Mesenchymal stem cells are important from the point of view of tissue engineering for therapeutic and clinical applications. However the Human Embryonic Stem cells Mesenchymal Progenitors (hES-MP) are better suited for tissue engineering owing to their reproducibility and good proliferation. The interactions between cells and the extra cellular matrix can be imbibed into cell culture by coating the scaffolds with growth factors and extracellular matrix proteins. Osteoinductive proteins such as Bone morphogenic protein 2 and osteopontin when coated on scaffolds may promote cell attachment, development and differentiation and use of attachment substrate and soluble growth factors could be avoided.
Foetal Bovine Serum (FBS) has been widely used in cell culture. However many problems are associated with its use such as its animal origin and batch variability.
Many substitutes for FBS are available and various companies have also developed serum free media which could be used as an alternative.
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In this project I shall study the differentiation of Human Embryonic Stem cell Mesenchymal Progenitors using the scaffolds with specific arrangements of BMP-2 and Osteopontin, developed by Oral Protein Technologies, using serum free and serum containing media to determine which best supports the development of these cells.
Mesenchymal stem cells are derived from the Bone marrow. These cells are multipotent having the ability to differentiate into osteoblasts, chondrocytes and adipocytes. However they are scarce and lose their capacity to proliferate and differentiate in prolonged cultures. Thus for tissue engineering purposes the use of Human embryonic stem cell derived mesenchymal progenitor cells (hES-MP) is beneficial. These cells are known to be reproducible, showing good proliferation and are easy to culture.( Hematti, 2011; de Peppo et al, 2010)
For tissue engineering it is necessary to provide the cells with an artificial environment which closely mimics the environment in vivo. Cells interact with the extracellular matrix(ECM) and these interactions are important for their growth and development. Culturing of cells on surfaces coated with the extracellular matrix proteins and growth factors would help mimic the environment in vivo and also enhance cell attachment without the use of attachment substrates. This would possibly eliminate the need for the addition of soluble growth factors. Bone and cartilage formation is induced by the growth factors like Bone Morphogenic Proteins (BMPs). Differentiation of mesenchymal stem cells in rat and mouse is also promoted by BMPs (Rickard et al, 1994). Osteopontin is an ECM protein which plays an important role in osteogenesis influencing the differentiation of mesenchymal stem cells. Polymeric osteopontin is also known to enhance the adhesion of mesenchymal stem cells (Hakimzadeh et al, 2010).Thus BMP-2 and Osteopontin can be immobilized on gold plated culture surfaces such that they form a monolayer comprising of well oriented peptides exposing only the motifs required for cell attachment. Such scaffolds developed by Orla Protein Technologies would be used in this project.
Cell cultures are generally practised with media containing Foetal Bovine Serum (FBS).FBS contains growth factors, proteins and hormones which help in maintenance of cells in culture. However batch variation of FBS leads to variability in results. Its use also possesses the risk of contamination and transmission of viruses due to its animal origin. Its use in culturing cells on protein coated surfaces would also affect cell attachment. Various substitutes for FBS have been used for cell culture. Many companies are developing Serum free media which are now available and can be substituted for FBS. I shall be using MesenCultÂ®-ACF by Stem cell Technologies which has been previously used for studying the proliferation of hES-MPs showing better results than FBS (Steven Slack, BEng Dissertation, Uni of Shef, 2010). I shall also use STEMPROÂ® MSC SFM developed by Invitrogen and compare the two to determine which best supports the survival, growth, proliferation and differentiation of hES-MPs
Stem cells are unspecialized and have the ability to self-renew and differentiate into lineage specific cells. They can be further classified as embryonic and adult stem cells. The embryonic stem cells are pluripotent and harvested from the inner cell mass of the blastocyst. These cells can also be obtained from the umbilical cord blood. Adult stem cells are found in various parts throughout the body. They divide in particular organs and replace damaged tissue serving as a repair system.
