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Potentially novel markers for chondrogenesis to speedup the work in the design and development of regenerative therapy for osteoarthritis

| March 14, 2015

Introduction
Treatment of cartilage damage presents one of the significant problems in orthopaedics. Cartilage damage, though not life threatening, affects the quality of life significantly. In most cases patients suffering from degenerative disorders such as osteoarthritis are forced to undergo expensive joint replacement surgeries. With advancements in biomedical science there is an increasing focus on the possibilities of nonsurgical and regenerative therapies for cartilage damage. It is well known that both bone as well as cartilage tissues are derived from the same Mesenchymal stem cells (MSC). Already research has established many known markers for bone formation such as BMP’s and the transcription factor Runx2, etc. Runx2 is important for bone growth plate formation. Runx2 mediates several protein- protein interactions that are connected with bone formation. Similarly there are well established markers for chondrogenesis such as aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP) etc. Also several micro RNA’s have been shown to impact protein synthesis and Chondrogenic differentiation. For instance, miR-145 is shown to have a negative impact on chondrogenesis Mouse based experiments have also confirmed that the presence of growth factor beta 3 (TGF-β3) promoted chondrogenic differentiation. The role of miR-145 and many chondrogenic marker genes such as (Col2a1), (Agc1), cartilage oligomeric matrix protein (COMP) and (Sox9) are being studied intensely and results clearly suggest miR-145 as a controlling factor of chondrogenesis. (Yang B, 2011)
However we are still a long way in the treatment of arthritis and more research is needed to quicken the development of cartilage regeneration therapy. This would help us understand chondrogenic proliferation differentiation and their maturing in to hypertrophic chondrocytes. Understanding the biological mechanisms and the various signalling pathways that underlie chondrogenic differentiation of MSCs is vital to developing effective cartilage regenerative therapy. The current research work is focussed on finding potentially novel markers for chondrogenesis as this would speedup the work in the design and development of regenerative therapy for osteoarthritis which is currently an intractable condition.
Markers across the Stages of Chondrogenesis
A brief overview of the various stages of chondrogenesis would provide better insight into the process. Already from previous studies we know that several markers are known to be specific for the various stages of chondrogenesis. For instance, the Sox9, COL2A1 (IIa) , Ncad, tnc , Ncam1 are some of the markers that are highly expressed in proliferative mesenchymal chondrocyte cells. A low expression of Ptc1, Fgfr3 and the NKX3-2 was indentified during this stage. (Zuscik et.al ) Sox9 belongs to the family of transcription factors. This protein is usually used as a marker for cell differentiation along the stages of development. The Col2a1 belongs to the fibrillar Collagen family and the gene controls the production of apha 1 chain of type II collagen. The Ncam1, a cell adhesion molecule, belongs to the immunoglobulin (Ig) superfamily. In the highly proliferative flat columnar chondrocyte cells there is an observed over expression of NKX3-2 , Ptc1, Fgfr3 while there is low expression of Vegf, Runx2 and Osx. Sox9 and Col2a1(IIb) and Agc1 were also found to be common markers at this stage of mesenchymal cell condensation. (Zuscik , 2008) The aggrecan gene (Agc1) belongs to the proteoglycan family and the encoded protein constitutes an important part of the extra cellular matrix. The Fgfr3 gene belongs to the fibroblast growth receptor family and is identified to play a vital; role in chondrogenesis. Mutations of this gene is an identified cause for skeletal dysplasia. The prehypertrophic chondrocyte stage is marked by increased expression of Ihh and Fgfr3. Studies have also revealed expression of Col2a1(IIb), Agc1 and Col10a1. (Lefebvre & Smits, 2005) A relatively high expression of Runx2, Vegf and Col10a1 is observed in the hypertrophic chondrocytes. The vascular endothelial growth factor (Vgf) belongs to the family of platelet growth factors. Runx2 belongs to the family of transcription factors known as RUNX. These markers were chosen based on their high P values in the array expression database suggesting their important role in the chondrogenesis process. (Array Expression Database).

