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Osteoclasts are derived from haemopoetic stem cells and differentiate from a mononuclear cell into a multinucleate bone-resorbing osteoclast. Many important factors participate in this signalling pathway, such as Macrophage Colony Stimulating Factor (M-CSF), Osteoclast Differentiation Factor (ODF) and Osteoprotegerin (OPG). One of the key mechanisms within the pathway is the interaction between ODF and its receptor-RANK. This leads to a series of signals which further the differentiation and activation of osteoclasts. OPG reduces this interaction by acting as a decoy receptor to ODF. Osteoblasts play a key role in the regulation of this pathway as they secrete M-CSF, ODF and OPG and therefore osteoclastogenesis is dependent on these cells. A variety of other factors and hormones influence the pathway indirectly by stimulating or inhibiting osteoblasts and affecting both their differentiation and activation. Knowledge of the interaction between osteoblasts, osteoclasts and the signalling molecules between them has given rise to a variety of treatments for bone disease but this project focuses on its use in osteoporosis drug therapy and tissue engineering development. Examples of osteoporosis treatment include monoclonal antibodies that target ODF, drugs that increase OPG secretion, or drugs that modulate the activity hormones that suppress the pathway, such as oestrogen. Some drugs work by a combination of mechanisms in addition to inhibiting osteoclast differentiation, such as increasing osteoblasts and bone forming activity and inhibiting reactions involved in the bone resorption process.
New techniques in bone tissue engineering make use of the pathway by culturing osteoclasts together with osteoblasts to form bone tissue substitutes of higher quality compared to those made solely from osteoblasts. Furthermore, studies have recently tried to combine the use of drug therapy with tissue engineering for better delivery of treatment.
Osteoclasts are large, multinucleate bone-resorbing cells which are important in remodelling bone to function more efficiently under certain pressures. They work by releasing hydrogen ions and hydrolytic enzymes such as Cathepsin K which are used to break up the organic content of the bone as well as the hydroxyapatite mineral portion. This mineral matrix contains Calcium and Phosphate ions which are then released into the blood.
Consequently, the regulation of osteoclast activity is very important as an increase in bone resorption will lead to an increase in calcium and phosphate ion concentrations in the blood. This is achieved with hormones such as parathyroid hormone (PTH stimulates osteoclasts indirectly via osteoblasts), Calcitonin (inhibits osteoclast activity) and a number of cytokines such as Interleukins 1 and 6. Studies have shown that these cytokines not only have a positive effect on osteoclast activity but also on their differentiation (Roodman, 1992). This is also the case with other local factors such as Osteoprotegerin (OPG) , Osteoclast differentiation factor (ODF or RANK Ligand) and many others.
Many studies in vitro and on mice have been conducted in order to determine the actions of specific substances which help develop an activated bone-resorbing osteoclast from its haemopoetic precursor. Osteoclasts begin with macrophage characteristics (Kurihara et al, 1990, Hattersley et al, 1991) and are then recruited to the bone surface where these factors play a part in inducing the formation of the osteoclast. In particular, the discovery of the RANK Ligand pathway in the mid 1990's by Amgen, saw a breakthrough in the understanding of osteoclast formation and the profound therapeutic implications of these substances were realised.
Many bone diseases characterised by excessive bone resorption by osteoclasts, such as osteoporosis or hyperparathyroidism can potentially be treated with knowledge of how to inhibit the pathway. Similarly problems with osteoclasts and the role of RANK in inflammatory diseases such as periodontitis and rheumatoid arthritis have also been studied (Gravallese et al, 2000). Conversely osteopetrosis, a genetic disease resulting in osteoclast dysfunction, could be treated by identifying factors missing from the pathway and replenishing them using gene therapy (Askmyr et al, 2009). Using stem cell transplantation and addition of factors that stimulate osteoclast phenotype acquisition has also been studied in order to develop high quality bone for fractures (Pirraco et al, 2009).
The osteoclast differentiation process and its impact on the pharmacology of drugs used to treat osteoporosis will be looked at in more detail, in addition to more recent studies into its potential in tissue engineering therapeutics for the future.
