osteoblasts. Bone development could either happen through intramembraneous ossification or through endochondral ossification. In a healthy individual bone remodelling is a natural process where the amount of bone lost is balanced by the amount of bone formed. But in case of disease or trauma an abnormal bone loss may require additional intervention to fix the defect. 56 In theory, hESCs remain ideal candidates for bone-tissue engineering. However, there are still many limitations in its use which have to be overcome. These limitations will be discussed in the following chapter. 57 CHAPTER 5 CONCLUSION 58 5 CONCLUSION In our study, we aimed to assess the in vivo osteogenic differentiation potential of hESCs and compare it with hFOB. For this, hESCs and hFOB were seeded on to PLGA scaffolds and directed into osteogenic lineage in vitro. When implanted subcutaneously for seven weeks into the dorsum of SCID mice, H1 derived osteoblasts generated mineralized tissue with rich blood supply. The newly formed tissue from hFOB did not confirm the presence of calcium salts in von Kossa staining. However both the groups staining positive for antibodies such as bone specific ALP and osteonectin. From this study, we concluded that mineralisation occurred at a faster rate in H1 generated osteoblasts when compare to hFOB. After seven weeks in vivo, hFOB could have been at an earlier stage of differentiation when compared to the H1 derived osteoblasts. 59 5.1 Limitations and future direction The methods described in this thesis demonstrate that hESCs can be directed towards osteogenic lineage. However it was important to obtain pure population of osteoblasts from hESCs for this comparison. Also, presence of undifferentiated cell remains in cell-scaffold constructs when implanted could lead to teratoma formation. The pluripotency exhibited by hESCs though a major advantage could also result in a heterogenous population of the differentiated cells. Future studies should be directed towards obtaining a pure population of osteoblasts from hESCs. This would enhance osteogeneis in vivo as well. Quantitative analysis of both H1 derived osteoblasts and hFOB before implantation could not be done. Also due to time constraints, the duration of the study could not be extended to further assess the mineralization in hFOB. Quantification of the cells in the gel before implantation is important for accurate comparison of osteogenesis between both the cell types. Thus more investigstions on the cell number and its effect on osteogeneis have to be carried out in the future. In our current study osteogenesis in an ectopic site was compared. Future work could be aimed at comparison of osteogenesis of H1 generated osteoblasts and hFOB in bone defects. An assessment of the effect of an ideal environment on osteogenesis in both H1 derived osteoblasts and hFOB could be investigated. 60 CHAPTER 6 APPENDIX 61 6 APPENDIX Table 1 Primers used for conventional PCR Gene β-actin Nanog Oct 4 Sox 2 OC BSP Primer sequence β-actinF: 5’ -CCAAGGCCAACCGCGAGAAGATGAC-3’ β-actinR:5’ -AGGGTACATGGTGGTGCCGCCAGAC-3’ NanogF:5’-GGCAAACAACCCACTTCTGC-3’ NanogR:5’-TGTTCCAGGCCTGATTGTTC-3’ Oct4F:5’-CGRGAAGCTGGAGGAGGAGAAGCTG-3’ Oct4R:5’ -AAGGGCCGCAGCTTACACATGTTG-3’ Sox2F:5-CCGCATGTACAACATGATGG-3’ Sox2R:5’-CTTCTTCATGAGCGTCTT-3’ OsteocalcinF: 5’--CTCACACTCCTCGCCCTATT 3’ osteocalcinR: 5’--CAGGAGGGAGGTGTGTGAGC -3’ BonesialoproteinF:5’-CAGTAGTGACTCATCCGAAG 3’ BonesialoproteinR:5’-CTCCTCTTCTTCTTCATCAC-3’ Product size (bp) Annealing Temperature ( C) 650 58 493 55 247 55 591 55 211 62 280 55 62 CHAPTER 7 REFERENCES 63 7 REFERENCES Ahn SE, Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, Lee YJ, Park KH, Cha KY, Chung HM. 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(2003) In vitro differentiation of embryonic stem cells into ineralized osteoblasts. Differentiation 71: 18-27. 69 [...]