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Fig 1: The expression of different markers during different stages of chondrogenesis. Zuscik et.al (2008)

Research into MSC derived chondrocyte cell constructs while encouraging, however, are not functionally comparable to differentiated chondrocyte cells because it is not easy to mimic the actual cellular environment. Currently researchers, who are bent on finding genetic therapies for degenerative disorders such as arthritis, are focusing on how phenotypic transition impacts the mechanical function of these cells. Understanding the molecular basis for these functional differences between chondrogenically differentiated MSC’s and tissue engineered chondrocytes is therefore vital to further progress in the treatment of degenerative and crippling disorders such as osteoarthritis. In this study we use the array expression database extensively to obtain a variety of chondrogenic and osteogenesis specific gene expressions. Some of these genes and transcription factors are already well researched and identified to be key to chondrogenesis. Already studies have identified some of the key markers by using experimental observations of MSC seeded and chondrocyte seeded constructs and periodic observations of the expression profiles over several days.
AIM
This research aims to identify gene markers that are involved in both cartilage as well as bone differentiation by thoroughly studying the gene expressions involved with chondrogenesis and osteogenesis. This is done by screening the Array express database for all genes related to bone formation and cartilage formation. This would be followed by thoroughly searching research material pertaining to the selected markers. The research hopes to highlight the signalling pathways that are involved with genes responsible for osteogenesis or chondrogenesis. Also by including a brief explanation of the various stages of chondrocytic differentiation and the associated markers this research aims to understand the signalling pathways and the end products of the various markers involved in each stage. It is hypothesized that such a comprehensive study of genes related to chondrogenesis and osteogenesis and the action mechanisms including the upregulation and down regulation of vital mediators involved in the modulation of cartilage and bone differentiation would help find new and novel markers.

Method
To identify information pertaining to various important gene markers concerned with chondrogenesis and osteogenesis the array express database (http://www.ebi.ac.uk/arrayexpress/) of ‘European Bioinformatics’ was used for searching for chondrogenic specific and osteogeneic specific genes. Of the hundreds of markers that were listed in Arrayexpress only fifty each were included for further research. These genes are listed in the tables. Several transforming growth factors including Bone Morphogenetic Proteins (BMP) such as BMP 12, 13 and 14 were listed and results suggest a positive correlation between the expression of these genes and chondrogenesis. The P values listed in the gene database was used as a guide in selecting the 50 genes as it relates to their expression levels gathered from several assays performed by researchers. Based on the P values the selected genes were tabulated and these 50 genes, are further screened to exclude markers that are already well established. Finally only one gene is picked up from each table (chondrogenesis and osteogenesis) and explored further.
Since complex biological processes involve subtle interaction between lots of genes and protein molecules involving many mediators and transcription factors, one of the main ways to improve our understanding of these processes is to start with a subset of related genes and then to explore them for further and more thorough analysis using available literature that are derived from direct laboratory experiments. The current research purely relies on such a strategy and the idea is to search for a novel gene as a marker that has not been studied thoroughly giving scope for further exploration. For example one of the recent research studies showed that polyhydroxyalkanoates (PHA) induce chondrogenesis and formation of chondrocytic extra cellular matrix (ECM). This implies the possibility of using PHA for therapeutic research for osteoarthritis. Also, miR-29a and miR-29b are found to directly affect chondrocyte differentiation. (Yan et.al, 2011) Research is also focusing on genetic manipulation of the MSCs to promote chondrogenetic differentiation. In particular, adenovirus mediated expression of the human fibroblast growth factor 2 (hFGF-2) and its effect on chondrogenesis are being studied. (Cucchiarini, et.al 2011) One recent study that followed the approach discussed above is Huang et.al. (2010)

Huang et.al (2010) found that there were 1202 genes which were highly expressed in mesenchymal seeded constructs by day 28 when compared to undifferentiated MSC’s. The study also reported that 730 of these genes were similarly expressed in the chondrocyte derived constructs by the same 28 day period. Genes such as COMP and SERPINA1showed significant over expression (300 times) between undifferentiated MSC (M0) compared to the MSC construct cells M(28). Also, the expression level of Chondroadherin was > 200 time at M(28) compared with M(0), while other well known genes such as aggrecan, Collagen types II were (>40-fold at M (28) compared with M(0). Some 317 genes were found to be under expressed in the MSC chondrogenesis compared to the chondrocyte derived constructs. In all, this study which used real time PCR analysis found that around 252 genes were under expressed and 72 genes over expressed during MSC chondrogenesis. Further analysis of 18 of these genes revealed that 14 of these (for example, Calpain 6 (>30), PRG4(>25), EVA1(>15), Thrombospondin 4 (>15), etc, were well expressed in chondrocytes while they were poorly expressed in MSC derived cells. Furthermore, 4 genes were found to be down regulated in chondrocytes while they were up-regulated in the MSC cell lines at day 28. These include Leiomodin 1,(<-2) Caspase 4 (<-2), Homeobox (<-5), Fas.(<-5) ( Huang et.al, 2010) Such comparative studies wherein both the MSC seeded cell constructs and the chondrocyte seeded cell constructs are analyzed, reveal a wealth of information about the genes that are both up-regulated and down regulated and provide us the vital clues as to the important markers that are involved in the conversion into chondrogenic phenotype. This study provides the opportunity to examine these critical factors in more detail and to help us expand out understanding of the complex biomechanics involved in chondrogenic differentiation.