The Osteoclast Differentiation Pathway
Osteoclasts are derived from haemopoetic stem cells and begin as circulating mononuclear cells which are then recruited to the bone surface where they form preosteoblasts. These cells then fuse to form a polykaryon which then develops into an activated osteoclast. In order for these stages to occur, various factors are needed and are therefore generated by macrophages in the blood or by the stromal and preosteoblast cells in the bone microenvironment, depending on the phase of differentiation.
The two main and essential factors that are secreted by the preosteoblasts are Macrophage Colony Stimulating factor (M-CSF) and Osteoblast Differentation Factor (ODF). Both of these have a "direct role in osteoclast differentiation, survival and proliferation" (Gilbert, 2003). M-CSF allows c-Fos and Mi transcription factors to be expressed in the preosteclast, which are needed in order to activate genes which convert the preosteoclast into an osteoclast (Gilbert 2003). These genes are responsible for expressing osteoclast associated markers such as the calcitonin receptor (CTR) and encoding tartrate resistant acid phosphatase (TRAP) and cathepsin K (CATK). The presence of these typical osteoclast properties are tested in experiments, to determine whether differentiation has taken place and hence they act as indicators of the presence of active osteoclasts. ODF binds to its receptor and activates the transcription factor NF-Kb which also plays a role in typifying osteoclast lineage through the expression of these genes. See figure 1 for a diagrammatic representation of this part of the pathway.
Many studies have taken place confirming the role of osteoblasts in supporting and stimulating osteoclast differentiation once it has reached the stage of a preosteoclast. For example this has been done by Takahashi et al, (1988) using co cultures of mouse spleen cells (which contain the preosteoclasts) with osteoblasts and comparing it with other cultures where osteoblasts are cultured separately from the spleen cells or with macrophages instead, and comparing the results for signs of osteoclast activity (such as TRAP positive cells or resorption lacunae). In this study osteoclast activity was only seen when osteoblasts were cultured with preosteoclasts in the spleen cells, highlighting the fact that osteoblast factors are vital in the process of developing an active osteoclast. More specifically , studies have been carried out to prove that it is in fact these identified osteoblast derived factors which are crucial to the process and not merely the presence of the osteoblasts themselves. This was shown in vitro by culturing preosteoblasts with only M-CSF and a soluble form of ODF which produced active osteoclasts that expressed TRAP and CTR. (Quinn et al 1998).
Furthermore experiments have shown that the factors released by these osteoblast cells such as M-CSF are essential in osteoclast differentiation and proliferation to the extent that an absence in osteoclasts occurs in mice with mutations in the genes which code for this factor (Yoshida et al 1990) and they therefore develop osteopetrosis as a result of a lack of bone resorption and remodelling. In vitro studies have also pointed out that many chemicals that increase the formation of osteoclast-like cells and pathological bone resorption such as nicotine, work via increasing the levels of M-CSF (Tanaka et al 2006), showing the direct effect this factor has on osteoclastogenesis. These results emphasize the importance of maintaining physiological levels of this M-CSF as it has a direct effect on the number of active osteoclasts.
As can be seen from figure 1 (page 6), in order for an osteoclast to be able to function properly after differentiation, it has to become polarised and form its specialised cell membrane or ruffled border for bone resorption (Takahashi et al, 2007). This process depends on a kinase enzyme known as C-src, which studies have also established, is an important messenger in M-CSF signalling pathways (Faccio et al, 2007). Subsequently murine models of a failure of this secondary messenger molecule develop osteopetrosis (Abu-Amer et al, 1997). Although this is mainly due to a failure in osteoclast activity, C-src can still be considered as an essential protein in the completion of a fully differentiated and active osteoclast.
Figure 1: Showing the pathway for osteoclast differentiation and the role of osteoblasts. (Figure taken from Developmental Biology, 8th Edition by Scott F. Gilbert 2006, drawn by M. Steinback,2000)
RANK, which stands for Receptor Activator of Nuclear Factor kB, is a membrane bound TNF receptor, which is expressed on osteoclast precursor cells and recognises the TNF related factor ODF. Consequently ODF is also known as RANK Ligand (RANKL) and as stated before, mediates differentiation of precursor cells in the presence of M-CSF, through cell to cell interactions with RANK (Roux & Orcel, 2000).
