Introduction

The recent surge of interest and information in genetic research, sparked by the Human Genome Project, have fostered the development of new techniques that have impacted basic molecular biology and clinical medicine. Information gained from these studies has enabled researchers to identify genes which are involved in specific disorders and diseases. By identifying which genes are expressed, researchers can better understand the biochemical pathways of disease. This basic level of understanding of the disease process has enormous potential for the treatment and prevention of disease. Furthermore, this information provides tremendous opportunities in the field of gene-based therapies, as highly specific, small molecules can be developed to target genes involved in the biochemical process for specific illnesses. If research indicates that a gene or group of genes is activated by the onset of a particular pathology, a therapy can be developed that will interfere with the biochemical pathway and prevent that activation, thus rendering the disease asymptomatic.

A variety of molecular biology techniques have enabled us to delve into the human genome. Some of these techniques are limited in the amount of genes that can be simultaneously studied. Northern analysis and RNase protection assay (RPA) can be used to study upregulation of gene expression, but require relatively large amounts of sample RNA and are restricted to investigations of a few genes at each time. Reverse transcriptase- polymerase chain reaction (RT-PCR) requires small samples, but the results are also restricted in the number of genes that can be studied, and this technique is affected by contaminations. In contrast, transcriptional profil-ing using microarray technology permits examination of the effects of experimental manipulation on the expression pattern of vast numbers of genes and gene families that control cell responses.(1) Depending on the system, anywhere from 500 to 10,000 genes can be studied simultaneously in each experi-ment. In addition, microarrays require only a small amount of sample RNA (~2 g) through the use of a primer mixture, spe-cific for every gene on the array, as opposed to non-specific oligo dT priming used in other molecular techniques. The microarrays can use either fluorescent or radioactive labeling to visualize the genetic expression. The intensity of the expres-sion can be quantified using software, and the transcriptional profile can be used to illustrate the gene pathways effected by the disease process or experimental treatment. With this new technology, it is possible to study the temporal expression of genes as they are activated in the transcription process .(1,2) Technology is currently readily available to permit investigators to develop their own arrays or disease-specific arrays or chips.

The cDNA microarray consists of a charged nylon mem-brane on which specific DNA sequences that encode the indi-vidual genes are immobilized in a matrix. The sample RNA is reverse transcribed to yield the complementary DNA (cDNA). This cDNA is then replicated through PCR by using primers specific for all the genes on the array, to produce a cDNA probe that can be labeled either radioactively or with fluorescence. This probe is hybridized to the nylon membrane and the tran-scriptional profile can be visualized by exposure to radiographic film or by fluorescence microscopy, depending on the labeling. Array specific image analysis software is used to quantify the expression and allows for temporal and/or treatment com-parison of transcription. In this report, we have presented our preliminary studies using microarrays to dissect the mechanism of wear debris-mediated osteolysis, in a common orthopaedic pathology effecting joint replacements (see Figure 1).(3) In the present studies, we have used microarray technology to characterize the pattern of gene transcriptional profiles in macrophages cultured with ultrahigh molecular weight polyethylene (UHMWPE) and titanium alloy (TiAlV) wear debris. (3) It is well documented that particulate wear debris phagocytozed by macrophages initiate an inflammatory response leading to osteolysis and aseptic loosening in joint replacements. These results demonstrate distinct patterns of gene response to particle treatment and provide initial insights into the complex regulatory pathways responsible for particle-mediated cell activation.

Figure 1: Particulate wear debris generated at the articulating surfaces is phagocytized by macrophages in the interfacial tissue and secrete inflamma-tory mediators and enzymes such as IL-1 , IL-1 , TNF-�Í, prostaglandin E 2 , collagenase, and gelatinase. These mediators stimulate osteoclastic bone resorption in the peri-implant area, which in turn leads to osteolysis and aseptic loosening 4,5,6 .

Monocytes were harvested from 400 ml peripheral blood using sequential Percoll gradients. (4) After an overnight adher-ence depletion, the monocytes were cultured with submicron UHMWPE (mean diameter 1.6 Ö m; 66% < 1 Ö m), TiAlV (mean diameter 1 Ö m) or with no debris as controls .(4,7) Based on prior optimization studies, the particle dose represented two times the surface area of cells, equivalent to challenge with approximately 40 particles per cell. The cell culture was inter-rupted at 30 min, 1 h, 4 h, 8 h, and 24 h, and the total RNA extracted.

Figure 2: Schematic Transcription Profiling Process

Figure 3.: A comparison of representative segments of the gene array repre-senting gene expression from non-stimulated (NS, left), UHMWPE-stimulated (center) and TiAlV-stimulated (right) monocytes after 4 hours in culture. Unmarked pairs of spots represent other genes either up- or down-regulated by different particles

Results

Both UHMWPE and TiAlV particle treatments produced marked increases in multiple pro-inflammatory cytokines including IL-1 , TNF-�Í, and IL-8. Figure 3 illustrates hybridization patterns for IL-8, TNF-�Í, and IL-1 after particle treatment. mRNA levels of many other genes of interest showed striking changes in steady-state levels, including message for several different chemokines and kinases involved in the regulation of cell responses. Most striking were the differences in the diversity and level of gene expression in UHMW-PE compared to TiAlV.

Discussion

These studies show the utility of the microarray technology for exploring the complex regulatory pathways and gene products involved in particle induced macrophage activation. This technology has the advantage of providing semi-quantitative screening of multiple classes of genes, including the gene regulation of cytokine expression, cell proliferation, and apoptosis. Furthermore, by comparing data from several different time periods, we have illustrated the temporal activation of these genes. Of particular interest was the finding that the gene expression was critically dependent upon the composition of the challenging particles. This approach provides a powerful technology for dissecting the molecular mechanisms by which wear debris regulates transcription of cytokine genes, related pro-inflammatory mediators, and contributes to aseptic loosen-ing of orthopaedic implants. By identifying which genes are involved in aseptic loosening and osteolysis, we can begin to approach joint arthroplasty from a different perspective. One possibility is to geno-stratify patients and develop drug thera-pies based on the genes patients exhibit and the response observed in vitro. If the activity of a gene or group of genes is successfully identified, therapies can be synthesized to target that gene and suppress its expression or target its product and inactivate it. Through such clinical studies, class ifications can be made as to which geno-stratified group would be more appropriate with which biomaterial (or its modifica-tion), thus increasing the success of the joint arthroplasty and reducing the need for future revision surgeries. Similar strate-gies may hold promise for other areas of orthopaedics such as fracture healing and tumor classification and treatment. In the near future, transcriptional profiling holds distinct opportunities to improve patient care and treatment in orthopaedics.

Acknowledgments

The authors would like to thank Jennifer Kenney for her technical assistance. The study was supported by NIH Grant DK 46773 (SRG) and the Department of Orthopaedic Surgery, Massachusetts General Hospital.