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The adult stem cells are further classified on the basis their origin. Hematopoietic and Mesenchymal stem cells are present in the bone marrow. Hematopoietic stem cells form the blood cells such as the myeloid and the lymphoid cells whereas Mesenchymal stem cells are capable of differentiating into osteoblasts, chondrocytes and adipocytes and are multipotent. The extent to which differentiation occurs may vary depending on the differentiation potential of the cells and whether it is carried out physically or mechanically.
Mesenchymal Stem cells
Morphologically, mesenchymal stem cells are long and thin and have a large nucleus. These cells also contain small amounts of mitochondria, golgi apparatus, endoplasmic reticulum and a prominent nucleolus. However these cells are scarce in the bone marrow. Scientists have argued that this scarcity of mesenchymal stem cells makes them less beneficial for tissue engineering (Sullivan et al, 2007). Mesenchymal stem cells are known to express markers such as CD 105, CD 73, CD44 and CD 90 but do not express haematopoietic markers like CD45 , CD34 , CD14 or CD11 or costimulatory markers CD80 , CD86 or CD40.These help in the identification of the MSCs (Campagnoli et al, 2001)
Mesenchymal stem cells have shown to retain their growth, proliferation and differentiation potentials both in vitro as well as in vivo. These cells expand easily in culture but are not immortal. They also migrate to the sites of injury when injected systemically. They find applications in tissue engineering particularly in bone and cartilage repair owing to their poor regenerative capacities. Mesenchymal stem cells are shown to be important in the treatment of osteogenesis imperfect, in dentistry for the implant in teeth and also in the treatment of inherited disorders (Horwitz et al, 1999; Shi et al, 2005; Reiser et al, 2005). They have also been studied in the treatment of infarcted myocardium and in other experiments have shown to mimic neural cells when injected in the brain of mice(Matsumoto et al, 2005; Kopen et al , 1999) Thus MSCs have a wide range of clinical applications, most of which still are under investigation.
. UndifferentiatedÂ MSCsÂ grown in monolayer culture (A) and after differentiation along the osteogenic (B), adipogenic (C) and chondrogenic pathways (D) and (E).Â Cell differentiationÂ is these cultures was observed following staining with von Kossa (B),Â Nile redÂ O (C),Â SafraninÂ O (D) and by immunostaining with an antibody specific forÂ type II collagenÂ (E).
Barry, F. P. and J. M. Murphy (2004). "Mesenchymal stem cells: clinical applications and biological characterization." The International Journal of Biochemistry & Cell Biology 36(4): 568-584
Human Embryonic Stem Cell derived Mesenchymal Progenitors.
Human mesenchymal stem cells after prolonged culture lose their proliferation and differentiation capacities and the use of embryonic stem cells involve the risk of formation of tumours. The alternative would be to human embryonic cells which are progenitors for a particular lineage(de Peppo et al , 2010).In this project I shall be using Human embryonic stem cell derived mesenchymal progenitor cells(hES-MP).These cells are easy to culture and show good proliferation without the use of feeder cells .They are reproducible and their safety and clinical application were tested in severely combined immunodeficient (SCID) mice resulting in the formation of homogenous and well differentiated tissue without the production of teratoma. hES-MP can be cultivated even in the absence of animal derived components thus avoiding contamination in the cell cultures. They are also are important from the point of view of tissue engineering. Though derived from human embryonic stem cells, the hES-MP showed high expression of genes similar to the mesenchymal stem cells and low level expression of genes characteristic of embryonic stem cells (de Peppo et al, 2010)
Foetal Bovine Serum and its substitution
In vitro, cell culture is mainly done with a basal medium such as MEM or DMEM along with the use of Foetal Bovine Serum (FBS). The FBS comprises of many constituents including growth factors and proteins like the bovine serum albumin (BSA) which help in the maintenance of cells in culture (Pérez-Ilzarbe et al, 2009). While FBS is most widely used in cell culture, sera from other animals may also be used. Cell growth, proliferation and differentiation are stimulated by the various hormones provided by the serum. It is also important for cell attachment and spreading and maintaining the pH of the culture media. However many problems are associated with the use of serum. Serum is not well defined and batch variation in the composition may lead to variability. It may also cause microbial contamination of the culture with the possibility of disease and viral transmission on transplantation. Due to its animal origin it may also cause immune response in the patient (Chevallier et al, 2009) If cell culture scaffolds are protein coated then the serum proteins may interact with the scaffold proteins and inhibit the proper binding of cells. Moreover there are ethical concerns with the use of FBS as it is obtained from the blood in the bovine foetus. The main concern is the suffering of the animals in harvesting and processing of FBS.