As previously stated, once ODF binds to RANK, there are a series of transduction signals, which lead to the formation of a mature osteoclast by activating specific genes. In addition to this, ODF is involved in the fusion of preosteoclasts to form a polykaryon and eventually an activated multinucleate cell though its NF-Kb activation (Woo et al 2000). As well as this, it was discovered that ODF is also responsible for increasing the activity of the osteoclast once it has differentiated. This was shown by combining mature osteoclasts with ODF, and observing an increase in osteoclast stimulation and bone resorption (Lacey et al 1998). This suggests that an intervention at this level which can block ODF binding to RANK may provide possible therapy for diseases of low bone density due to increased osteoclast action.
The human body must have its own endogenous antagonist of RANK activation in order to regulate the number of differentiated osteoclasts and maintain their activity at the physiological levels seen in people with normal bone density. This regulator is known as osteoprotegerin (OPG), which is also a member of the TNF family, and was actually identified before RANK and its ligand. In fact the discovery of OPG by Amgen led to the identification of RANK . It "lacks a transmembrane domain and represents a secreted receptor" (Roux & Orcel 2000). It has also been described as a "decoy receptor" (Roux & Orcel 2000) because it recognises ODF and binds to it instead of RANK, thus inhibiting the differentiation and activation pathway of the osteoclast. Many studies have taken place in vitro and in vivo in order to test the relationship between OPG, RANK and ODF.
In vivo, mice which have been administered with OPG develop a form of osteopetrosis due to a lack of differentiated osteoclast cells (Simonet et al 1997) whereas mice which have a reduction in OPG by knocking out the specific gene show decreased bone density and more fractures (Bucay et al 1998). This can be seen clearly in figure 2.
Lack in OPG Overexpression of OPG
Figure 2 showing osteoporotic bone on left and osteopetrotic bone on the right. (Taken from Bolon et al 2002)
These early studies focussed on the potential role of OPG in osteoporosis therapy, whereas more recent articles have shown other possible implications for the protein. For example OPG gene therapy has been looked at in order to decrease osteoclast bone remodelling after a fracture (Ulrich-Vinther et al 2005). Other areas that have been looked into are the local and hormonal factors that regulate bone remodelling by adjusting the ratio of RANKL to OPG expressed by osteoblasts. It is this ratio which determines osteoclast differentiation and activity (Gogakos et al, 2009).
Factors that stimulate osteoclastogenesis can be seen in Figure 1 labelled as osteotrophic factors such as Vitamin D3 and parathyroid hormone which increase osteoclast activity and IL-6 which stimulates osteoclast differentiation as well as activity (Roodman, 1992). Other cytokines are also involved (see figure 3) such as IL-11, IL-1 and TNF and these may all play a part in the bone destruction in rheumatoid arthritis (Duff, 1993). Studies have shown that IL-6 in particular can regulate bone turnover in vivo when administered in immunologically deficient mice (i.e. mice that did not secrete IL-6 as this is a T cell cytokine) by increasing osteoclast numbers (Rozen et al, 2000). This suggests that increasing or decreasing the level of these cytokines can help treat osteopetrosis or osteoporosis respectively.
Figure 3 (Roux and Orcel, Arthritis ResÂ 2000Â 2:451) shows local biological factors that affect osteoclast (OC) differentiation and activation. ODF is labelled RANK. Stimulators of OPG, particularly 17Î² estradiol, act as inhibitors of the osteoclastogenesis pathway as OPG intercepts the RANKL/RANK interaction and therefore stimulating this blocks the progression of a preosteoclast to a mature osteoclast.
The Role of Oestrogen and Osteoporosis Treatment
Oestrogen plays a role in modulating both osteoclast differentiation and activation and acts via osteoblasts, osteoclasts and immune cells, in order to produce an overall inhibition of excess bone turnover and resorption. Several experiments have been done in vivo and in vitro in order to determine oestrogen's specific effects but it is sometimes difficult to assess whether oestrogen is working via osteoclasts directly or via other cells in the culture which indirectly affect osteoclasts, given that the culture will contain the multiple cell types found in bone (Oursler, 2003).
