Age-related proliferative behavior of mandibular osteoblasts [Elektronische Ressource] : an in vitro study / vorgelegt von Mohammad Samer Juma'a

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• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 18
• Poids de l'ouvrage: 2 Mo

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Aus der Universitätsklinik für Zahn-, Mund- und Kieferheilkunde der Albert-Ludwigs-Universität Freiburg i. Br. Abteilung für Kieferorthopädie          

Age-related Proliferative Behavior of Mandibular Osteoblasts - An in vitro study -

      Inaugural-Dissertation zur Erlangung des Zahnmedizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br.         vorgelegt 2004 von Mohammad Samer Juma’a geboren in Damaskus / Syrien

                                                                 Dekan:Prof. Dr. J. Zentner 1. Gutachter:Prof. Dr. I. E. Jonas 2. Gutachter:   Prof. Dr. Dr. J. Düker  Promotionsjahr:2005

 

 

 

 

 

Table of Contents       1. Introduction…………… ……………………………………………...……1 1.1. Osteoblasts’ Growth Factors and Mediators…………………………………………3 1.2. Characteristics of Growth Factors and Mediators………..…….......... ...................5 1.3. Proliferation Tests……..………………………………………………………………..9 1.3.1. MTT-Test..…… ……………………………………………………… .......................9 1.3.2. Easy For You (EZ4U)-Test..……………………………………………………….10   2. Literature Review…...……………………………………………………11 2.1. Orthodontic Aspects of Ageing…...…… ...………… ………………………………..15   3. Aim of the Study… …..…………………………………………………..18   4. Materials and Methods………………………………………………….1 9 4.1. Samples………...………………………………………………………………………19 4.2. Materials………………………………………………………………... .....................19 4.3. Preparation Procedures……………………………...……………………………….20 4.4. Culturing of Samples (first passage)……...…… ..… …..….…… …………………..22 4.5. Feeding of Cultured Biopsies (first passage)………….….…………………..……23 4.6. Transferring of Cells (second passage)……....…… … ...… .…… ………………….23 4.7. Feeding of Osteoblastic Cells (second Passage)…..……….…........ ...................24 4.8. Cell Preparation for Proliferation Tests……………….....………………………….24 4.9. MTT-Test…………………….…..……………………………………………………..24 4.10. Easy For You-Test (EZ4U)………………………………………………………....25 4.11. Calk-Proof Experiment According to van Kossa….……………………….……..25 4.12. Light Microscopic Examination……………….…….…………..... ........................26 4.13. Scanning Electron-Microscopic (SEM) Examination..…………………....…......28 4.14. Separation – Thin Section – Technool gy (Hard Cross Section Technology) …28

  5. Results………………… …………………………………………………..32 5.1. MTT-Test……………………...………………………………………………………..32 5.2. Easy For You (EZ4U)-Test...…………………………………………………………33 5.3. Statistic Analysis……...……………………………………………………………….34 5.3.1. EZ4U-Test..………………………………………………………………………….34 5.3.2. MTT-Test...………...………………………………………………………………...35 5.3.3. Cell-Counting by Using the Separation – Thin Section – Technology (Hard Cross Section Technology)………..………………………………………………36 5.3.4. Measurement of Cell Surface……………...………………………………………37   6. Discussion…………… …………………………………………………...38 6.1. EZ4U (easy for you)-Test…………………………………………………………….38 6.2. MTT-Test……...………………………………………………………………………..39 6.3. Comparison…………………………………………………………………………….39 6.4. Cell Number and Surface…………………………………………………………….40 6.5. Orthodontic Aspects of Morphological and Histological Osteoblast-Ageing…….43   7. Summary……………… …………………………………………………..45   8. Zusammenfassung………………………………………………………46   9. Appendix….……………………………………………………………….47 9.1. EZ4U-Test...……………………………………………………………………………47 9.2. MTT-Test.………………………………………………………………………………49 9.3. Number of Cells per View-Field…….………………………………………………..51 9.4. Surface of the Cells per View-Field………………………………………………….52   10. References…..…………………………………………………………..54   11. Curriculum Vitae………………………………………………………..61

