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Research Article
 

Gene Expression Profile of Synovial Cells in Experimental Post-Traumatic Arthritis of Knee in Swine



O. Baatartsogt, K. Choi, P.K. Mandal, Hee-kyong Lim, Guan-Hao Li, Hong-Sik Kong, Dae-Hyun Hahm, Chi-Ho Lee and Jun-Heon Lee
 
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ABSTRACT

To get insights into pathological pathways of Post Traumatic Arthritis (PTA) this study was done on gene expression profile of synovial cells of knee using gene chip analysis developing experimental post traumatic arthritis (ETA) in swine. ETA was induced by transection of the Anterior Cruciate Ligament (ACL) of left knee in 3 piglets. Articular cartilage and synovial tissues were obtained after 0, 2, 5 and 8 weeks for histopathologic examination. Synovial cells collected after sacrificing the piglet at 8 weeks, were used at 5 passage for gene expression profiling using Affimetrix Gene Chip. Histopathologic examination showed overt chronic inflammation indicating the development of ETA. Through genome analyses it was observed that 87 known genes were up-regulated and 76 known genes were down regulated. By analyzing, it was found that many genes with differential expression are related to inflammation, immune response, lipid binding, cell adhesion, growth activity and muscle development. The present study provided an insight into the TA related gene expression pattern. The genome analysis of synovial cells provided us new candidate molecules which may be useful to understand the pathogenesis of Post Traumatic Arthritis (PTA). The established porcine model may serve as in vivo disease model for further research on traumatic arthritis to elucidate molecular pathogenesis.

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  How to cite this article:

O. Baatartsogt, K. Choi, P.K. Mandal, Hee-kyong Lim, Guan-Hao Li, Hong-Sik Kong, Dae-Hyun Hahm, Chi-Ho Lee and Jun-Heon Lee, 2009. Gene Expression Profile of Synovial Cells in Experimental Post-Traumatic Arthritis of Knee in Swine. Biotechnology, 8: 70-77.

DOI: 10.3923/biotech.2009.70.77

URL: https://scialert.net/abstract/?doi=biotech.2009.70.77
 

INTRODUCTION

Post traumatic arthritis is the syndrome of osteoarthritic joint degeneration that develops after joint injuries (Buckwalter and Brown, 2004). A knee fracture or severe tears of the knee ligaments may damage the articular cartilage over time, causing knee pain and limiting knee function. Post Traumatic Arthritis (PTA) develops after articular injury, is one of the most common causes of secondary osteoarthritis (D`Lima et al., 2001). Recent estimates suggest that PTA is responsible for 12% of 21 million cases of OA in US (Brown et al., 2006). Primary osteoarthritis predominantly affects the elderly, but PTA usually affects younger people, especially athletes who are likely to have joint injury due to vigorous physical activities (Buckwalter and Brown, 2004; Buckwalter and Martin, 2004). Older patients of osteoarthritis can often be treated effectively with reconstructive procedures and restriction of activities but these approaches are not acceptable for young and athletic people and also have poor long-term outcomes (Ahlberg and Henricson, 1981; Ellingsen and Rand, 1994; Sullivan et al., 1994). Therefore, management of these cases of PTA is a difficult clinical problem.

The pathogenesis of PTA has not been fully understood yet. Clinical experiences show that joint injuries including intra-articular fracture may induce considerable incongruities and ligament tears that may lead to unstable joint, increase the risk of progressive joint degeneration that causes PTA (Buckwalter and Lane, 1997; McKinley et al., 2004a; Trumble and Verheyden, 2004). Basic experiments have shown how mechanical forces damage articular surfaces and how it respond to the injury (McKinley et al., 2004b; Trumble and Verheyden, 2004). Direct impact damage at the time of injury (Vrahas et al., 2004; Ewers et al., 2001), increase of cartilage contact stress resulting from residual articular incongruity (D`Lima et al., 2001; Trumble and Verheyden, 2004; Newberry et al., 1998; Lefkoe et al., 1993) and pathologic loading resulting from articular instability (McKinley et al., 2004b; Delamarter et al., 1990) are thought as the major etiologic factors of PTA. However, there is no method for prediction or an early diagnosis of PTA incases of trauma or injury of joints.

The synovial membrane is a thin lining within the joint cavity that is responsible for maintaining normal joint function and homeostasis. Synovial cells are the primary source of articular hyaluronic acid and other glycoproteins such as lubricin. There is growing evidence that proinflammatory cytokines such as interleukin-1 (IL-1) or tumor necrosis factor α (TNF α) are up-regulated in the joint following trauma and thus play an important role in the pathogenesis of PTA (Guilak et al., 2004; Furman et al., 2006) similar to their role in primary OA (Goldring, 2000; Fernandes et al., 2002). A recent study reported lower levels of IL-1a and higher levels of anti-inflammatory cytokines IL-4 and IL-10 in MRL/MpJ mice which were found to be resistant to PTA by intraarticular fracture model study (Ward et al., 2008). But incase of PTA the role of synovial cells is not yet known.