Many studies in the early 90's showed that mature osteoclasts express oestrogen receptors and this is how the hormone induces many of its direct effects, such as reducing the number of these cells. Oestrogen brings about this outcome by repressing the activation of proteins that are needed for osteoclasts to differentiate in response to ODF. For example the protein c-Jun and the kinase enzyme which activates it (JNK) are both down-regulated by oestrogen (Srivastava et al 2001) and this reduces ODF-induced differentiation, thus reducing the number of newly formed mature osteoclasts.
A decrease in the number of osteoclasts is not solely achieved by direct action on the cells themselves, but by a combination of this with osteoblast action. For example, an increase in osteoclast apoptosis is also an effect of oestrogen (Faloni et al, 2007) and studies have shown that this may occur via stimulation of both osteoclast and osteoblast TGF-Î² production (Akatsu et al, 1998).
The main differentiation inhibiting effects of oestrogen are carried out via its actions on osteoblasts and immune cells (Zallone et al, 2006). These include reducing the secretion of cytokines such as IL-1, IL-6, TNF-Î±, which as established earlier, all increase osteoclastogenesis or bone destruction, and thus a reduction in these factors helps to maintain bone density (Gogakos et al, 2009). Oestrogen also reduces the expression of M-CSF and ODF which are essential for the formation of mature bone resorbing osteoclasts. Furthermore, as seen in figure 3 (oestrogen represented as 17Î²-estradiol), oestrogen stimulates the secretion of OPG, and therefore the combined effect is a decrease in the ratio of ODF to OPG and consequently less osteoclast differentiation.
Given that ODF, OPG and the various cytokines have effects on osteoclast activity, oestrogen's regulation of these factors also plays a part in altering this activity. For example in vivo studies showed that an increase in TRAP and bone loss occurred when oestrogen was lowered (Bell et al, 1997). Similar studies in vitro obtained similar results (Rissanen et al, 2008), highlighting the crucial role of this hormone in bone homeostasis. It is not known exactly how this occurs but there is a lot of evidence suggesting that in addition to the effects of the ODF/OPG ratio, oestrogen may have a direct effect on osteoclast gene expression of these bone degrading proteins plays (Oursler, 2003). In vivo models of oestrogen-treated rats showed suppression of TRAP and carbonic anhydrase gene expression in osteoclasts compared with ovariectomised rats (these rats were deficient in oestrogen) that had higher levels of these bone resorbing enzymes (Zheng et al, 1995).
Overall it is clear that oestrogen has a major osteoprotective role in the body and that a deficiency would lead to an excess in bone resorption and a decrease in bone density as seen in osteoporosis.
Osteoporosis and poor bone health is a disease which affects one in two women and one in five men over the age of 50 (National Osteoporosis Society) and can be the cause of fatal fractures. It is a disease characterised by low bone density and disruption of bone microarchitecture. There are many risk factors associated with osteoporosis, such as age, being on long term steroids, smoking, and many others. Osteoporosis may occur in many hormonal disorders where levels of hormones that are associated with bone homeostasis are affected such as PTH, testosterone or other steroid hormones. Examples of such conditions are hyperparathyroidism or Cushings disease where an excess of PTH and cortisol (a glucocorticoid) is produced respectively. One of the biggest associated risks however, is being a woman who had her menopause at an early age, due to its effects on oestrogen levels. During menopause, the woman's body produces less oestrogen from her ovaries and therefore there is an increase in osteoclast differentiation and activity, resulting in weaker bones.
Age related and postmenopausal osteoporosis is the most common forms of the disease and many of the available treatments target the osteoclast differentiation pathway. The main therapies and the molecular mechanisms behind them will be discussed further.