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Many scientists have tried using different substitutes for FBS for culturing cells. In some instances where human serum was used as a substitute cell proliferation rates improved but early cell death was observed(Shahdadfar et al, 2005).Cells were then initially cultured in FBS followed by transfer into serum free medium which promoted the differentiation of MSCs into bone cells for transplantation. Other substitutes included the use of human serum along with thrombin activated platelet releaste in plasma and pooled human platelet lysate in culturing of MSCs. Of these, the most suitable substitute for FBS was found to be pooled human platelet lysate for MSC expansion.
Human platelet lysate (HPL) was used extensively in various experiments as a substitute for FBS. It has shown to increase the cumulative cell numbers and cause rapid expansion. The cells thus generated met the all the criteria's for MSC (Lange et al, 2008). Human platelet lysate, like FBS, provides essential proteins and growth factors to the cells. HPL also caused an upregulation of gene expression and comparison between cells grown in FBS and HPL showed that cells cultured in HPL grow at a faster rate than those in FBS. The use of HPL as a substitute for FBS has also been shown in other studies (Kocaoemer et al, 2007).
In studies involving the allo-antigen specific immune responses of MSCs, cells cultured in FBS were found to be more suitable for preventing such complications however it was also found that culturing cells in human platelet lysate is also an alternative (Bernardo et al, 2007). In other experiments serum was replaced by transforming growth factor Î²1 (TGFÎ²1), leukaemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix.Â The cultured cells showed normal growth and differentiation thus providing and effective alternative for FBS. (Amit et al, 2003)
Several companies are developing serum free culture media which can be used as a substitute for FBS. However very little is known about development and response of cells to such media. MesenCultÂ®-ACF is a serum free media developed by Stem Cell Technologies. It lacks animal derived components is known to support the growth and proliferation of hES-MP better than FBS(Steven Slack,BEng Dissertation, Uni of Shef, 2010). MSCs have shown to retain their differentiation capacities with reduced contamination by hematopoietic cells as compared to serum containing media (Wagey,2010).
STEMPROÂ® MSC SFM is a serum free media for the culture of Mesenchymal stem cells developed by Invitrogen. MSCs showed increased proliferation and expansion in this media as compared to MEM with 10% serum. These cells were smaller in size and expressed CD105++Â andÂ CD146dim. . Though these cells showed lower alkaline phosphatase activity, they showed successful bone formation in vivo. MSCs have also shown to retain their multi-lineage differentiation potentials beyond the fifth passage of cells. Thus studies show that in vitro expansion and differentiation of MSCs using serum free media is feasible and comparable to that in media containing serum (Agata et al, 2009).In this project I shall be using MesenCultÂ®-ACF and STEMPROÂ® MSC SFM and comparing them to see which best promotes cell survival, proliferation and differentiation.
While culturing cells in a medium, the attachment substrate is first coated on to the surface prior to the plating of cells. It helps in the attachment of cells and also in their expansion and enumeration. In this project the use of attachment substrate will be excluded as the integrin -peptide interactions would facilitate cell attachment and development , mimicking the environment in vivo.