The Two Selected Markers (MIA and IL-1β)
This research strives to identify new gene markers that are specific to chondrogenesis by using the expression profile from previous studies reported in the array express database and by screening of appropriate literature from the pub med database for related research. It is already well established that collagen 2, and other proteoglycans are some of the major extracellular matrix components. In the present study, however, the quest was to research more about some of the less studied genes that could potentially be novel markers. The exercise involving screening of over 50 genes was done to pick out markers that are less thoroughly studied and yet identified as potential biomarkers for cartilage formation and bone formation respectively.

In this study, melanoma inhibitory activity( MIA) was selected because it is a unique protein that is synthesized in the body only by the chondrocytes and the malignant melanocytes. Also research is only slowly progressing on the role of MIA in chondrogenesis. It makes an excellent candidate as the main marker for chondrogenesis among the 50 other genes that were screened. The unique secretion of this marker by the chondrocytes would constitute an excellent biomarker for chondrogenesis. A simple ELISA test is all required for the identification for the secreted MIA , which would be an ideal marker for chondrogenesis. Previous studies that focused on MIA as a potential marker for chondrogenesis have also suggested positive results.
Bone formation involves the complex interplay of osteoprogenitors and several cytokines that interact in the recruitment and the regulation of these precursor cells. The activity of one of the important bone growth factors (BMP) is itself mediated by several protein molecules. For instance, Runx2 is one of the well known transcription factors involved in regulating the activity of the BMP. Many other cytokine molecules are thought to impact the osteoinductive ability of BMP’s. Diemerization of specific receptors by the activity of BMP is crucial for its osteoinductive ability. The Smad1/5 signalling pathway is critical for osteogenesis. BMP activity leads to the phosphorylation of SMAD1/5 and promotes the osteogeneic differentiation. However this BMP activated SMAD pathway is well controlled by several BMP antagonists including pseudo receptors, cytoplasmic binding protons etc. Understanding the significant other novel molecules that control the activity of the BMP’s is vital in the development of clinical therapies for accelerating bone repair and growth.

While lot is known about the BMP and its role in inducing osteogenesis there is also research into other cytokines and their mediating roles in osteogenesis. Recently some studies reported the antagonist noggin, its suppression and the activation of the BMP induced osteogeneic differentiation. (Wan et.al, 2007) Currently more research is focussed on finding other cytokines that might play an active role in osteogenesis. For the present study Interleukin-1β is selected as the novel marker because it has high expression and it has been not been thoroughly explored. Also the new pathways by which Interleukin-1β induces bone mineral deposits (other than ALP activity) provides us new outlook into understanding the osteogeneic differentiation and bone formation.