A simple way to treat the symptoms of menopause including osteoporosis would be to increase oestrogen levels with hormone replacement therapy (HRT) and this was mainly used as a preventative measure against osteoporosis. However, the Women's Health Initiative conducted a randominsed control trial which showed that HRT can increase the risk of diseases such as breast cancer and thromboembolism (R. Parks, 2008) and therefore this treatment is less commonly used. This trial however, did not assess whether giving lower doses or short term replacement played a part in eliminating these risks (R. Parks, 2008) and only looked at the results from one product and the outcome may have been different for other kinds of HRT (Anderson et al, 2002). An alternative to HRT is widely used now in the form of Selective Estrogen Receptor Modulators (SERMs). These drugs can act as agonists of the oestrogen receptor in certain tissues and antagonists in others. This tissue selectivity (Oursler, 2003) allows the drug to increase oestrogen activity in bone but decrease it in breast tissue for example to stop any of the adverse effects seen with HRT. Studies have shown that SERMS work by inhibiting excess bone resorprtion in a similar way to the oestrogen hormone. SERMs, like oestrogen, reduce the number of osteoclasts and have been shown to prevent the expression of IL-1Î² and IL-6 (Taranta et al, 2002). In a particular in vitro study on a specific SERM drug, Raloxifene, it was also shown that this class of drug work by stimulating osteoblasts and their production of osteoblast transcription factors, as well as inhibiting osteoclasts (Taranta et al, 2002). These combined effects that prevent bone loss have been confirmed in vivo by randomised control trials on women. In particular one was conducted on the use of Raloxifene in preventing vertebral fractures in women (Ettinger et al, 1999) and showed a significant reduced risk in women taking the drug compared with women given a placebo. This was a very useful study because it had a large sample size and women from 25 different countries, showing the drug's effectiveness for a variety of ethnicities. Nevertheless, assessments were only made on the bone mineral density of the spine and femoral neck and therefore the results may not necessarily mean Raloxifene is useful in decreasing bone loss significantly in other parts of the skeleton.
Although SERMs are useful for postmenopausal osteoporosis in women because they counter balance the loss in oestrogen, there have also been studies showing their efficacy in age related bone loss (Ke et al, 2001).
Another class of drugs that have been developed on the basis of the osteoclast differentiation and activation pathway are human monoclonal antibodies that inhibit the ODF/RANK signalling pathway. Initially this was based on the idea that OPG intercepts RANK activation by ODF and therefore a recombinant human fusion protein of OPG (Gogakos et al, 2009) was used. The effects of this molecule were discussed previously on page 7 and can be seen in figure 2. Â However, later monoclonal antibodies which targeted ODF were developed (Simonet, 2008) as an alternative to soluble OPG because "its clinical use was limited by the development of antibodies that resulted in a reduced effective half-life and the necessity for increasing doses and frequency of administration" (Gogakos et al, 2009 on a study by Boyce et al, 2008). Denosumab is a fully human monoclonal antibody which means there will be less side effects as opposed to chimeric or humanised monoclonal antibodies which can induce skin rashes, low blood pressure, vomiting etc. (Dixon S, 2009). It has been shown to work by binding to ODF with high affinity and high specificity, preventing it from binding to RANK and stimulating osteoclast differentiation (see figure 4 for diagrammatical representation). Consequently denosumab was shown to increase bone mineral density to up to 6.7 percent in the lumbar spines of post menopausal women (McClung et al, 2006). Here again it is clear that the knowledge of the essential factors needed for the differentiation and activation of osteoclasts has provided the foundations for the development of this effective treatment of osteoporosis.
Figure 4: The bottom part shows RANKL/ODF binding to the RANK receptor and therefore allowing proliferation and differentiation and the top part shows this chain of events inhibited by OPG, recombinant fused OPG protein and denosumab. Taken from Expert Rev Endocrinol Metab.Â 2009;4(6):639-650
While the drugs mentioned so far mainly target osteoclast differentiation, a wide variety of other mechanisms exist which still capitalise on the recent understanding of the formation of osteoclasts. A good example of this is strontium ranelate which not only decreases bone resorption but also significantly increases bone formation by stimulating osteoblasts. Studies were conducted in vitro on murine cells and a rise in the expression of osteoblast markers was seen as well as a reduction in osteoclasts (Bonnelye et al, 2008). The mechanism by which it reduces the number of osteoclasts is not certain but some studies have indicated that it is via increasing osteoblast secretion of OPG and decreasing osteoblast expression of ODF (Pierre J. Marie, 2007) and therefore reduces osteoclast differentiation. On the other hand, a randomised control trial which aimed to determine the relationship between the drug and OPG levels revealed that strontium ranelate did not significantly change OPG concentrations in post menopausal women (Ertorer, 2007). However, this was a very small trial with only 32 women taking part and measuring OPG levels only 3 months after treatment and hence a significant upregulation of OPG may have occurred had there been a bigger sample size, and if a longer duration for the drug to work was allowed before assessments were made. Strontium Ranelate's other effects on osteoblasts make it unique compared with alternative antiosteoporotic drugs in that it stimulates osteoblast cell replication via activation of calcium sensing receptors on preosteoblasts (Pierre J. Marie, 2007). Another mechanism of this drug is inhibiting osteoclast function in addition to differentiation by "disruption of the osteoclast actin-containing sealing zone" (Bonnelye et al, 2008). It has been demonstrated that this treatment works by various modes of action and is highly effective in counteracting the decline in bone density seen in osteoporosis. Despite this advantage, results of bone density increase should be interpreted carefully as there is "attenuation of X-ray when some of the calcium in bone is replaced byÂ strontium" (Blake et al, 2007) may exaggerate the benefit of the drug and this should therefore be taken in consideration and corrected for.