... sources of stem cells available for regenerative therapy include (1) the adult stem cells from the adult tissue (autologous or allogenic) and from the bone marrow (mesenchymal stem cells, hematopoietic stem cells) and (2) embryonic stem and embryonic germ cells derived from discarded human embryos and germ line stem cells Adult stem cells can be derived from various parts of the body including bone... like cells (Yu et al, 2007) concerns over immune rejection could be eliminated very soon Other challenges in the use of ESC include controlling lineage specific differentiation and elimination of residual stem cells Thus to obtain a purified population of osteoblasts from ESCs a good understanding of the mechanism underlying the lineage specific differentiation of hES cells is mandatory 1.5 Human Embryonic. .. capacity of the hES cells to differentiate into osteoblasts 19 and their ability to mineralize can be detected through staining procedures such as alizarin red or von Kossa 1.7 In vivo bone formation using Stem Cells Bone formation through in vivo implantation of the stem cells that are directed to osteogenic differentiation are being studied and reported First clinical reports of human MSC loaded onto... potential of hESCs was insufficient to form cartilage in vitro and bone in vivo Tremoleda et al in 2008 compared the in vivo osteogenic potential of hES cells with that of hMSC They demonstrated that bone formed by in vivo directed differentiation of hESCs in diffusion chamber model had no obvious qualitative difference to bone formed by hMSC A comparison of osteogenesis of hES cells within 2D and... Image processing was completed using software supplied by the confocal microscope’s manufacturer 2.6 2.6.1 In vivo experiments Culture of H1 cells in Matrigel For in vivo experiments H1 cells cultured in feeder-free culture system were used Confluent H1 cells grown on feeder layer were removed from the culture plastics after incubation with collagenase The H1 cells were replated into mTeSR1TM (Stemcell... signaling of hESCs have revealed various pathways that are crucial for the maintenance of pluripotency These include FGF, TGFβ/activin/ nodal, BMP, Wnt pathways (Sato et al, 2003) Fibroblast growth factor2 signaling is crucial for the self renewal of hES cells The mechanism of FGF2 signaling include induction of supportive factors secretion in MEF, up-regulation of major genes expressed in TGFβ/activin/nodal... grown in Extracel (Glycosan Biosystems, Salt Lake City, Utah) TM Extracel is made up of three biocompatible components: Gelin-S, Glycosil and Extralink Hydrogel was formed when the crosslinking agent, Extralink™ was added to a mixture of Glycosil™ and Gelin-S™ which also contained the cells This mixture was then placed in culture plates and allowed to gel at 37 C Gelation occurred in about twenty minutes... each of the wells holding the cell-gel construct Media was changed every day for the H1 cultures and once in two days for hFOB cultures The cultures were maintained in gel for a week and then assessed for cell viability Extracel is intended to be used as a cell carrier for seeding cells into the scaffolds for in vivo experiments 2.5.1 Assessment of cell viability in ExtracelTM 2.5.1.1 FDA/PI staining... Cell viability in Extracel was assessed by Fluorescein diacetate/ Propidium iodide (FDA/PI) staining 12 µM stock solution was prepared by dissolving of 5 mg FDA (Sigma) in 1 ml of acetone Then FDA working solution was prepared by adding 27 0.04 ml of stock to 10 ml of dulbeccos phosphate buffered saline (DPBS) 1.5 µM working solution was prepared by dissolving 1 mg of PI (Sigma) in 50 ml of DPBS for use... Cellular components used in tissue engineering encompass viable cells of autologous, allogeneic or animal origin While using viable cells, care is needed to control introduction of infectious diseases, cross-contamination from donors or introduction of infectious agents from materials used to process cells Also, consequences of immune and inflammatory responses after the implantation of the cell- scaffold ... staining (c) hFOB H & E staining (d) hFOB von Kossa staining Scaffold (e) hFOB H & E staining Figure At 2-week time point: H&E staining and von Kossa staining of sections showing scaffold and cells. .. time point: H&E staining and von Kossa staining of sections showing scaffolds devoid of cells 43 Figure At week time-point: (a) H&E staining showing newly formed tissue within the... understanding of the mechanism underlying the lineage specific differentiation of hES cells is mandatory 1.5 Human Embryonic stem cells As the name suggests, the human embryonic stem cells are