The rest of this paper will thoroughly research these two genes as important biomarkers for chondrogenesis and osteogenesis. A thorough research of these markers would help us better understand the transition into chondrogenic or osteogenic phenotypes which would in turn help us find novel genetic therapies for degenerative conditions such as osteoarthritis.
Discussion
MIA and Chondrogenesis
MIA is highly expressed during chondrogenesis. When HMSC cells are in the TGFβ medium there is an over expression of MIA as could be gathered from the following diagram. The following graph illustrates the secretion of MIA in human mesenchymal cell chondrogenesis. @amiWeber et.al (2007) studied the expression of MIA as a sole marker for chondrogenesis by using hMSC cells cultured under appropriate protocols to stimulate chondrogenesis. The hMSC cells for this study were obtained from an adult donor and the cells were cultured in DMEM-LG along with 10% solution of fetal calf serum (FCS). As a control, they also grew monolayer cultures. The researchers also optimized the cell culture factors including changing of the feeding density to promote faster cell doubling and effective chondrogenic differentiation. This allowed them to study chondrogenic differentiation up to 37 population doublings. Then the researchers measured the amounts of MIA in the passages (6, 12 , 15, 18 and 25) by harvesting the cells in each of these passages into four pellets. MIA- ELISA testing was performed for the different experimental specimens. Then the amounts of MIA in these pellets across different passages were compared with the MIA levels that were gathered from the measurements of the unstimulated monolayer culture references. Using dying and Neubauer counting chamber the researchers were able to measure the cell count. The data indicated that there was a significant increase (P<0.03) in MIA expression up to the 32nd population doubling. In particular, the MIA levels in the pellets under study were between 0.14+- 0.08 and 0.78 +-0.34 while the levels observed in the unstimulated monolayer cultures ranged between 0.03+-0.03 and 0.12+-0.06 ng/d/ 10.000 cells. The following table indicates the comparative MIA levels. (Weber at.al, 2010) This study shows that MIA expression could be used a marker for chondrogenesis@14_001

Another study by Sanyal et.al (2003) studied the effect of MIA on periosteal chondrogenesis. Since successful induction of chondrogenesis is critical for successful periosteal transplantation for cartilage damage this research was significant. The expression of MIA starts during the early stages of chondrogenic differentiation and continues well into mature cartilages. The researchers studied the mRNA specific temporal spatial expression of MIA in periosteal explants. For this they cultured the periosteal explants in conditions that were favourable for chondrogenic differentiation. A significant increase in the levels of MIA was noticed from day 7. In fact, MIA levels shot up to 40 times between days 7 and 10 of the experimental period. Furthermore, the researchers also found the temporal expression pattern of MIA to be similar to that of collagen type IIB mRNA. Safranin-O Staining indicated the colocalization of MIA mRNA and collagen type IIB mRNA clearly suggesting an active role for MIA in periosteal chondrogenesis.(Sanyal, 2003) This is research is particularly important from the perspective of treatment for cartilage damage as there is a potential for MIA to be directly used as a cytokine into human bone joints.
A more recent study by Schmid et.al (2010) studied the role of MIA in chondrogenesis using a mice knockout model. For the purpose of this study the researchers used a disruption of the MIA/CD-RAP gene and studied chondrogenesis in both in vivo and in vitro models. The authors noted significant chondrocyte generation and proliferation but there was delayed differentiation. Also, electron microscopic studies of the mice cartilage revealed defects in the fibrillar structure indicating that MIA/CD-RAP is essential for the proper formation of cartilage. Further analysis of the cartilage tissues from the knockout mice revealed a down regulation of the nuclear RNA binding protein p54nrb. Since p54nrb is known to be a modulator for Sox9 activity and since SOX9 regulates the differentiation and transition into hypertrophic chondrocytes (Lueng et.al, 2011), the researchers inferred a strong connection between MIA expression and chondrocyte differentiation. In a subsequent study the researchers showed that MIA , though not directly involved in chondrocytic differentiation, played an important role in influencing the differentiation mediated by (TGF)-β3. In particular, MIA negatively influenced osteogenic differentiation triggered by BMP2. This study clearly showed that MIA has a clear role in cartilage formation and more importantly chondrogenic differentiation.
To further study the effect of MIA in osteoarthritis the researchers induced OA in MIA/CD-RAP knockout mice by detaching the medial meniscotibial ligament. The researchers also used wild type mice(WT) as a control model to analyze the role of MIA. PCNA staining was used to observe the proliferation of chondrocytes. There was a marked increase in chondrocyte proliferation among the MIA/CD-RAP knockout mice compared to the control group or the WT. To more thoroughly analyze the effect of the loss of MIA/CD-RAP and its impact on chondrocyte proliferation the flow cytometric cell cycle analysis using BrdU/Hoechst quenching method was adopted. It was found that in MIA knockout mice chondrocytes upregulated Cyclin D2, a gene which is responsible for activating Cyclin dependant Kinase (CDK), which controls cell cycle progression from G1 to S phase. Cyclin D2 therefore accelerates the cell cycle and increases the proliferation of chondrocytes which is why the OA knockout mice models showed higher proliferation of chondrocytes compared to WT mice resulting in an enhanced calcified cartilage layer. Therefore it could be inferred that MIA, by mediating the Cyclin D2,controls the proliferation and triggers the differentiation. Thus this study showed that p54nrb by acting as a mediator of MIA/CD-RAP controls the SOX9 transcriptional activation of Col2a1. Schmid et.al (2010).The study also showed that the absence of MIA led to an increase in proliferation of cartilage precursor cells, an important conclusion that might give new direction and focus to the development of cartilage regeneration therapies.