Another class of drugs which are most commonly used in the treatment of osteoporosis are Bisphosphonates, which are nitrogen-containing compounds which target osteoclast function. These drugs bind to Calcium hydroxyapatite, of which there is a high concentration in bone and therefore increases the likelihood of uptake by osteoclasts. The main mechanism of action of bisphosphonates is by inhibiting an enzyme called farnesyl pyrophosphate synthase (FPPS) which is an essential part of the mevalonate pathway. Disruption of this pathway in turn affects chemical messengers needed for osteoclast bone resorption (Gogakos et al, 2009). Other mechanisms include stimulating osteoclast apoptosis (Frith et al, 1997) which therefore reduces the extent of bone resorption. There are many subclasses of Bisphosphonates such as those that are non-nitrogenous or those that bind to bone for longer and each have some different pharmacological properties (Russel et al, 2007) but share an overall effect of preventing the resorption activity of the osteoclast. The efficacy of these drugs such as Alendronate and Risedronate have been tested and have shown a relative risk reduction of bone fractures in post-menopausal women of up to 41% (J. Bilezikian, 2009) and are therefore widely used for both the prevention and treatment of osteoporosis.
Exploration of the biological effects of Bisphosphonates, which were first discovered more than 40 years ago, has facilitated other innovative treatment possibilities for the future. Recently the possibility of using Statins as a treatment for osteoporosis has been looked into because of their similar ability to inhibit the mevalonate pathway. Figure 5 shows a diagrammatic representation of the reasoning behind this theory. A meta-analysis of observational and randomised controlled trials of the effects of statins on bone mineral density showed "statistically significant beneficial effects of statins" on bone (Uzzan et al, 2007). Nevertheless they are not as effective as Bisphosphonates due to differences in their structures meaning Statins will not bind to bone as readily (Cruz et Gruber, 2002). More research is needed to further evaluate their use as a potential treatment.
Figure 5 showing the link between Statins, Bisphosphonates and osteoclast activation. Statins inhibit the pathway earlier that Bisphosphonates so should theoretically produce the same osteoclast inhibiting effects. (Figure taken from Cruz AC, Gruber BL. Statins and Osteoporosis: Can these Lipid-Lowering Drugs also Bolster Bones?Â Cleve Clin J MedÂ 2002;69:277-278)
Other developments in the therapy of osteoporosis include C-src kinase inhibitors. The action of C-src in activating a newly formed osteoclast was discussed previously and therefore the development of a drug which prevents this taking place will reduce the excessive bone resorbing activity seen in patients. Experiments have been carried out both in vitro and in vivo on rats and both showed a reduction in bone resorption using the C-src inhibitors (Missbach et al, 1999). There are many ways of inhibiting the C-src protein such as interfering with the substrate binding site, the ATP binding site or changing the synthesis and degradation of the protein (Missbach & Green, 2000). Most research on this class of drug have been done on animal models and little has been done to show the effects on humans. Nevertheless recently it has been tested in phase 1 trials and has had encouraging results (Rucci et al, 2008).
A recent interesting development of a potential class of drugs for the future, known as NFkappaB decoy oligodeoxynucleotides have a direct impact on the osteoclast differentiation pathway and their activation. As mentioned earlier, the binding of ODF to RANK leads to the activation of the chemical Nuclear Factor-kappa B which then leads to cell fusion and a continuation of the differentiation process. The NFKappaB decoy oligodeoxynucleotides inhibits NFKappaB's ability to work and therefore, with administration of the drug in vitro, there is a reduction in M-CSF induced differentiation, in addition to a reduction in bone resorption (Shimizu et al, 2006). Other studies have shown that inhibiting this transcription factor also leads to an increase in osteoclast apoptosis and therefore less bone resorption (Penolazzi et al, 2003).
