Gene Expression Analyses Of Subchondral Bone In Early Experimental Osteoarthritis By Microarray

Osteoarthritis (OA) is a complex degenerative joint disease that affects millions ofmiddle-aged and older individuals. It is characterized by progressively cartilageerosion, osteophyte formation, subchondral bone modification, and synovialinflammation, following alterations in the biomechanical and biochemical propertiesof the joint.OA is generally considered to be a cartilage disease, but increasing evidenceindicates that it is also a bone disease. Changes in the subchondral bone may occurprior to the onset of cartilage degeneration. Subchondral bone consists of thesubchondral bone plate and the underlying trabecular bone and bone marrow space.Alterations of the subchondral bone increase with the progression of OA. It has beenshown that subchondral bone is an effective shock absorber and nutrients or cytokinescan be transported from the subchondral bone to the overlying cartilage via clefts orchannels in the tidemark. Subchondral bone cells influence cartilage metabolism;however, whether changes in subchondral bone precede or follow cartilagedestruction is still debated. It is, therefore, crucially important to understand themolecular characteristics of subchondral bone changes in vivo, especially during theearly stages of OA. This information would be important because any identifiedalterations in this period could contribute to the development of new diagnosticmarkers or therapeutic targets for OA.With the development of microarray technology, changes in the expression levels of thousands of genes can be examined simultaneously, and integral analysis of thedysregulated genes can be performed to obtain information regarding the cell-levelpathogenic mechanisms of OA. Furthermore, microarray data can be used to discovernovel molecular diagnostic markers and therapeutic targets. A small number of studieshave reported the gene expression profiles of articular cartilage and bone from humanor animal OA samples. These reports have provided important diagnostic markers andtherapeutic targets for OA, however, gene expression profiles and the chronology ofOA-induced changes in the subchondral bone that underlie cartilage degenerationremain poorly understood.It is currently impossible to obtain adequate subchondral bone samples fromhumans to study the initiation and early stages of OA. Moreover, studies of ex vivocultures from osteoarthritic subchondral bone have not precisely identified themolecular changes that occur in vivo. It is, therefore, necessary to examine the earlystages of OA in subchondral bone using animal models. By performing medialmeniscectomy and medial collateral ligament transection, a surgically induced ratmodel of OA was used to address the questions posed by this study. This animalmodel is consistent with the post-traumatic OA in humans and can be used for thegenetic analysis of OA.For the first time, we describe a simple and effective method for isolating RNAfrom the subchondral bone from the knee joint in a rat model of OA. This procedurecan be used for genetic analyses and effectively avoids RNA degradation in thesubchondral bone samples during the operation. In addition, this work the first studyto use microarray technology to elucidate the time-course of the molecular changesthat occur in the subchondral bone just beneath the damaged cartilage in the earlystages of experimental OA.1. Objective1) To study the gene expression profiles of subchondral bone in the rat OA model atmultiple post-surgery time points.2) To elucidate the time-course of the molecularchanges that occur in the subchondral bone in the early stages of experimental OA.3)To analysis differentially expressed genes and pathways in the subchondral bonerelated to OA and find some important diagnostic markers and therapeutic targets forOA. 2. Material and MethodsPart1Rat model of osteoarthritisMethods: Ninety10-week-old male Sprague-Dawley rats were used in the study. Theanimals were equally divided into two groups: the experimental group (E-Group) andthe sham-operated group (S-Group). Rats in the E-Group underwent open surgery.This surgery involved both a medial meniscectomy and a medial collateral ligament(MCL) transection. The S-Group rats were subjected to a sham operation in which asimilar incision was made but no changes were made to the medial meniscus or themedial collateral ligament. Following the surgery, all animals were immediatelytreated with penicillin and tramadol. The animals were sacrificed at1,2, and4weekspost-surgery. Fifteen animals were used for each time point in each treatment group.Five animals were used for histological analysis and immunohistochemistry; theothers were equally used for subchondral bone separation and total RNA extraction.Results: The rat model of OA was validated by gross morphological and histologicalanalysis. Similar to the disease progression observed in humans, the grossmorphological and early histological events associated with OA appeared to occur in atime-dependent manner in the animal model. These findings confirmed that a medialmeniscectomy and medial collateral ligament (MCL) transection effectively inducedOA-like early changes in the cartilage.Part2Subchondral bone separation and total RNA extractionMethods: The femur condyles frozen in liquid nitrogen were placed in insulatedboxes, which were filled with liquid nitrogen. The articular cartilage and epiphysealplate tissue of the femur condyle sample were cleared away with a micro-electricburnish instrument.Total RNA from the subchondral bone was extracted using anE.Z.N.A Total RNA Kit Ⅱ.Results:(1) After the sepharose electrophoresis, the RNA extracted with thecombination of Trizol protocol and glass fiber filter method showed clear18S/28Sbands and integrated18S/28S RNA ratios, as well as5S band, which suggested theintegrity of RNA. However, RNA samples extracted with traditional Trizol protocolcame out with blurry18S/28S bands but bright5S band, which suggested high degeneration of RNA.