Table 1: The Role of Interleukins 1to10 (IL-1 to IL-10) in Healing INTERLEUKIN ROLE SOURCE IL-1 Induces fever, ACTH release, enhances TNF-, IFN-; activates granulocytes, endothelial cells, stimulates haematopoiesis. Macrophages, mast cells, keratinocytes, lymphocytes. IL-2 Activates macrophages, T lymphocytes, NK cells, stimulates differentiation of B lymphocyte; induces fever. Macrophages, mast cells, keratinocytes, T lymphocytes. IL-3 Stimulates bone marrow stem cells T lymphocyte IL-4 Early: Stimulates fibroblast proliferation; Late: Downregulates cytokine expression Mast cells. IL-5 B-cell differentiation, eosinophil and IgA production TH2 lymphocytes
IL-6 Released in response to IL-1; induces fever; enhances release of acute-phase reactants by the liver; inhibits ECM breakdown during proliferation. Macrophages, mast cells, keratinocytes, lymphocytes. mediator) IL-7 Lymphocyte maturation Stromal cells of red bone marrow and thymus IL-8 Enhances neutrophil adherence, chemotaxis and granule release; enhances epithelialisation. Macrophages, mast cells, keratinocytes, lymphocytes. IL-9 Stimulate mast cells T lymphocytes IL-10 Downregulates cytokine expression in latter stages (anti-inflammatory). Mesenchymal stem cell. (Cross and Mustoe, 2003; Stratford et al., 2006) Colony Stimulating Factor (CSF) Colony Stimulating Factor (CSF) is a member of a group of cytokines that stimulate wound-healing. Macrophages secrete CSF-1, an autocrine mediator, in order to ‘reproduce’ itself in an act of ‘self-preservation’. The macrophage releases granulocyte-macrophage CSF, which has chemotactic, as well as cellular proliferation and activation abilities (Stratford et al., 2006). Interferon (IFN) The interferon (IFN) group is made up of three subtypes; IFN-, IFN- and Each group is named according to their capability to ‘interfere’ with viral
2002; Gerstenfeld et al., 2003b). They are referred to as positive acute-phase proteins and are secreted by macrophages, inflammatory cells and also cells of mesenchymal origin that are found in the periosteum (Wang et al., 2004). The function of these cytokines is to promote chemotaxis, thereby attracting other inflammatory cells to the site of injury. This mechanism stimulates extracellular matrix synthesis, angiogenesis and the recruitment of endogenous fibrogenic cells to the affected area. There is peak concentration of these proteins within the first 24 hours of the fracture occurring, followed by a drop in levels during the period of cartilage formation. Cytokine quantities are later restored, and become markedly increased again during the remodelling phase (Gerstenfeld et al., 2003a; Wang et al., 2004). The formation and remodelling of endochondral bone is regulated by cytokines (Barnes et al., 1999). For example, TNF- stimulates the migration of MSCs, evoking apoptosis of hypertrophic chondrocytes and stimulating osteoclastic activity. Indeed, absence of TNF- causes delayed resorption of mineralised cartilage, thereby inhibiting bone formation. Levels of IL-1, IL-6 and TNF- are increased again during fracture callus reshaping in the later stages of fracture healing and remodelling (Tsiridis et al., 2007). Transforming Growth Factor Beta (TGF-) There are many members of the TGF- superfamily, consisting of numerous growth and differentiation factors. More than 30 TGF- superfamily members
BMP-2 Day 1 to 21 Recruitment of MSCs, chondrogenesis, role in fracture healing cascade, regulates expression of BMPs; differentiation of MSCs BMP-3, 8 Day 14 to 21 Regulation in osteogenesis BMP-4 Day 14 to 21 Involvement in early stage of callus formation BMP-7 Day 14 to 21 Regulation of ossification GDF-10 BMP-5, 6 Day 3 to 21 Regulation in ossification, BMP-6 initiates chondrocytes maturation GDF-5, 1 Day 7 to 14 GDF-5 involved in chondrogenesis; Stimulation of MSC aggregation, angiogenesis, chemotaxis, degradation of matrix proteins TGF-1 TGF-2 TGF-3 Day 1 to 21 Day 3 to 14 Day 3 to 21 Chemotactic for bone forming cells and macrophages; proliferation of MSC, osteoblasts, chondrocytes (Russell et al., 1980; Murray et al., 1993; Dong and Canalis, 1995; Aspenberg et al., 2001; Cho et al., 2002; Devlin et al., 2003b) Platelet-Derived Growth Factor (PDGF) Platelet derived growth factor (PDGF) is a dimeric polypeptide composed of  and  chains; it is a ligand of the tyrosine kinase receptor, and its binding is
affected by IL-1, TNF- and TGF-1 (Solheim, 1998). It is chemotactic for MSCs, and is synthesised by platelets, monocytes, macrophages, endothelial cells and osteoblasts (Andrew et al., 1993). There are potentially beneficial clinical implications for PDGF, and Nash et al., (1994) showed that PDGF increased callus density and volume in the tibial fractures of rabbits (Nash et al., 1994). Fibroblast Growth Factor (FGF) There are nine structurally-related polypeptides of the FGF family, which are bound to the tyrosine kinase receptor. In normal adult tissue, FGFs are both acidic and basic in nature, and there are varying degrees of potency between members. In fracture healing, FGFs are synthesised by monocytes, macrophages, MSCs, osteoblasts and chondrocytes. The growth and differentiation of all of these factors are further promoted by FGFs (Zimmermann et al., 2007; Jemal et al., 2008). Fibroblast growth factors are active in the initial period of bone repair and are, therefore, crucial in angiogenesis and MSC mobilisation. There is also an instrumental involvement in chondrocyte proliferation and maturation, and evidence suggests that FGF can have an advantageous influence on callus size (Matzuk et al., 1995; Lieberman et al., 2002). Insulin-Like Growth Factors (IGFs)