Amodel for the recognition of the ï¬bronectin RGDsite and auxiliary sites by an integrin
Ruoslahti, E. (1996). "RGD AND OTHER RECOGNITION SEQUENCES FOR INTEGRINS." Annual Review of Cell and Developmental Biology 12(1): 697-715.
Tissue Engineering involves understanding the mechanism of cell growth and survival in the body and applying these principles to produce tissues in vitro for therapeutic use. The culturing of cells requires the optimization of various factors to provide the cells with an artificial environment which closely mimics the one in vivo allowing cell survival, growth and proliferation. For this it is important to consider various parameters such as the culture media, scaffolds for cell growth and growth factors to suit a particular cell type.
In tissues, the extracellular matrix provides support to the cells and is also important for intercellular communication. It is known to regulate cell behaviour and also contains growth factors (Kumar et al, 2004). The ECM and cell interaction is important for survival, growth and differentiation of cells ( Adams and Watt, 1993). For tissue engineering purposes surfaces are coated with molecules from the ECM to enable these interactions in vitro. Proteins of the extracellular matrix such as collagen and fibronectin display certain binding sites or motifs to which the integrin's of the cell attach. Cell culture surfaces are thus coated with ECM proteins to mimic the environment in vivo and to enhance the attachment of cells (Kleinman et al, 1987).
Mesenchymal stem cells can adhere and grow on glass as well as plastic.Surfaces coated with growth factors and proteins have also been used in culturing of MSCs. The importance of extracellular matrix proteins like fibronectin, type 4 collagen, laminin and elastin in cell signalling leading to cell differentiation has been has been shown(Balaschke et al,1994). Fibronectin, albumin and collagen have been used to test attachment and spreading of MSCs. For the differentiation of MSCs into osteoblasts the dishes coated with fibronection were found to be more suitable than those coated with albumin. Improved cell attachment and spreading were observed along with increase in cell number in dishes coated with fibronectin. (Ogura et al, 2004)
MSCs have also been studied for their osteogenic differentiation on biodegradable sponges made of gelatin and beta tricalcium phosphate (BTCP). The attachment, spreading and proliferation of the cells was found to be dependent on the amount of BTCP and the culture method. (Takahasi et al, 2004). The various parameters of the extracellular matrix such as matrix structure, organization, composition and mechanical and physical properties required for the arrangement of cells and their differentiation into specific lineages have been described by Reilly and Engler, 2009.
The peptide motifs of the ECM proteins, which are required for biological reactions as well as for cell attachment, can be immobilized on cell culture surface by a number of methods. Adsorption of the protein may result in its denaturation and it is difficult to control the density and orientation of the peptide motifs when coated on surfaces which may lead to inconsistency. Covalent coupling may cause protein denaturation and loss of its activity. Thus immobilization of proteins by these techniques shows variability and reproducibility is reduced.