Another study by Schubert et.al (2010) focussed on understanding the transcriptional mechanisms of the MIA/CD-RAP gene and how it influences chondrogenic differentiation. Using both in vivo and in vitro models the researchers analyzed the molecular functions of MIA/CD_RAP that are particularly connected with chondrogenic differentiation. In particular, the researchers wanted to unravel the underlying molecular mechanisms pertaining to the MIA mediated inhibition of the osteogeneic differentiation promoted by bone morphogenetic protein (BMP). The point of this research was to shed more light into the significance of MIA for chondrogenic differentiation. To rule out the possibility of MIA’s direct physical interaction with BMP an ELISA test and Co-immunoprecipitaiton tests were done. The results clearly negated the possibility. Since BMP is connected with the initiation of the SMAD as well as the ERK signalling pathway, the researchers were then prompted to analyze the effect of MIA in either of these pathways. Western blotting tests for phosphorylation of SMAD1/5 for chondrocytes that were treated with BMP alone and with BMP and MIA/CD-RAP, indicated that MIA did not significantly impact SMAD1/5 regulation. Therefore the researchers focussed on the ERK signalling pathway. The following graph illustrates the effect of SMAD and ERK signalling on chondrogenic differentiation. .

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Fig 4 : ‘Influence of SMAD and ERK activity on HMSC differentiation’ Cells co-treated with either
dorsomorphin (2 µM) or chordin (2 µg/ml) (Schubert et.al, 2010)

ERK activity has been known to negatively impact chondrogenic process. Previous studies have already confirmed that ERK down regulates cartilage related gene expression. The researchers performed western blot test again on dedifferentiated chondrocytes by treating it with MIA (100 ng/ml). A considerable drop in ERK activity was observed. Treatment with MIA resulted in increased expression of aggrecan, a protein molecule that is associated with cartilage. Furthermore when treated with JBS5 antibody there was a marked decrease in aggrecan expression. Both SMAD and ERK activity result in an upregulation of the osteogeneic markers such as osteopontin and osteocalcin. This led the researchers to assume that MIA by interfering with the ERK signalling pathway decreases the osteogenic differentiation. By binding with integrin α5 MIA reduces its availability and consequently hampers the ERK signalling. Thus by negative regulation of integrin α5 MIA promotes chondrogenic differentiation. This research also attests to the potential role of MIA in the treatment of cartilage damage. MIA introduced into the cartilage matrix could help to maintain the chondrogenic phenotype by binding with and reducing the availability of the integrin α5 for the ERK activation. (Schubert et.al, 2010)
Berg et.al (2010) studied the expression of MIA/CD-RAP in the synovial fluids of horses to understand the connection between MIA and joint diseases. For the purpose of the study the researchers collected synovial fluid samples from 25 healthy horses. They also induced inflammatory arthritis in 5 horses and MIA concentrations from their synovial fluids were also measured. Elisa tests were performed to measure the MIA concentrations. For healthy horses the concentration remained at 8.2–52 ng/ml while there was a significant decline in MIA concentrations in those horses that were induced to have inflammatory arthritis. This study of MIA/CD-RAP concentrations obtained from horse joints clearly indicated a large variation proving that MIA is a clear marker for cartilage differentiation. ( Berg et.al, 2010)

One recent study by Kuijk et.al (2010) focussed on identifying soluble serum biomarkers for early identification of treatment efficacy in patients undergoing therapy for psoriatic arthritis. For this purpose the researchers studied several cartilage and bone biomarkers and their serum level changes post treatment with adalimumab, which is a standard treatment for psoriatic arthritis. 24 patients were randomly chosen to be treated either with adalimumab 40 mg for a week or with placebo over a four week period. Measurements of serum levels of markers such as CPII, PINP (type 1 and 2 procollagen) , MIA, MMP-3, cartilage oligomeric matrix protein (COMP) and osteocalcin (bone formation) were undertaken first at baseline then 4 weeks and at 12 weeks. The results revealed a great reduction (P<0.005) in serum matrix metalloproteinase (MMP)-3 levels while the levels of the marker remained unchanged in the placebo group. Also an increase in serum MIA level (P<0.005) was noticed in the treatment group while there was no such increase in the placebo group. These changes in serum marker expressions could be noticed from the following diagram.