As has been established, the osteoclast differentiation pathway has facilitated the development of many drugs which interfere with its processes at different stages and have an overall protective effect on bone. Other drugs have been manufactured which simultaneously target the activation pathway or other cells in bone to also produce a similar defensive effect. Knowledge of the osteoclast differentiation pathway can not only help develop new drugs to combat disease but can also assist in the improvement of tissue engineering techniques with therapeutic implications in the future.
Tissue Engineering and the Role of Osteoclasts
Tissue engineering is a relatively new process by which fresh tissue is formed to improve, repair or replace biological tissue. It requires the use of cells, biological and chemical factors and additional material to form 3-dimensional scaffolds or matrices for the tissue. The need for bone tissue engineering is considerable given that bone substitutes are necessary in a variety of cases. For example when there is healing delay or non union of fractures, the bone substitutes are used in restoring and maintaining bone function (BTEC of Carnegie Mellon University). These types of cases represent a significant proportion of fractures, specifically 5-10% reported in the US (Dawson et al, 2008). Tissue engineering can also be used to repair other bone defects caused by tumours or infectious disease (Braddock et al, 2001).
The main approach used to construct these bone substitutes is to culture the relevant cells, such as osteoblasts in this case and add growth factors and other signalling molecules to the culture so the cells differentiate and proliferate to form tissue. Figure 6 represents this technique.
Figure 6 from http://www.btec.cmu.edu/tutorial/bone_tissue_engineering/bone_tissue_engineering.htm
BTEC section of Carnegie Mellon University website
The scaffold is required for large defects in order to act as a temporary extracellular matrix in which the cells attach (BTEC of Carnegie Mellon University, as well as providing the correct shape for the implant. There is not simply one way to carry out tissue engineering and there are many choices at each step in the process. For example the cells that are cultured need to be chosen and the origin of these cells also. They could be taken from the patient or a donor for example and two types of cells could be cultured instead of one. The properties of the scaffold material need to be considered such as its degradability and how well the cells can proliferate within it. Other factors of significance are when to implant the scaffold. Sometimes the scaffold can be implanted after the cells have been harvested and attached to it or on the other hand, the cells and matrix can be implanted together at separate sites (BTEC of Carnegie Mellon University). There is also the issue of whether to use stem cells which are an "exceptionally promising tool in tissue engineering" (Pirraco et al, 2009) because of their ability to differentiate into a variety of lineages.
The most important aspect of the process is the addition of the signalling molecules and understanding which growth factors need to be added in order for the cells to develop into a functioning and useful tissue. Cells will not grow in an organised fashion if simply cultured without these specific factors as it is the precise signalling pathways found in the body which interact with cells and cause their activation. The osteoclast differentiation pathway described earlier can be applied in this context.
Up to now, most of the experiments that have been carried out on bone tissue engineering have been done using only osteoblasts which are cultured from osteoprogenitor or stem cells. However, this forms a static environment which does not represent normal bone well (Z. Xia, 2004). This is because bone tissue is dynamic and contains a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Other studies have come to similar conclusions and found that there are fundamental differences between bone in vivo and the osteoblast cultures whereby there is an "inability of cultured osteoblasts to form lamellar bone-like structures and deposit the characteristic mineral of native bone" (Han & Zhang, 2006). The same study reported that the cultured osteoblasts fail to deposit collagen fibres and cannot mineralise osteoid but instead forms "woven bone-like...immature" bone. Moreover, this study identified the need for osteoclasts as they have a regulatory effect on osteoblasts (Phan et al, 2004) as well as an overall role in bone homeostasis. This has been recognised by many other reports which agree that the introduction of osteoclasts in the tissue matrix can lead to "high quality" engineered bone (Pirraco et al, 2009).
