Abstract:The development of crack patterns is a serious problem affecting the durability of orthopedic implants and the prognosis of patients. This issue has gained considerable attention in the medical community in recent years. This literature focuses on the five primary aspects relevant to the evaluation of the surface cracking patterns, i.e., inappropriate use, design flaws, inconsistent elastic modulus, allergic reaction, poor compatibility, and anti-corrosiveness. The hope is that increased understanding will open doors to optimize fabrication for biomedical applications. The latest technological issues and potential capabilities of implants that combine absorbable materials and shape memory alloys are also discussed. This article will act as a roadmap to be employed in the realm of orthopedic. Fatigue crack growth and the challenges associated with materials must be recognized to help make new implant technologies viable for wider clinical adoption. This review presents a summary of recent findings on the fatigue mechanisms and fracture of implant in the initial period after surgery. We propose solutions to common problems. The recognition of essential complications and technical problems related to various approaches and material choices while satisfying clinical requirements is crucial. Additional investigation will be needed to surmount these challenges and reduce the likelihood of fatigue crack growth after implantation.Keywords: fatigue crack growth; fracture; internal fixation; alloys; absorbable materials; design flaw; elastic modulus; compatibility; allergic reaction
Cultural Heritage constructions of twentieth century consist largely of mortar and concrete substrates. These concrete structures have suffered different types of decay processes. One of the most widely used consolidants is the Tetraethoxysilane (TEOS), which forms the basis of most existing commercial strengthening agents to protect porous building materials against deterioration. A novel, non-toxic strengthening and protective agent for mortar and concrete substrates was synthesized in a one-pot sol-gel procedure, incorporating in TEOS, Polydimethyl siloxane (PDMS), and nanoparticles of synthesized calcium oxalate (CaOx). PDMS provided hydrophobicity and reduced surface tension that causes cracks on the surface of produced xerogel. The synthesized nanocomposite both in sol and xerogel form was assessed with a variety of analytical techniques (FTIR, XRF, SEM, Optical Microscopy, Dynamic Light Scattering, Thermogravimetric analysis). The excellent physical properties of the produced colloidal solution of the nanocomposite, such as low viscosity and density, allow a penetration up to 2 cm from the surface in the treated cement mortars. This involved improvement of the mechanical and physical properties, such as the dynamic modulus of elasticity and increased water repellency. The treated cement mortars exhibited well-preserved aesthetic surface parameters and significant maintenance of the treatment. Furthermore, no harmful byproducts were identified indicating the nanocomposite compatibility to the siliceous and carbonate nature of the treated cement mortars.
Use 1 μl of bacterial solution as a template for PCR using a standard Taq enzyme in a 25 μl reaction, with 1 μM T7 oligo, 0.2 mM dNTPs and the following cycling conditions: 95°C 50s, 52°C 30s, 72°C 90s for 25 cycles. For a good transcription reaction in the next step, the PCR reaction should yield ~200ng/ul of product.
Typically, 24 hours post-injection is a good starting point for a good RNAi effect. For many genes, the strength and penetrance of RNAi phenotypes are increased in progeny laid more than 24 hours post-injection, especially for genes with a strong maternal contribution. It is a good idea to do a time course to find the optimum time of scoring post-injection, looking for a time when the phenotype is strongest and most penetrant. For some genes, shorter times post-injection will give a stronger effect, particularly for genes with a zygotic but not a maternal function. If antibodies are available, it can be helpful to stain progeny at different times post-injection to see when the protein is maximally reduced. In order to maintain progeny production at later time points, mate the injected hermaphrodites with N2 males after injection. Using an RNAi supersensitive strain can increase the strength and penetrance of phenotypes. rrf-3 and eri-1 both display sterility at 25°C and smaller broods than N2 at lower temperatures (Simmer et al., 2002; Kennedy et al., 2004), but this can be overcome by mating with N2 males after injection, as they are cross fertile at all temperatures (J. Ahringer, unpublished).
Use 7μl of the PCR reaction mixture (above) for in vitro transcription with T7 RNA polymerase (e.g., Thermo T7 polymerase: TOYOBO (#TRL-201)) in a 100μl scale reaction. Both sense and antisense RNAs are transcribed from the PCR product in this single reaction. An annealing step is unnecessary. Typical yields are ~0.4 μg/μl of RNA (40μg total).