Orla protein technologies use a novel technique for ECM protein immobilization. The outer membrane protein from Escherichia Coli, OmpA, is allowed to self-assemble and attach to the gold coated surface via the thiol bearing cysteine. The outer loops of OmpA have the peptide motifs engineered into them. Thiol alkane molecules are used to fill the gap between the OmpA molecules such that only the motifs are exposed. Thiol alkane contains sulphur molecules which bind to gold forming strong gold thiolate bonds ( Bain et al, 1989).This method allows the formation of a stable monolayer of protein displaying only the peptide motifs for cell attachment (Tamm et al, 2001). Scaffolds with motifs from Collgen 1, collagen 4, fibronectin and laminin have been used to test the attachment and growth of Pc12 cells. These cells showed an increase in cell attachment on surfaces coated with the active motifs as compared to uncoated glass. It was also seen that cell attachment was proportional to the concentration of the ECM proteins adsorbed on the surface. Cell attachment was also tested on gold surfaces with only OmpA without the peptide motifs. As compared to surfaces with peptide motifs, low levels of cell attachment were observed which were weak and the cells were easily dislodged (Cooke et al, 2008).In my project I shall compare the gold coated surfaces with the active peptide motifs of Bone morphogenic protein-2 with those coated with osteopontin to see which ideally promotes cell survival and possibly induces differentiation of hES-MP
Bone Morphogenic Proteins
Bone Morphogenic proteins (BMP) are growth factors which induce the formation of bone and cartilage. In animals like rat and mice they promote the differentiation of mesenchymal stem cells (Locklin et al, 1995 ;Rickard et al, 1994) BMPs belong to the transforming growth factor beta superfamily of proteins. They are important for regulation of embryonic development as well as development of organs.(Hogan , 1996)They are also known as metabologens as they are involved in maintaining homeostasis and in metabolism.(Reddi and Reddi, 2009).There are seven types of BMPs (BMP1 to BMP-7). Certain studies have shown that human MSCs are not differentiated by BMP in the presence of serum (Diefenderfer et al, 2003). Whereas BMP induced differention of Human MSCs into osteoblasts was seen in serum free conditions (Osyczka and Leboy,2005). As compared to growth factors such as bFGF , FGF -8, FGF -10, IGF-1 etc , Bmp -6 has shown to play an important role in the regulation of mesenchymal stem cell differentiation in serum free conditions.(Friedman et al ,2006).BMP -2 along with TGF b1 have shown to induce chondrogenic differentiation of embryonic stem cells (Kuske et al, 2011) Similar studies have been carried out on human embryonic stem cells with BMP 7 and TGF b1 and have shown to induce chondrogenesis without the formation of embryoid bodies (Nakagawa et al , 2009) It has also been shown that BMP 2 stimulates the production of Alkaline Phosphatse (ALP) and osteocalcin in stromal cell lines indicating its role in differentiation of these cells (Yamaguchi et al , 1996). Calcium phosphate coatings with recombinant human bone morphogenic protein 2 stimulated the production of alkaline phosphatase in rat stromal cells (Liu et al, 2004). BMPs are also known to modify integrins to act as a regulator for cell adhesion (Nissinen et al, 1997).
Oseteopontin belongs to a family of non-collagenous proteins called SIBLING (smallÂ integrin-bindingÂ ligand,Â N-linkedÂ glycoprotein). It is important for the mineralization of bone and dentin and is present in their extra cellular matrix. Osteopontin was first found in osteoblasts and is also known as Bone sialoprotein 1 (BSP 1). Though osteopontin is mainly produced by osteocytes, osteoclasts and osteoblasts it is also synthesized by fibroblasts (Ashizawa et al,1996) , myoblasts (UaesoontrachoonÂ et al ,2008) , macrophages ( Murry et al , 1994) and smooth muscles (Ikeda et al , 1993). The process of bone remodelling involves osteopontin secreted by the bone cells (Denhardt and Noda, 1998). Osteopontin also plays an important role in in proliferation, differentiation of rat fetal cells into osteoblasts (Zohar et al, 1998). Chicken osteopontin shows similarities with mammalian osteopontin and is involved in osteoblast development and differentiation both in vivo as well as in vitro (Moore et al,1990). Proteins such as fibronectin , fibrinogen , collagen and osteopontin have Arg-Gly-Asp (RGD) as sites for cell recognition. These are recognized by receptors that are structurally related called Integrins. This system of RGD- integrin mediated adhesion has shown to improve cell anchorage, growth and differentiation (Ruoslahti and Pierschbacher,1987). Studies have also shown that wells coated with osteopontin promote cell adhesion and spreading of smooth mucle cells as well as endothelial cells (Liaw et al , 1994).Polymeric osteopontin is involved adhesion of human mesenchymal stem cells.(Hakimzadeh et al ,2010).On introducing site directed mutations in the RGD sequence of osteopontin, it did not support the adhesion of cells even under high concentration or longer duration thus indicating the importance of the conserved RGD in the attachment of cells(Xuan et al , 2004).