1. Introduction       Successful orthodontic treatment is dependent upon the adaptive potential of human hard and soft tissues. At a cellular level, bone remodeling is related to the proliferation capacity of osteoblasts and osteoclasts beside the muscular tissues and vascular system. It has been thought that the proliferation capacity of bone cells decreases with age due to cellular and extracellular factors. At a microscopic level, even normal action of daily activities can induce cracks in bone. With age these microscopic cracks appear to accumulate exponentially and are associated also with an increase in bony porosity (Frank et al. 2002). It has yet to be fully elucidated whether this is largely due to a reduction in the process of repair. After using histomorphometrical methods, the examination of the incidence and the localization of microcracks in human bone specimens have proven that the amount of microdamages increases dramatically with advancing age (Schaffler et al. 1995). It has been observed that at the same time as bone loss occurs on the endosteal surface, bone is being added to the periosteal surface, but much more slowly than during growth (Parfitt 1984). Even the fracture healing in young patients is faster than in the elderly, but it is unknown whether these changes are attributed to the number of osteogenic precursor cells of bone or whether they are connected with a change in the proliferative cellular kinetics (Shigeno & Ashton 1995). There are several possible mechanisms to explain the age-related reduction in bone formation. It could be related to the availability of fewer stem cells or the changing biological activy of local regulatory factors. The responsiveness of the osteoblast lineage may also decrease (King & Keeling 1994). It has been suggested that the number of proliferative precursor cells on trabecular bone surfaces is higher in younger subjects (Shigeno & Ashton 1995), and the decrease in bone amount is accompanied with a decrease in bone formation rate, which can be due to the reduction in the number and activity of osteoprogenitor cells, or the osteoblasts (Kabasawa et al. 1996). A human osteoblast is similar to other diploid cell types. It has a limited proliferative capacity and undergoes ageing and senesence (Kassem et al. 1997). Therefore age-related bone loss is partially accounted for by an increased

 

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osteoblastic maturation and a decrease in osteoblastic proliferation (Martinez et al. 1999). Enlow (1982)tissue itself does not have the capacity to reported that bone  has increase in size with senesence because the cellular components do not have any growth activity after maturation. However, there remains proliferative potential processes in the soft tissues of the periosteum, endosteum and the surrounding muscular, vascular, and nerval tissue.                                                        

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1.1. Osteoblasts Growth Factors and Mediators ’   The interaction of the human osteoblast with its surrounding local and systemic environment is complex. It has influences from the extracellular matrix, cytokines and systemically derived hormones. Some of these interactions have been clearly established in the recent literature, others remain to be defined. The extracellular matrix is composed of various products which are from local or exogenous resources. Many of these proteins are known to be growth factors like; transforming growth factor-ß (TGF-ß), insulin-like growth factors (IGFS) and fibroblast growth factors (FGF). These concentrate in mineralized bone and probably play a role in bone regeneration after injury (Termine 1990). Growth factors and cytokines are important mediators of communication between cells and also between the cell and its extracellular matrix. Furthermore they mediate locally the effects of several hormones on bone cells (Rifas 1999). An example of these communication factors can be seen in the osteoblast which synthesizes four attachment proteins and molecules to mediate the interaction of a connective tissue cell with its extracellular environment. These are; fibronectin (FN), thrombospondin (TSP), osteopontin (OP), and bone sialoprotein (BSP). The later three all bind ionic calcium and exist in the bone matrix to influence the processes of cellular development and differentiation. With advancing age they degrade to lower molecular weight fragments, probably this degradation is associated with deactivation of their bioactivities. At present it is unknown if this alteration leads to a decrease in cell function in adult individuals (Termine 1990). Other systemic factors such as parathyroid hormone, vitamin D metabolites, and calcitonin mediate the process of bone remodeling (Saito et al. 1994). Moreover it is well-known that osteoblasts exhibit estrogen- and parathyroid hormone- (PTH) receptors, and produce cAMP following PTH-stimulation and synthesis of type-I- collagen, and osteocalcin (Simmons et al. 1994). The developing bone matrix also contains two different small proteoglycans, Prostaglandins: PG-I and PG-II. While mature bone matrix contains primarily PG- II, the fetal connective tissue contains more abundant concentration of PG-I (Termine 1990). In addition to the above-mentioned growth factors, one of the most distinct characteristics of osteoblasts is the synthesis of alkaline phosphatase and other

 

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secretory proteins like osteonectin, osteopontin, and fibronectin (Pavlin et al. 1994). Alterations of the concentration of any of these factors, as well as the responsiveness of bone cell to these factors, might lead to a decrease in osteoblasts’ generation and consequently to an incomplete refilling of the resorption lacunae (Pfeilschifter et al. 1993) The cytokines IGF-I, IGF-II, TGF-ß and PGE-2 control the proliferative rate of the osteoblasts and their capacity to generate various components of bone extracellular matrix, but osteoblasts also produce constitutively osteoclastogenic IL- 6 and CSFs as well as osteoclastic inhibitory factors TGF-ß (Simmons et al. 1994). The bone formation process starts with the recruitment of osteoblasts in the vicinity of the resorption site as an initial step. This process takes place as a result of the proliferation of osteoblastic precursor cells. It has been proven that this proliferation is induced initially through local growth factors, such as TGF-ß, platelet- derived growth factor (PDGF) and IGFs, which may be released from bone during resorption (Pfeilschifter et al. 1993) The bone formative properties of the human osteoblast reduce with increasing age; this reduction is more obvious by comparing osteoblasts obtained from fetuses with others obtained from adult donors (Termine 1990).                            