Methods of establishing an osteoblast-osteoclast co-culture system have been developed by Jones et al in 2006. The technique used was to isolate monocytes from murine bone marrow and then induce differentiation by adding M-CSF and ODF. After 4-7 days multinucleate osteoclast cells formed and were tested for tartrate resistant acid phosphatase activity. This study again highlights the applications of mapping the osteoclast differentiation pathway. Although culturing the two cell type culture is a simple idea, it is important to establish the correct ratio of osteoblasts to osteoclasts for adequate bone replacement (Jones et al, 2006). In addition to this a suitable scaffold material which will allow the co-culture cells to attach is needed. These factors have been investigated and one particular report showed that using a ratio of 1:100 osteoblasts: osteoclasts on a silk fibroin scaffold support the growth of the co-culture and can be applied to this novel technique in bone tissue engineering (Jones et al, 2009).
Another innovative application of tissue engineering which is currently being explored is the combination of drug delivery with the scaffold implant. This involves loading the drugs onto the scaffold and then implanting it in the body. This has been tried with a variety of different drugs for different purposes and therefore a new field of tissue engineering termed Tissue Engineering Therapeutics (Baroli, 2009) is emerging. There is a range of benefits of administering drugs in this way, such as obtaining the desired time frame for the drug to act as well as the desired concentration (Mourino &Bouccaccini, 2009). Studies have also shown that using this technique can also improve the drug release profile (Habraken et al, 2007). These advantages are gained through altering the scaffold material as it influences the time it takes for it to degrade and release the drug (Nair & Laurencin, 2007). An added benefit of incorporating drugs into the scaffold is that it ensures local delivery of the treatment. This avoids any systemic side effects of the drug if it were to circulate in the blood and reach other parts of the body. It also increases the interaction between the drug and the target tissue (Mourino & Bouccaccini, 2009).
The types of agents that have been combined with TE so far are antibiotics, anti-inflammatory and anti-resorptive agents. Antibiotics are useful drugs in this application to treat osteomyelitis in addition to preventing infection from the implantation itself (Mourino & Bouccaccini, 2009). Often, infection can arise from contamination of the scaffold before implantation or bacteria already on the skin adhering to and colonising the scaffold, preventing it from implanting well. Subsequently many experiments have been carried out on a variety of antibiotics combined with TE to help solve these problems. For example Gentamicin has been tried with different scaffold materials in vitro (Shi et al, 2009) and tetracycline was tried in vivo and was shown to prevent bacteria proliferation without affecting the bioactivity of the scaffold (Domingues et al, 2004).
The use of anti-inflammatory drugs is valuable in this context because implantation of the scaffold can induce a host immune response, causing inflammatory cytokines to be released (Chevalier et al, 2009). Glucocorticoids and steroids such as Dexamethasone have been impregnated into scaffolds (Duarte et al, 2009), which is very useful considering the vast systemic side effects of steroids.
Furthermore the administration of bisphosphonates via tissue engineered implants has been looked into (Fauchex et al, 2009) as a way of preventing excess bone resorption in a variety of diseases including osteoporosis.
This field of tissue engineering is still in the early stages and most of the studies have been carried out in vitro only. There are still many obstacles such as the need for more accuracy in the concentration of drug released and the period of time it acts at the target tissue (Mourino & Bouccaccini, 2009). There is a lot of potential in this field for improving drug delivery as well as the production of bone tissue replacements with improved biocompatibility and mechanical properties (Quirk et al, 2004).
Osteoclast differentiation requires a variety of factors and depends heavily on regulation by osteoblasts. Studying the mechanisms behind the formation and function of these cells has been crucial to the development of extremely useful treatment. With an increasingly aging population bone health will play an important role as a major co-morbidity in patients with medical, orthopaedic and gynaecological diseases and therefore the significance of this research should not be underestimated. All the drugs used to combat bone resorption in osteoporosis are based on altering differentiation, proliferation or rate of apoptosis of these cells and this has only been achieved through understanding the molecular pathways associated with these processes. The role of osteoclasts is important in many other diseases other than osteoporosis, such as Paget's disease, osteopetrosis, but the vast amount of research that has been undertaken in each area has meant that only one of these was discussed in detail.
The direct use of the signalling molecules to induce osteoclastogensis seen in bone tissue engineering, a field of medicine with vast implications and increasing research, demonstrates the significance of osteoclast differentiation now and in the future.