Recently, genomic analysis has gained great interest for the determination of biochemical processes involved in diseases (Fung et al., 2000). The comparative characterization of gene expression patterns in tissues has the potential to serve as the basis for new diagnostic tools and in designing of disease specific therapies (Sinz et al., 2002). Genomics is gaining popularity in the research on arthritis such as rheumatoid arthritis, osteoarthritis using synovium or cartilage (Ward et al., 2008, Furman et al., 2007; Justen et al., 2000; Adreas et al., 2008; Ashwell et al., 2008; Haup, 2007). However, no detailed study on genome analysis of synovial tissue from PTA has been done. Therefore, we have studied the gene expression profiles of synovial cells of the knee joint in Experimental Traumatic Arthritis (ETA) in swine using gene chip analysis

MATERIALS AND METHODS

Inducing ETA: This study was conducted at School of Biotechnology, Hankyong National University, South Korea during 2006. Three male hybrid piglet of 2 months old, the first generation of female Yorkshire and male Landrace were used in this study. Surgery was done under sterile conditions using atropine (subcutaneously) 0.1 mg kg-1 and ketamine (intramuscularly) 5 mg kg-1.

Fig. 1: Operative procedures of experimental Anterior Cruciate Ligament (ACL) transection of left knee

Longitudinal skin and fascial incisions were made over the anteriolateral side of the left knee (Fig. 1). The Anterior Cruciate Ligament (ACL) was exposed by a capsulotomy, then it was transected at its tibial insertion area with a surgical blade and cartilage sample including synovial tissue were obtained. The joint capsule and skin incision were closed with nylon sutures and a dressing of povidone-iodine was applied to the wound. After surgery, penicillin 500 KIU was injected intramuscularly daily for a week to prevent post-operative bacterial infection and the piglet was allowed unrestricted activities in a clean cage. The right knee was used as control for comparison.

Follow-up experiments: At 2 and 5 weeks after surgery, cartilage tissues of the ACL- transected knee were obtained by needle biopsy, which was performed under the same anesthesia as described earlier, using ultrasonographic guidance. The piglet was sacrificed at 8 weeks, normal and experimental (ETA) knee joints were disarticulated for taking tissue samples to confirm the development of ETA by histopathologic examination and for genome analysis.

Histopathologic examination: All samples taken from synovium during the experimental period from both knee were immediately placed in 70% ethanol, then specimens were fixed in 10% neutral buffered formalin at room temperature for 24 h and embedded in paraffin. Thin sections were cut from the paraffin blocks, mounted on the slide and stained with Hematoxylin and Eosin as per standard procedure. Histologic study was done to confirm the development of PTA and comparison was made with the control specimen.

Isolation and culture of synovial cells: The synovial tissues collected from both knees after 8 weeks by sacrificing the piglet were used to prepare tissue samples for genome analysis. Synovial cells were isolated by digesting synovial tissue with 2.5 mg mL-1 collagenase type II (Gibco BRL) in Dulbecco`s modified eagle`s medium (Gibco BRL) with 10% heat-inactivated fetal bovine serum for 2 h at 37 °C, centrifuged at 1500 rpm for 10 min, washed twice in Phosphate Buffer Saline (PBS). Synovial cells were cultured in Dulbecco`s modified eagle`s medium (Gibco BRL) L-glutamine (4.00 mM), (4500 mg L-1) Glucose, 0.1 mM sodium pyruvate, 1% antibiotic-antimycotic (100X) (Gibco BRL), 1% MEM non essential amino acids solution 10 mM (Gibco BRL) and 10% heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. After overnight culture, the non-adherent cells were removed. The synovial cells were used in this study at five passage.

Affymetrix gene chip analysis: The generation of Gene chip data from the normal and ETA synovial cells was performed by Seoulin Bioscience Corporation (Seoul, Korea). Specifically, total RNA (about 5 μg) from the normal and ETA synovial cells was used for labelling.