In tissues, cells are supported by the extracellular matrix (ECM) The ECM contains proteins which help in the communication of cells and also confers mechanical properties to the tissues. Cells are anchored to the ECM by means of integrins (Abbott, 2010). Thus when cells are cultured in vitro they require an appropriate environment which mimics the one found in vivo for the attachment and proliferation of cells. If the peptide content is varied then it changes various properties like density, viscosity, stiffness and mechanical properties. These changes will affect the attachment of cells and subsequently their growth (Mujeeb et al, 2008). Generally cells are cultured in a two dimensional method but three dimensional culture of cells, which mimics closely the environment in vivo, is also being practised. In the two dimensional culture method cells are grown attached to the surface of a scaffold in a well or they may be grown attached to plastic surfaces like in tissue culture flasks. The cells are allowed to grow and divide to confluence depending on the available surface area. After this passaging of cells is done and again they are grown to confluence till a sufficient quantity of cells are obtained (Larson et al, 2006). For the design and fabrication of scaffolds it is important to understand cell interactions and the cues in the extra cellular matrix. Motifs of proteins such as Bone morphogenic Protein 1 and osteopontin have been immobilized onto scaffold proteins on gold surfaces. These motifs regulate adhesion of osteoblasts, their differentiation and bone formation (Mitchell et al, 2010).
Cell cultures are generally carried out using a defined media on glass or plastic surfaces using serum. Serum, though required by cells, contains growth factors in very low concentrations moreover serum also has a complex composition. In many cases soluble growth factors have been added to promote cell survival, growth and proliferation (Gospodarowicz and Moran, 1976).Studies using mammary epithelial cells cultured on a substratum containing collagen showed proliferation with minimal use of epidermal growth factor (EGF) in a serum free media (Salomon et al,1981) Laminin also promotes the attachment of epithelial cells and induces the production of neurites by neuronal cells (Terranova et al , 1980 ; Engvall et al ,1986) Likewise spreading of human keratinocytes and incorporation of thymidine has been facilitated by type 4 collagen and fibronectin without the addition of soluble growth factors (Woodley et al, 1990). In this project, osteoinductive proteins, Bmp-2 and osteopontin, will be coated on scaffolds with the aim to induce differentiation of hES-MP to osteoblasts in the absence of soluble growth factors.
In this project I will be comparing the biomaterial scaffolds coated with bone morphogenic protein 2, a growth factor, with those coated with osteopontin which is an extracellular matrix protein to study which is most suitable for hES-MP differentiation. The hES-MP will be cultured in 2D in both serum containing and serum free media to compare growth , proliferation and differentiation in both cases.
To test the proliferation of cells the MTS assay will be performed. The MTS assay is a calorimetric assay in which MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),which is a tetrazolium compound is reduced to a purple coloured formazan dye which can be read at 490-500nm. The formazan product is produced from MTS in the presence of phenazine methosulphate (PMS) or phenazine ethosulphate (PES) which acts as an electron coupling agent. This test can be used to test cell viability , cell proliferation and also toxicity of certain compounds as the toxicity would affect cell viability or inhibit growth. The number of living cells would be directly proportional to the absorbance read at 490 nm. Only in the presence of active reductase enzymes these reductions take place hence indicating the number of viable cells(Cory et al, 1991).
Production of ALP and its assay using p- Nitrophenol Phosphate as substrate.