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The other markers did not exhibit such a change. This study showed that MIA and MMP-3 could be useful as novel biomarkers in the identification of the efficacy of therapy for psoriatic arthritis.

Interleukin-1β and Osteogenesis

One recent study by Ferreira et.al, (2010) researched the inflammatory cytokine Interleukin-1β and its role in osteogenesis. To study the role of this cytokine in inducing osteogeneic differentiation in HMSC’s the researchers measured the expression of bone markers at protein and RNA, measured the bone mineral deposits and analyzed the IL -1β pathway. To the surprise of the researchers they found that IL -1β induced osteogeneic differentiation in HMSC’s but did not cause the expression of the familiar bone markers.
For this study the researchers gathered MSC’s from the intramedullary canal of patients at the Brigham’s Women’s hospital and the cells were cultured in low glucose medium along with 10% Fetal bovine serum. The culture medium was changed the next day with 50 μg/mL ascorbic acid-2-phosphate, 10mM β-glycerophosphate and 10 ng/ml recombinant human IL-1β and the cells were maintained in this growth medium for 21 days. This period provided enough time for osteoblastic differentiation. The researchers measured osteoblastic differentiation by measuring the calcium deposits, calorimetric measurement of Alkaline phosphotase activity of the monolayer cells, and the transcription factors that are usually associated with osteogenesis. By using Alizarin red and biochemical assay the researchers were able to detect calcium deposits at the end of the study period. Throughout the study the researchers also calibrated mRNA expression levels for several genes that are normally identified during the osteogeneic differentiation process. These include Bone sialoprotein, type I collagen, ALP, osteocalcin, osteopontin, runt-related transcription factor 2 (runx2) etc. These mRNA gene expressions were documented 3, 710, 14, 18 and 21 days after the addition of 10 ng/ml recombinant human IL-1β.
Furthermore, immunoblotting tests were used to detect the activation of various signalling pathways such as mitogen-activated protein kinase (MAPK), Phosphoinositide 3-kinase (PI-3K) and SMAD by the activity of Interleukin-1β. By using inhibitors specific to these various pathways the researchers were able to identify the role of these pathways in the osteoblastic differentiation triggered by IL- 1β. Real time PCR was used to identify the expression of bone specific genes during the various stages of the study period. In rhBMP-2 treated cells the expression of runx1, type 1 collagen, osteocalcin, bone sialoprotein and osteopontin were detected. However, rhBMP-2 treated cells did not express ALP, one of the important genes responsible for mineralization and this explained why rhBMP-2 treated cells could not form bone deposits even though they triggered the expression of the usual osteogeneic genes. The dexamethasone treated cells expressed ALP , runx2, type 1 collagen but did not express other osteogeneic transcripts. Interestingly enough the IL-1β treated cells did not express a single transcript related to osteoblasts. The following figure shows the ability of IL-1β and Dexamethasone treated cells in creating bone mineral matrix while the control cells and BMP-2 treated cells did not induce mineralization. This suggests that BMP, though it enables osetoblast transcripts, does not play a direct role in bone mineralization. .

@adxThe fact that IL-1β induces bone mineralization without triggering the expression of osteogeneic transcripts and that it does not induce ALP activity which is considered central to the bone mineralization process provides an opportunity of further study of this cytokine and its action mechanisms in inducing bone deposits. The following graph clearly indicates the ALP activity triggered by the various cell culture mediums. It is very visible from the graph that there is little if any ALP activity in the cells cultured under IL-1β medium. This opens up new scope for further research in to bone mineralization. @0