Probe synthesis from total RNA samples, hybridization, detection and scanning were performed according to standard protocols from Affymetrix. Briefly, cDNA was synthesized using the One-Cycle cDNA Synthesis Kit (Affymetrix). Single-Stranded (ss) cDNA was synthesized using Superscript II reverse transcriptase and T7-oligo (dT) primers at 42 °C for 1 h. Double-stranded (ds) cDNA was obtained using DNA ligase, DNA polymerase I and RNase H at 16 °C for 2 h, followed by T4DNA polymerase at 16 °C for 5 min. After cleanup using a Sample Cleanup Module (Affymetrix, Santa Clara, CA), ds cDNA was used for in vitro transcription (IVT). cDNA was transcribed using the Gene chip IVT Labeling Kit (Affymetrix) in the presence of biotin-labeled CTP and UTP. Then the biotin-labeled IVT-RNA was fragmented and hybridized to the porcine genome GeneChip array at 45 °C for 16 h, according to the manufacturer`s instructions. After hybridization, the arrays were washed in a GeneChip Fluidics Station 450 with a non-stringent wash buffer at 25 °C, followed by a stringent wash buffer at 50 °C. After washing, the arrays were stained with a streptavidin-phycoerythrin complex. After staining, intensities were determined with a Gene chip scanner, controlled by GeneChip Operating Software (GCOS; Affymetrix).

RESULTS

Experimental post-traumatic arthritis was induced in 3 pigletss by anterior cruciate ligament transection and studied the gene expression by gene chip analysis. The results are described below

Ultra-sonography: Ultrasonography performed at 2 and 5 weeks post ACL-transection to study the pathologic changes in the joint and to confirm arthritis. It was observed that there was no change after 2 weeks, however, inflammatory changes were observed at 5 weeks which confirmed the development of arthritis due to ACL transaction (Fig. 2).

Histology: Articular cartilage at 5 weeks post-ACL transection showed a proliferative state and this change might be due to the response of articular chondrocytes to some kind of abnormal stimulations such as mechanical overload, inflammatory process etc. Articular cartilage at 8 weeks post-ACL transection showed findings similar to that of cartilage at 5 weeks and chondrocytes were relatively stabilized (data not shown). Joint synovium at 8 weeks post-ACL transection showed overt chronic inflammation that supported the development of ETA (Fig. 3).

Fig. 2: Ultrasonographic findings of experimental post-traumatic arthritis of swine knee joint following anterior cruciate ligament transection (a) Right knee at 2 weeks, (b) Left knee at 2 weeks and (c) Left knee at 5 weeks

Fig. 3: Joint synovial cells with H and E stain from 8 weeks post-ACL transected swine knee. (a) Normal x 100 and (b) post anterior cruciate ligament transection synovium x100

Gene chip analysis: The gene expression profiles of synovial cells using affymetrix Gene chip® Porcine Genome Array on 24123 probes including 124 control probes were analyzed and results are explained (Fig. 4). Genome-wide microarray analysis showed differential expression of distinct genes which are known in porcine synovial cells. However, a large number of differentially expressed genes have not yet been described in porcine genome database. GCOS and RMA statistical analysis showed reproducibly differentially expressed genes. The differentially expressed genes were functionally annotated with reports from literature. Visualization of these differentially expressed genes by hierarchical clustering demonstrated that expression of normal and ETA synovial cells were similar to each other. Gene Ontology (http://www.geneontology.org; GO) annotations were determined for each gene product represented. GO terms are consistent descriptions of gene products on terms of the biological processes they are involved in, the cellular components in which they exist and molecular functions they perform.

Fig. 4: (a) Affymetrix Gene Chip of normal and (b) experimental traumatic arthritis, synovial cells

A total of 1696 genes were found to have differential (2 fold change) expression of which 1057 genes were up-regulated and 639 genes were down regulated. Among the up-regulated genes 87 were known (Table 1) and among the down regulated genes 76 were known (Table 2). A total of 163 known genes were found to have differential expression. From the porcine genome database they may be categorized for molecular function, biological process and cellular component.

Table 1: List of genes up-regulated in synovial cells of porcine experimental traumatic arthritis

Table 2: List of genes down-regulated in synovial cells of porcine experimental traumatic arthritis

Important differentially expressed genes: The important genes with differential expression belongs to immune response, inflammatory response, apoptosis, genes of cell adhesion, genes of lipid binding, genes of growth factor activity and genes of muscle development. The important up-regulated expression of genes observed in synovial cells are FAS receptor, IL 18, TLR 4 and secreted phospho protein I related to immune and inflammatory response. Important down regulated genes are IL 1α, IL 6, IL 7, Mx protein, proteosome subunit LMP 7, beta 2-microglobulin, RANTES protein, monocyte chemoattractant protein 1, oligoadenylate synthetase and chemokine ligand 2 related to immunity and inflammation.