 

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1.2. Characteristics of Growth Factors and Mediators   a) Alkaline phosphatase   Alkaline phosphatase exists in high concentrations in cells which initiate the mineralized nodule formation in vitro. Furthermore it is well known that periodontal ligament (PDL) cells containing high levels of alkaline phosphatase show more rapid or increased nodule formation when compared with PDL-cells with lower alkaline phosphatase concentrations (Areco et al. 1991).     b) Osteonectin   Osteonectin is a bone glycoprotein, which has its highest concentration levels between the non-collagenous proteins in the developing bone; moreover it has a high affinity to bind calcium-hydroxyapatite, collagen, and thrombospondin. Osteonectin has been found in various non-bone cells, but only in the phase when the individual undergoes rapid growth and proliferation. Osteonectin production is highest in bone when compared with the other connective tissues in the human body. There is a relation between the existence of osteonectin in high levels in osteoblasts and its proliferative potential. This is increased in cells with greater content of osteonectin. In bone matrix this protein seems to be more structural and may act through its mineral and protein-binding properties (Termine 1990).     c) Type-I-collagen   Type-I-collagen is the prototype of the collagen family, and is known to be the most abundant component in the extracellular matrix. This is responsible for several significant biomechanical properties of the connective tissue (D’Souza & Litz 1994). Type-I-collagen protein is encoded by two genes, COLIA1 and COLIA2 which are expressed in all collagen-producing cells in a coordinated manner (Pavlin et al. 1994).  

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Osteoblasts synthesize relatively more type-I-collagen when compared with other collagen-producing cells. This can be considered as one of the distinctive characteristics of the osteoblastic phenotype. It has been suggested that this production of type-I-collagen is modulated by; 1,25-dihydroxyvitamin-D3 (vitamin D), hormones like (PTH), cytokines, and growth factors such as insulin and (IGF-I). There are all mechanisms that are partly mediated at the transcriptional level (Pavlin et al. 1994,D’Souza & Litz 1994). After synthesizing high levels of type-I-collagen, the osteoblasts deposit it to create the osteoid layer. This process is followed by a mineralization phase to form the bone matrix (Pavlin et al. 1994).     d) Vitamin D   Vitamin D is an important calcium-regulating hormone. It has several effects on the bone cells, including the stimulation of osteopontin and osteocalcin synthesis. It also inhibits the synthesis of type-I-collagen in osteoblastic cell lines (Pavlin et al. 1994) An adequate response to several hormones such as 1,25 dihydroxyvitamin D3 (calcitriol) is considered to be a normal function of osteoblast cells (Kveiborg et al. 2001).     e) Parathyroid hormone (PTH)   Several tasks of PTH have been reported, such as the stimulation of cell proliferation, DNA-synthesis in osteoblast cells in vitro, and bone apposition when combined with vitamin D. According to its concentration, it can also have bone resorbing effects by interacting with the regulation of calcium-homeostasis; PTH may cause inhibition of cell proliferation when it is applied in high doses (Carvalho et al. 1994). It has been proven that PTH-inducible cAMP formation increases with ageing, which seems to be more associated with an increase in PTH-receptors than with their decrease (Pfeilschifter et al. 1993).

 

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f) Interleukin-6 (IL-6)   Interleukin-6 is a cytokine produced by many cell types including the stromal cells and the osteoblasts which both contain large amounts of it. Some believe that IL-6 is not a regulator of osteoblast function (Saito et al. 1994, De Vernejoul et. al. 1993). Others however despute this and it has been reported that IL-6 has an inhibitory effect on bone formation through the stimulation of osteoclastic activity and subsequently bone resorption (Saito et al. 1994) The high production of interleukin-6 by osteoblasts is associated with high bone resorption rate in mice after ovariectomy (De Vernejoul et al. 1993).     g) Interleukin-1 (IL-1)   Saito et al. (1994)(IL-1) as a potent biological responsehave defined the cytokine modifier, which has a wide range of biological activities, and plays a central role in the inflammatory process. It stimulates bone resorption and induces osteoblast proliferation, but shows an inhibitory effect on bone formation in vitro.     h) Transforming growth factor-beta 1 (TGF-ß1)   The cytokine (TGF-ß1) has many effects on osteoblast activities. For instance, its influence on the net-structure accumulation of the cellular matrix causing either more degradation or in contrast increase of its synthesis. Moreover, it could act as an activator of type-I-collagen gen transcription (D’Souza & Litz 1994).     i) Prostaglandin E (PGE)   It has been proven that Prostaglandin E stimulates bone resorption in vitro and causes more calcium release when applied in high doses (Saito et al. 1994).    

 

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