Wehling et.al (2010) is another comprehensive study that focussed on the possible role of IL-1β in the inhibition of chondrogenesis and promoter of osteogenesis. Since IL-1β is an inflammatory cytokine that floods the region during an injury the researchers of this study were particularly focussed on the effects this would have in cartilage regenerative effects in inflamed joints. For the purpose of this study the researchers focussed on two inflammatory cytokines namely tumor necrosis factor-α (TNF-α) and IL-1β and their impact on the chondrogenic potential of HMSC’s. There researchers obtained the HMSC’s from the femoral intermedullary canal and the cells were cultured in a low glucose invitrogen medium along with 10% Fetal bovine serum for 2 weeks and then further cultured in a 10 ng/ml recombinant human (rh)FGF-2 growth medium. The culture cells were then transduced with Ad.srIκB. Real time PCR was used to identify the expression of chondrocytic transcripts. Also ALP activity was assessed using p-nitrophenyl phosphate as the substrate medium. The researchers found that both IL-1β as well as (TNF-α) had a pronounced inhibitory effect on chondrogenesis. There was also a significant increase in NF-κB activity which the researchers measured using the Ad.NF-κB-Luc reporter construct. The researchers inferred that Il-1β activity might be mediated by NF-κB. When they introduced srIκB, a known inhibitor of NF-κB, there was a increase in chondrogenic response. Furthermore the study revealed that the expression of Matrix metalloproteinase-13 was induced by IL-1β mediated by NF-κB. The study also attested to the previous findings that IL-1β inhibited alkaline phosphotase activity suggesting the existence of alternative bone mineralization pathways other than ALP. This study assumes great significance from the perspective of cartilage regeneration therapy in trauma cases as the therapies for cartilage growth could be impaired under inflamed conditions where IL-1β and other inflammatory cytokines are over expressed. (Wehling et.al, 2010)

From the other perspective, this study also chimes with the findings of recent research that IL-1β promotes osteogeneic differentiation and inhibits chondrogenesis. From a clinical approach, understanding the workings of these new cytokines, related intercellular mediators and their synergistic effects on osteogenesis and chondrogenesis will help to speed up the development of regenerative therapies for people suffering from cartilage and bone disorders.

Conclusion
Osteoarthritis presents as an intractable disease impairing the normal functioning of people. An aging population is only adding to our problem as more and more people suffer from such chronic conditions. At the moment regenerative therapy is the only ray of hope for the millions of people suffering from osteoarthritis and other forms of cartilage and bone disorders. Naturally occurring chondrogenesis and osteogenesis are governed by a complex mesh of genes, proteins, mediators and transcriptional factors that interact dynamically. Understanding some of these important biochemical mechanisms and the vital pathways are crucial for our success in speeding up the research into regenerative therapies. The present research was motivated by the goal of finding new and novel markers that might play an active role in chondrogenesis and osteogenesis. To this end, the current work focussed on indentifying potential gene markers based on their high expression values pertaining to chondrogenesis and osteogenesis gathered from the numerous gene assays reported and grouped in the Array express database.
Two markers were selected based on their high expression scores and their less explored status making them ideal candidates for future research. The selected markers, MIA for chondrogenesis and IL-1β for osteogenesis, were further explored by searching peer reviewed literature that examined their roles in cartilage and bone formation. This helped in identifying new pathways and mediators that could lead us to furthering our future research in chondrogenesis and osteogenesis. For instance the present study found that MIA has a negative effect on the ERP signalling. From a clinical perspective introducing MIA into the cartilage matrix could help to maintain the chondrogenic phenotype by binding with and reducing the availability of the integrin α5 for the ERK activation. This would improve and speed up cartilage repair. Similarly, the role of IL-1β in osteogenesis, and in particular, its pathway that does not include the familiar ALP activity suggests the existence of alternative, less studied bone mineralization pathways that could be further researched and developed in the context of fracture healing therapies. Therefore the study of Interleukin-1β provides a new avenue for osteogeneic research that could be very useful in the bone repair and regeneration therapies.

References
1) Amano K, Hata K, & Muramatsu S et.al (Apr 2011), Arid5a cooperates with Sox9 to stimulate chondrocyte specific transcription, Molecular Biology of the CELL 15;22(8): pg 1300-11
2) Anja K. Bosserhoff, Reinhard Buettner (2003), Establishing the protein MIA (melanoma inhibitory activity) as a marker for chondrocyte differentiation, Biomaterials, 24, 3229-3234.