Important genes with up-regulated expression in synovial cells are related to cell adhesion (link protein precursor, protocadherin 11 X-linked, putative cartilage oligomeric matrix protein and scavenger receptor class B member 1), lipid binding (lipopolysaccharide-binding protein), growth factor activity (keratinocyte growth factor, bone morphogenic protein) and muscle development (troponin T slow type isoform sTnT1). Other genes found to have down regulation in synovial cells are related to cell adhesion (type I transmembrane endothelial adhesion molecule, collagen type V alpha 3), lipid binding (plasma phospholipid transfer protein, adipocyte fatty acid binding protein, similar to apolipoprotein A-I and lipoprotein lipase), growth factor activity (fibroblast growth factor 2) and muscle development (muscle-specific intermediate filament design).

DISCUSSION

This is the first study that has determined the genome-wide molecular expression pattern of synovial cells from traumatic arthritis in a porcine model and thus provided comprehensive insight into joint degeneration in TA. In case of osteoarthritis, earlier the diagnosis, greater the chance of preventing irreversible damage. It has been reported that traumatic arthritis often leads to secondary osteoarthritis (D`Lima et al., 2001). Hence, to understand the pathogenesis at molecular level the gene expression profile in experimental PTA was studied.

In the present study experimental traumatic arthritis was successfully induced by anterior cruciate ligament transection in three piglets after 5 weeks and the same has been confirmed by ultrasonographic and histological evidences. Histologic analysis demonstrated progressive joint degeneration with loss of proteoglycan in articular cartilage and subchondral bone thickness was also observed, therefore ACL transection generated a reproducible and clinically relevant joint injury that progressed to osteoarthritis like changes (Furman et al., 2007). If the ligaments are torn or attenuated, an increase slide or rotation occur, causing excessive sheer force to the articular cartilage, then progressive chondromalic change is inevitable. A torn ACL may cause rotatory instability and it may be in single or multiple planes (Trumble and Verheyden, 2004).

In this study, large number of important genes were found that showed differential expression using Affymetrix Genechip analyses on 24123 probes including 124 control probes many of them are related to immune and inflammatory response system. Besides, synovial cells showed a distinct expression of genes associated with lipid binding, apoptosis, cell adhesion, growth factor activity.

The association of instability with PTA has been reported by McKinley (2004a, b) and Delamarter et al. (1990) and the presence of instability and incongruity severely affect cartilage loading. Patients sustaining ligament injuries in the knee without fracture also have a significant incidence of PTA (Daniel et al., 1994; Gillquist and Messner, 1999; Kannus and Jarvinen, 1989). Patients sustaining ACL tears, followed up prospectively for more than 5 years, had a significantly increased incidence of PTA (Daniel et al., 1994). In a review of ACL tears, injured knees had a 10-fold increase of degenerative changes, compared with uninjured limbs (Gillquist and Messner, 1999).

The search for differentially expressed genes is an important means to find markers of disease for diagnosis, to understand pathological pathways and for treatments. There are recent efforts towards this direction for osteoarthritis and PTA using synovium or cartilage (Furman et al., 2007; Justen et al., 2000; Ashwell et al., 2008). As far as we know it has not been tried before for understanding the PTA using synovial cells in porcine model, hence it is difficult to compare our observations.

Differential expression of genes associated with growth, apoptosis, cell adhesion and inflammation in in vitro study of human synovial cells derived from rheumatoid arthritis are reported and synovial cells reflected the disease-related pathophysiology (Haup, 2007). A latest report confirmed the differential expression of genes associated with matrix molecules, iron and phosphate transport, protein synthesis, skeletal development, cell proliferation, lipid metabolism and the inflammatory response in chondrocytes from a porcine injury model (Ashwell et al., 2008). The differential expression and proliferation of synovial cells observed in the present study may be an attempt to repair themselves. Expression of adhesion molecules facilitate the trafficking of T-cells and other leucocytes into the synovial cells and play a major role for initiation of joint degeneration (Justen et al., 2000).

In summary, a total of 163 known genes were found to have differential expression in ETA in porcine knee. It was observed that the differential expression of genes related to inflammation, immune response, apoptosis, lipid binding, growth factor and muscle development. Result from this study has contributed further evidence that synovial cells play important role in the pathology of arthritis and for recovery from the condition.

CONCLUSION

PTA induced by transection of ACL of porcine knee showed expression of cytokines related to inflammation, immune response and lipid binding. The molecules already reported to be involved in TA is confirmed and new molecules which may be useful in further research is reported. Thus porcine model was established as useful for clinical research related to PTA. Further, characterization of the gene products may help to understand pathological mechanisms in post traumatic arthritis.

ACKNOWLEDGMENTS

This study was partly supported by the GRRC program of Gyeonggi province (2008(4-2), Development of Techniques for Applications of complex materials in Bone and Joint disease and Growth of Bone of Pig using natural and chemical products).

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