birth. This was consistent with a low bone mass, osteopenia and abnormal osteoblastic function. Humans with NOG mutations can present frank skeletal and joint impairment, whilst NOG knockout mice die shortly after birth and have dense, deformed long bones, implicating the mediatory role of noggin (Brunet et al., 1998; Devlin et al., 2003a). The rare genetic bone disease Fibrodysplasia Ossificans Progressiva (FOP) is characterised by abnormal hallux development and progressive heterotopic endochondral ossification of the muscles. This disorder is due to a lack of expression of the NOG gene (Fontaine et al., 2005). Chordin The antagonist chordin (Chd) binds with BMP-2, BMP-4 and BMP-7 in a similar way to noggin, thereby antagonising the actions of BMP by blocking the binding action to its receptors. Chordin also has a role in embryonic development, especially in skeletogenesis, as high levels are expressed in cartilage and in the epiphyseal regions of developing long bones (Bostrom, 1998; Balemans and Van Hul, 2002; Marshall et al., 2004). Expression of Chd in vitro has been found to be inversely related to chondrocyte maturation and differentiation due to BMP inhibition and is considered to be a negative regulator of endochondral ossification. The secreted protein twisted gastrulation (TSG) is a cofactor that attaches to Chd, thus facilitating the inhibition of BMPs by sensitising binding. Twisted gastrulation can also act as an independent inhibitor, and levels are highest in the early stages of osteoblast differentiation (the preosteoblast stage) (Oelgeschlager et al., 2003; Petryk et al., 2005).
are more at risk of injury is unclear, although general weakness, inferior balance and poorer neuromuscular function have been identified as possible reasons (Kyro et al., 1993; Nelson et al., 1994; Sacks and Nelson, 1994). The adverse effects of smoking on fracture healing were reported in patients that underwent spinal fusion (spondylosyndesis or arthrodesis), with smokers generally having a higher rate of pseudoarthritis than the non-smoking control groups (40% vs. 8%) (Sloan et al., 2010). Several clinical studies have been carried out on human subjects regarding fracture healing and smoking (Whitesides et al., 1994; Silcox et al., 1995). Carpenter et al. (1996), reported that outcomes of lumbar fusion operations were significantly more favourable for non-smokers (P<0.02). The group also found that non-smokers were more likely to return to work on time than smokers following this surgical procedure (Carpenter et al., 1996). Another trial, reported by Adams et al. (2001), involving 140 smoking and 133 non-smokers, found that the mean time to union of open tibial fractures was 32 weeks for smokers, compared with just 28 weeks for non-smokers. The authors also found that, in the same groups of patients, bone grafting was necessary in 36 smokers, but only 24 non-smokers required the procedure in order to stimulate bone healing (Adams et al., 2001). These results were statistically significant (P<0.05), and showed that smoking is deleterious to human fracture healing. The findings were consistent with other experimental studies, which also demonstrated
Table 7: Expression Summary of Mesenchymal Stem Cell Markers throughout 10 Passages Marker 1st Passage 2nd Passage 3rd Passage 4th Passage 5th Passage 6th Passage 7th Passage 8th Passage 9th Passage 10th Passage CD29 99.95% 100% 93.18% 88.13% 98.22% 99.78% 99.80% 99.85% 99.63% 100% CD44 100% 100% 98.27% 97.29% 99.49% 99.79% 99.83% 100% 99.65% 98.87% CD73(PE) 100% 100% 97.38% 100% 99.82% 100% 100% 100% 100% 100% CD105 35.72% 47.58 71.96% 83.21% 90.91% 94.44% 91.75% 92.48% 81.40% 69.43% CD166 99.36% 97.33% 100% 98.86% 98.33% 100% 99.82% 99.65% 99.49% 100% CD34 69.39% 34.42% 25.35% 19.29% 5.98% 6.85% 6.45% 5.85% 7.68% 6.62%
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Ethical Approval Documentation EA2 Ethical Approval Form: Human Research Projects Please word-process this form, handwritten applications will not be accepted This form must be completed for each piece of research activity whether conducted by academic staff, research staff, graduate students or undergraduates. The completed form must be approved by the designated authority within the Faculty. Please complete all sections. If a section is not applicable, write N/A. 1 Name of Applicant ANDREW STEPHEN SLOAN Department: NATURAL AND APPLIED SCIENCES Faculty: FACULTY OF HEALTH, LIFE AND SOCIAL SCIENCES 2 Position in the University Postgraduate Researcher 3 Role in relation to this research PhD Student 4 Brief statement of main Research Question The key question that is hoped to be answered is; what are the biological adverse effects of smoking on the microenvironment of healing bone fractures?