3) Alice H. Huang, B.S.,Ashley Stein, B.S. et.al (2010), Evaluation of the Complex Transcriptional Topography of Mesenchymal Stem Cell Chondrogenesis for Cartilage Tissue Engineering, Tissue Eng, 16(9): 2699–2708.
4) Alice H. Huang, Megan J. Farrell, and Robert L. Mauck, (2010), Mechanics and Mechanobiology of Mesenchymal Stem Cell-Based Engineered Cartilage, Journal of Biomechanics, ;43(1) pg. 128-36

5) Arno W. R. van Kuijk1, Jeroen DeGroot,& Rishma C. Koeman et.al, Soluble Biomarkers of Cartilage and Bone metabolism in early proof of concept trials in Psoriatic arthritis: Effects of adalimumab vs. Placebo, PLoS ONE 5(9)

6) Christian Weber, Stephanie Gokorsch & Peter Czermak, (2007), Chondrogenic differentiation of human Mesenchymal stem cells during multiple subcultivation, ESACT proceedings, Volume 4, Part 1, 29-34.

7) Derrick C. Wan, Jason H. Pomerantz & Lisa J. Brunet et.al (2007), Noggin suppression enhances in vitro osteogenesis and accelerates in Vivo bone formation, The Journal of Biological Chemistry Vol. 282, NO. 36, pp. 26450–26459

8) Elisabeth Ferreira, James W. Wells, Christopher H. Evans, (2010), Inflammation adn Osteogenesis: a paradoxical effect of interluekin1 on the osteogeneic differentiation of human, bone marrow derived mesenchymal stem cells. Harvard Medical School , viewed Apr 18th 2012, < http://www.inflammationresearch.org/2010vanarman/ferreira.pdf>

9) Yang B, Guo H, Zhang Y et.al (2011), MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS One. ;6(7):e21679

10) Yan C, Wang Y, Shen XY (2011), MicroRNA regulation associated Chondrogenesis of Mouse MSCs grown on Ployhydroxyalkanoates. Biomaterials. 2011 Sep;32(27):6435-44

11) Cucchiarini M, Ekici M, Schetting S, Kohn D, Madry H.( Aug 2011) , Metabolic activities and chondrogenic differentiation of human Mesenchymal stem calls following recombinant adeno associated virus- mediated gene transfer and over expression of fibroblast growth factor 2. Tissue Eng Part A. 17(15-16) pg. 1921-33.

12) Jonason JH, Xiao G, Zhang M, Xing L, Chen D. (2009) Post-translational Regulation of Runx2 in Bone and Cartilage. J Dent Res

13) L.C. BERG*, J. LENZ &, M. KJELGAARD et.al (Jan 2010) , Cartilage- derived retino acid-sensitive protein in Equine synovial fluid from healthy and disease joints, Equine Veterinary Journal, Vol 40, Iss 6. Pg 553-557.

14) Leung VYL, Gao B, Leung KKH, Melhado IG, Wynn SL, et al. (2011), SOX9 Governs Differentiation Stage-Specific Gene Expression in Growth Plate Chondrocytes via Direct Concomitant Transactivation and Repression. PLoS Genet 7(11)

15) Michael J. Zuscik, Matthew J Hilton, Xinping Zhang et.al (2008), Regulation of Chondrogenesis and Chondrocyte differentiation by stress, J Clin Invest. Vol 118, Issue 2.
16) N. Wehling,G.D. Palmer,C. Pilapil et.al (2010), Interleukin and tumor necrosis factor α inhibit chondrogenesis by human mesenchymal stem cells through NF-kB dependent pathways. Arthritis & Rheumatism, 60: 801–812

17) R Schmid, S Schiffner & A Opolka et.al (Nov 2010), Enhanced cartilage regeneration in MIA/CD-Rap deficient mice, Cell Death and Disease, Vol 1(11).

18) Sanyal A, Clemens V, Fitzsimmons JS, Reinholz GG, (2003), Induction of CD-RAP mRNA during periosteal chondrogenesis, Journal of Orthopaedic research, 21 (2), pg 296-304

19) Thomas Schubert, Jacqueline Schlegel & Rainer Schmid et.al, (2010), Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage –derived retinoic acid- sensitive protein (Mia/CD-RAP), Experimental and Molecular Medicine, Vol 42 , 3. Pg 166-174.

20) Ve´ ronique Lefebvre and Patrick Smits, (2005), Transcriptional control of Chondrocyte fate and Differentiation, Birth Defects Research (Part C) 75:200–212

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