Background The applications for rapid prototyping have expanded dramatically over the last 20 years. In recent years, additive manufacturing has been intensely investigated for surgical implants, tissue scaffolds, and organs. There is, however, scant literature to date that has investigated the viability of 3D printing of surgical instruments. Materials and Methods Using a fused deposition manufacturing (FDM) printer, an army/ navy surgical retractor was replicated from polylactic acid (PLA) filament. The retractor was sterilized using standard FDA approved glutaraldehyde protocols, tested for bacteria by PCR, and stressed until fracture in order to determine if the printed instrument could tolerate force beyond the demands of an operating room. Results Printing required roughly 90 minutes. The instrument tolerated 13.6 kg of tangential force before failure, both before and after exposure to the sterilant. Freshly extruded PLA from the printer was sterile and produced no PCR product. Each instrument weighed 16g and required only $0.46 of PLA. Conclusions Our estimates place the cost per unit of a 3D printed retractor to be roughly 1/10th the cost of a stainless steel instrument. The PLA Army/ Navy is strong enough for the demands of the operating room. Freshly extruded PLA in a clean environment, such as an OR, would produce a sterile, ready to use instrument. Due to the unprecedented accessibility of 3D printing technology world wide, and the cost efficiency of these instruments, there are far reaching implications for surgery in some underserved and less developed parts of the world.

Changes in DNA methylation patterns are a common characteristic of cancer cells. Recent studies suggest that DNA methylation affects not only discrete genes, but it can also affect large chromosomal regions, potentially leading to LRES. It is unclear whether such long-range epigenetic events are relatively rare or frequent occurrences in cancer. Here, we use a high-resolution promoter tiling array approach to analyze DNA methylation in breast cancer specimens and normal breast tissue to address this question. We identified 3,506 cancer-specific differentially methylated regions (DMR) in human breast cancer with 2,033 being hypermethylation events and 1,473 hypomethylation events. Most of these DMRs are recurrent in breast cancer; 90% of the identified DMRs occurred in at least 33% of the samples. Interestingly, we found a nonrandom spatial distribution of aberrantly methylated regions across the genome that showed a tendency to concentrate in relatively small genomic regions. Such agglomerates of hypermethylated and hypomethylated DMRs spanned up to several hundred kilobases and were frequently found at gene family clusters. The hypermethylation events usually occurred in the proximity of the transcription start site in CpG island promoters, whereas hypomethylation events were frequently found in regions of segmental duplication. One example of a newly discovered agglomerate of hypermethylated DMRs associated with gene silencing in breast cancer that we examined in greater detail involved the protocadherin gene family clusters on chromosome 5 (PCDHA, PCDHB, and PCDHG). Taken together, our results suggest that agglomerative epigenetic aberrations are frequent events in human breast cancer. [Cancer Res 2008;68(20):8616-25]

position of guanine in a reaction that inactivates one MGMT molecule for each lesion repaired (reviewed in reference 24). Cells deficient in or depleted of MGMT are therefore sensitive to the mutagenic and cytotoxic effects of carcinogens and chemotherapeutic agents that produce O 6 -alkylguanine lesions (9, 12, 32). MGMT expression has been found to vary widely between normal and tumor tissues (3,10,30) and between individual cells within a tumor (21); additionally, 20% of human tumor cell lines lack MGMT activity altogether (8). Loss of MGMT expression is rarely, if ever, due to deletion, rearrangement, or mutation of the MGMT gene (27,31). This observation, taken together with the relative ease with which MGMT gene expression can be lost in cell culture, suggests that the gene is under epigenetic control (2, 23).The human MGMT gene possesses a CpG island, as defined by Gardiner-Garden and Frommer (13), which extends from approximately nucleotide (nt) 480 to 1480 of the 5Ј end of the gene and spans approximately 500 bases 5Ј and 3Ј of the transcription start site at nt 956 (nucleotide numbering based on references 15 and 26). Inappropriate 5-methylation of CpG cytosines within the MGMT CpG island is a likely epigenetic mechanism of MGMT inactivation. The existence of this mechanism is supported by the demonstration that a cell line lacking MGMT activity (MGMT Ϫ ) was capable of transcribing a human MGMT promoter-chloramphenicol acetyltransferase construct, leading to the conclusion that all of the necessary transcription factors were present in the MGMT Ϫ cell line. Additionally, in vitro methylation of the same MGMT promoter-chloramphenicol acetyltransferase construct prevented its transcription in transient-transfection assays (16). Finally, analysis of the MGMT CpG island promoter in glioma cell lines has provided perhaps the strongest support for CpG methylation and inactivation of the MGMT gene. Linker-mediated PCR analysis of the methylation status of individual CpG cytosines in the MGMT promoter of glioma cell lines with variable levels of MGMT gene expression showed that increasing levels of methylation are associated with corresponding decreases in MGMT expression (6).We have previously reported that the human multiple myeloma cell line 8226/S selected with verapamil and doxorubicin, either in parallel or serially, became phenotypically MGMT Ϫ (11). Reselection of 8226/S with verapamil alone led again to an MGMT Ϫ cell line, which we designated 8226/V. We have used this isogenic model to investigate the role of cytosine methylation in the control of MGMT expression. Analysis of the entire MGMT-associated CpG island, which includes the minimal promoter, the first untranslated exon, and the minimal enhancer (17), indicates that the loss of MGMT expression in 8226/V is associated with a marked increase in methylation of CpG cytosines within discrete regions that bracket the transcription start site. Furthermore, restriction enzyme accessibility assays showed that the MGMT ϩ 8226/S

Using an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes-the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to f100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion-epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion. (Cancer Res 2006; 66(22): 10664-70)

p53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2 0 -deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.