Alkaline Phosphatase (ALP) is an enzyme which removes phosphate groups from different molecules. Isoform of Alkaline phosphatase which is produced by the bone cells is known as Alkaline Phosphatase 2. It is produced by osteoblasts. Isoforms of alkaline phosphatase are also expressed in placenta, liver, kidney and intestine (Posen and Doherty, 1981). They differ in nature and their post-translational modification but are the products of the same gene. Bone specific ALP and Liver ALP have similar structures; however both can be differentiated based on their electrophoretic mobility and thermostability. Serum levels of alkaline phosphatase can be measured and help in diagnosis of various bone diseases ( Garnero and Delmas , 1993). Bone specific ALP serves as a biomarker indicating formation of bone (Goldstein et al, 1980).It is also helps to monitor the activity of osteoblasts (van straalen et al, 1991).Studies have also shown that ALP is also involved in bone mineralization. Abnormal Mineralization of bones was seen in cases of hypophosphatasia characterized by a defective production of ALP (Cole and Cohen, 1991). ALP produced by cells in culture can be can be detected indicating the differentiation of mesenchymal stem cells into osteoblasts. A rapid and reproducible and sensitive technique to measure the activity of ALP is using p-Nitrophenol phosphate as substrate. p- Nitrophenol phosphate substrate which is colourless in alkali is hydrolysed by alkaline phosphatase produced by the cells to produce a yellow coloured p- Nitrophenol and inorganic phosphate which can be measured at 405nm. At higher cell concentrations increased absorbance is observed along with rapid reaction. This technique helps in ALP detection of concentration 0.05mM/min/ml (Sabokbar et al, 1994)
Production of Calcium - Alizarin Red Assay
Bone tissue if formed by the process of ossification by osteoblasts. In the cartilage calcification occurs at the matrix vesicles. Ca2+ and inorganic phosphate accumulate and serve as nucleating agents for the formation of the inorganic component of bone , hydroxyapatite. Von Kossa staining, fluorescent calcein binding and Alizarin Red S (ARS) staining can be used as mineralization assays. Alizarin Red S is an organic compound also known as 1,2-dihydroxyanthraquinone (C14H8O4 ). ARS staining has been widely used to measure calcium deposits by bone forming cells. As compared to the other assays ARS statining is know to be versatile as the ARS dye assayed by its extraction from the monolayer of cells. After microscopy visualization of the stained cells , the dye can be extracted for quantification. Extraction using acetic acid has been shown to be more sensitive with improved signal to noise ratio as compared to the cetylpyridinium chloride extraction. When extracted with acetic acid the dye is read at 405nm and with cetylpyridinium chloride it is read at 550nm (Gregory et al, 2004 ; Stanford et al, 1995)
Sirius Red Assay for Collagen
Collagen is a naturally occurring basic protein and is a major component of bone, cartilage , tendons and ligaments. It's a fibrous protein providing mechanical strength, support and flexibility to the body. Thus production of collagen by cells in culture shows their differentiation into osteoblasts. Collagen produced by cells can be detected by means of an anionic dye, Sirius Red. Reaction between the basic groups of collagen and sulphonic acid groups of Sirius Red helps in the detection of collagen. Birefringence is produced by the parallel arrangement of the dye molecules and the collagen fibres. This helps in detecting collagen produced by a technique which is simple yet sensitive and specific. Large quantities of samples can be screened by this method as quantification can be done in the micro titre plates. Collagen in tissues can also be also be assayed based on the content of hydroxyproline or by incorporating proline which is radioactively labelled into collagen (Tullberg-Reinert and Jundt, 1999; Junqueira et al, 1979)
Immunostaining and Confocal Microscopy
Immunostaining includes various techniques for the detection of proteins in samples using antibodies. It has applications in research and diagnostics. It can be used to detect the presence of certain proteins in tissues, their sub cellular localization and distribution (Coons et al, 1941). Confocal microscopy is am imaging technique used to obtain sharp images by using point illumination in which light other than that from the focal plane is excluded. High resolution images with better contrast are obtained as compared to those from the conventional microscope. The images obtained are suitable for minute observations (Semwogerere and Weeks, 2005).Both immunostaining and confocal microscopy will be used to identify and localise proteins of the bone extracellular matrix such as bone sialoprotein, osteocalcin and osteonectin.