L1s can be used instead of L4s in either of the above protocols. An advantage of using L1s is that some phenotypes can be scored in the fed worms instead of the progeny, allowing an easily scored synchronized population to be used. However for some genes, inherited maternal product will be sufficient for gene activity, preventing induction of a phenotype in the fed worms. Also, as many genes are required at multiple times in development, different phenotypes may be seen when using L1s compared to L4s. For example, RNAi of some genes induces sterility of the fed L1s whereas L4 feeding induces embryonic lethality of the progeny. This will preclude scoring of progeny for these genes if L1s are fed. In contrast, using L1s is beneficial if the assay is for any form of lethality (e.g., sterility, larval lethality, or embryonic lethality).
A key feature of these protocols is that they include quantitative quality control assessments at each stage of library construction. Thus the success of each part of the procedure can be verified along the way, and any unsuccessful batches can be discarded immediately if a failure occurs. The production of a successful deletion library can be essentially guaranteed as long as the individuals constructing the library are committed to meeting all the quality control criteria. Every failure we are aware of resulted from individuals failing to take the quality control targets seriously and forging ahead without meeting them.
It is recommended to start by just setting up one batch and processing it from beginning to end to make sure all the technical steps work before beginning full-scale production. Proceed to full-scale production by setting up five batches, one per day for five days, and then spending five days harvesting those batches, one per day. Repeat this procedure once more to produce enough batches for a complete library. At the end, complete the processing of the pooled DNA preps for all the batches. This schedule distributes the work over a three-week period in a fairly even fashion.
Before scaling up to production level batches of 24 plates, run pilot worm cultures to make sure the target of 1500 starved L1 F2 animals per well is being met or exceeded. During the piloting, ensure that 20 F1 animals are placed in each well. Also, try culturing worms by varying the concentration of bacteria up and down within a two-fold range. It is critical to empirically optimize the culturing step, and also to empirically determine that the target of 1500 F2 per well is met. Please note that psoralen-mutagenized animals grow very differently from non-mutagenized animals. It is thus critical to optimize growth conditions using animals mutagenized with psoralen following the same procedure as will be used for the library. This will also provide an opportunity to optimize the UV dose during psoralen mutagenesis.
The yield should be at least 3.4 μg of genomic DNA, contaminated by a small amount of RNA. It is absolutely essential that a good yield is achieved, so piloting the DNA preps and quantitating the yield is essential before proceeding at production scale. The yield of some of the preps can be checked by running a bit (3 μl) on an ethidium bromide stained agarose gel and comparing to a larger, purer prep of genomic DNA of known concentration (determined by OD260). When things go well, it is possible to achieved yields of up to 15 μg.
The goal of the screening procedure is to identify pooled DNA samples that contain DNA from a deletion mutant for a gene of interest. By PCR amplifying a region of the gene of interest and running the products on a gel, in principle the smaller deletion amplicon will be observed on the gel as a band with faster mobility than the larger wild-type amplicon. However, deletion mutant DNA is present at only 1 part in 4,000 in the pooled DNA sample, so the challenge is to increase the abundance of the deletion amplicon to the point where it can be visualized on a gel.
A template that has suffered an internal deletion has a small advantage over the wild-type template during each cycle of PCR due to the shorter length of the deleted template. Suppose that the chance of completing an extension across a deleted template is 10% better than for the wild type. After 35 cycles of amplification there will be a 28-fold enrichment of the deleted amplicon relative to the wild-type amplicon. After 70 cycles the enrichment will be 790-fold. As can be seen from this example, a key to detecting rare deletion mutations is to send the PCR through a very large number of productive cycles. Even if the PCR machine is programmed to run through 70 cycles, if the amplification reactions run out of reagents and reach their endpoints after only a small number of cycles, rare deletion amplicons will not be enriched sufficiently to detect them. The goal is thus to set up PCR reactions under relatively inefficient conditions that will allow amplification to continue productively for a full 70 cycles. The poison primer method, described below, is a simple way to do this. 2b1af7f3a8