(2)RNA samples extracted by both two protocols showed highpurity.Part3Study of gene expression profiles of subchondral bone of OA bymicroarrayMethods: The RNA was further purified using RNeasy spin columns. One microgramof total RNA was isolated from the subchondral bone of5E-Group and5S-Groupknee joints for each time point. The RNA was amplified and labeled using the AgilentQuick Amp One-Color labeling kit. The RNA was then hybridized to Agilent WholeRat Genome Microarray (Agilent,4x44K, G4131F). Following microarrayhybridization and washing, the processed slides were scanned with an Agilent DNAmicroarray scanner. The raw gene expression data were extracted from AgilentFeature Extraction Software (Version10.5.1.1) and imported into Agilent GeneSpringGX software (version11.0) for further analysis-DE genes analysis, Unsupervisedhierarchical clustering, Gene ontology (GO) analyse and Patway analysis.Results:1)2,234DE genes was dentified at1week post-surgery,1,944DE geneswere identified at2weeks post-surgery,1,517DE genes were identified at4weekspost-surgery.Specifically, the number of up-regulated and down-regulated genes weresimilar at1and2weeks post-surgery. At4weeks, however, a large number of DEgenes with down-regulated expression (approximately67.7%) were identified.2)Clustering of the results at each time point resulted in E-group samples clusters andS-group samples clusters, which indicated that the E-Group samples haddistinguishable gene expression profiles relative to the S-group samples at each timepoint.3) The distributions of the annotated DE genes involved in the three functionalcategorizations at each time point were similar. Many common representativebiological processes were found both at1and2weeks post-surgery. The onlycommon biological process to all three time points was bone development, whichdemonstrate that subchondral bone remodeling occurred throughout OA pathogenesisin this model.Of the annotated DE genes involved in the cellular component category,the greatest number were predominantly involved in the extracellular componentcategory at both1and2weeks post-surgery. At4weeks, however, the greatest number was involved in the intracellular component category. The classifications ofthe molecular functions that were enriched for in the set of DE genes were quitedifferent among the three time points.4) Pathway analysis revealed that fiverepresentative pathways were involved in subchondral bone of OA at1weekpost-surgery, including cytokine-cytokine Receptor Interaction Pathway, ECMReceptor Interaction Pathway, Endocytosis Pathway, TGF-beta Signaling Pathwayand Chemokine Signaling Pathway, two representative pathways at2weekspost-surgery, including Tryptophan Metabolism Pathway and TGF-beta SignalingPathway, three representative pathways at4weeks post-surgery, including Valine,Leucine and Isoleucine Biosynthesis Pathway, Porphyrin and ChlorophyllMetabolism Pathway and Ribosome Pathway.Part4Validation of the microarray gene expression data by real-timepolymerase chain reaction (PCR) and immunohistochemical analysisMethods: To quantitatively examine gene expression in subchondral bone RNAsamples, ten genes involved in osteoclast, osteoblast and bone remodeling in OAobtained from the microarray analysis were selected as targets. GAPDH was used asthe internal control.To validate the microarray gene expression data at the proteinlevel, Mmp3and Aspn proteins were selected for immunohistochemical analyses.Results:1) Gene expression patterns for Mmp3, Acp5and Igf1precisely matched theexpression patterns obtained from microarray analysis. Gene expression patterns forPostn, Aspn, Bmp5, Tgfβ1and Tnfsf11matched the expression patterns obtainedfrom microarray analysis at two time points. Gene expression patterns for Ihh and Alponly matched the expression patterns obtained from microarray analysis at one timepointsexpression.2) Mmp3protein expression levels were markedly increased in theE-Group compared to the S-Group at1and2weeks whereas no significant differencewas observed between these two groups4weeks post-surgery. These results are incomplete agreement with the expression level of this gene determined using real-timePCR.Nevertheless, differential Aspn protein expression between the E-and S-Groupswas not observed in the subchondral bone at any time point.3. Conclusions 1) Medial meniscectomy and medial collateral ligament (MCL) transection effectivelyinduced OA-like early changes in the cartilage.2) It is a simpler, reliable and practical method that micro-electric burnish implementis used to clean up all the articular cartilage and epiphyseal plate tissues in liquidnitrogen and obtain subchondral bone sample from knee joint of rat.3) Time-dependent molecular changes occurred in osteoarthritic subchondral bone.4) Alterations of the subchondral bone increase with the progression of OA andsubchondral bone may be a therapeutic target for OA.5) The DE genes and pathways found in subchondral bone of the early stages ofexperimental OA may be new important diagnostic markers and therapeutic targetsfor OA.