Bone Sialoprotein(BSP), like osteopontin, is a SIBLING protein and is found in tissues like bone cartilage and dentin. It is found in their extracellular matrix and has high content of sialic acid. In humans its variant is present, called Bone Sialoprotein 2 (Fisher et al, 1990).Bone sialoprotein is important for the formation of bone and for the development of osteoclasts. BSP mutant mice have shown impaired bone formation and impaired bone growth (Malaval et al ,2008). BSPs play an important role in cell attachment as they possess RGD motifs which bind to integrins on cell surface. In osteoblasts, the BSP gene is expressed in order to induce bone formation. Thus cultured hES-MP cells producing BSP would indicate differentiation and formation of bone. BSP also plays a role in hydroxyapatite nucleation and cell signalling and is also known to bind to hydroxyapatite, calcium and collagen (Ganss et al, 1999) BSP is expressed in osteoblasts at late stages of development , that is in mature osteoblasts, in young osteocytes and osteoclasts. However it is not found in all connective tissues (Bianco et al, 1991).
Osteocalcin is produced by osteoblasts and is known asÂ bone gamma-carboxy glutamic acid-containing proteinÂ or Bone GLA Protein (BGLAP). It is primarily present in bone and is non-collagenous protein. It is required for the formation of bone and its mineralization. Osteocalcin is also known to play an important role in the release of insulin and the hormone adiponectin. It also serves as a biochemical marker indicating bone formation and osteoblast differentiation. Serum levels of osteocalcin are used in the diagnostics of various bone diseases. Osteocalcin knockout mice have shown improved function and bone formation. The absence of osteocalcin did not repair bone resorption. This study indicated that osteocalcin is important for bone formation (Ducy et al, 1996). On treating osteoblast like cells with growth factors such as BMP-7 and Deminaralized Bone Matrix (DBM , no significant difference was seen in the production of osteocalcin as compared to the control(Davis et al , 2006). Previous studies have shown that adult stem cells, derived from the adipose tissue, express markers for bone formation like alkaline phosphatase, collagen, osteocalcin and osteopontin (Hicok et al, 1998).
Osteonectin is an extracellular matrix protein found in bone. It is a glycoprotein secreted by osteoblasts and is acidic. During the formation of bone and mineralization, osteonectin is secreted by osteoblasts. It binds to collagen, sodium and also has affinity for calcium. Osteonectin aslo plays an important role in interactions between the cell and the extra cellular matrix, mineralization of bones, angiogenesis, wound repair and morphogenesis (Bradshaw and Sage, 2001;Reed and Sage, 1996). Increased expression of osteonectin is seen in many types of cancers such as melanomas, carcinomas and those affecting breast, pancreas and colon. Development of cancerous growth relates the role of osteonectin in cell proliferation and migration. On the other hand in some cells it does not allow proliferation to occur. Studies in melanoma cells have shown that when the expression of osteonectin is inhibited, no tumors are formed (Ledda et al, 1997). In studies involving human mesenchymal stem cells constitutive expression of osteonectin is seen throughout osteogenesis along with the expression of other bone related proteins (Bruder et al, 1998). However according to some studies, osteopontin though present in osteoblasts, is also present human mesenchymal stem cells to the same extent and thus presence of osteopontin does not necessarily indicate differentiation (Nuttelman et al, 2003).
Although the differentiation of cells using protein coated surface has been studied, the arrangement of osteoinductive proteins in monolayer enabling only the exposure of essential peptide motifs is unique and maximises the exposure of the membrane to the peptide which is likely to promote cell attachment and osteogenesis. The use of Dexamethasone as a soluble growth factor is inappropriate owing to its steroidal properties.BMP being expensive cannot be widely as a soluble growth factor in cell culture. The addition of such soluble growth factors can possibly be avoided by using the protein coated surfaces. The use of serum free media would facilitate the use of the cultured cells for therapeutic and clinical applications thus minimising the problems associated with the use of FBS.
This project is important to study the growth and differentiation of hES-MPs on protein coated surfaces using serum free media without the addition of soluble growth factors.