By Pauline M. Doran
This quantity goals to explain in actual fact and intimately the main useful talents fascinated by cartilage tissue engineering. equipment are defined for isolation and growth of chondrocytes and stem cells; differentiation; synthesis and alertness of 3-dimensional scaffolds; layout and operation of bioreactors; in vivo trying out of engineered constructs; and molecular and practical research of cartilage cells and tissues. often used applied sciences are lined, in addition to newer advances in inspirational components comparable to ‘smart’ biomaterial improvement, novel bioreactor layout, -omics research, and genetic manipulation of matrix synthesis. the foremost techniques mentioned both underpin the growth already completed in cartilage tissue engineering or are indicative of the course of destiny examine within the sector. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, simply reproducible step by step laboratory protocols, and tips about troubleshooting and fending off identified pitfalls.
Authoritative and state of the art, Cartilage Tissue Engineering: tools and Protocols is an invaluable source that informs the clinical neighborhood in regards to the experimental paintings overlaying a wide diversity of targets for cartilage synthesis and regeneration.
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Extra resources for Cartilage Tissue Engineering: Methods and Protocols
Prepare a reagent master mixture for each primer pair. Each test reaction should contain 25 μL of Platinum® SYBR® Green qPCR SuperMix-UDG, 1 μL of ROX reference dye, 2 μL of forward primer, 2 μL of reverse primer and 10 μL of autoclaved Milli-Q water. Load 40 μL of master mixture per PCR reaction. Dilute aliquots of cDNA samples 1:100 with autoclaved Milli- Q water. Add 10 μL of each diluted sample to their Fetal and Adult Chondrocytes 35 esignated wells and seal using cap strips to prevent contamid nation and evaporation.
Each peak corresponds to the melting temperature of the amplified product (see Note 24). Plot efficiency curves as the measured increase in Ct after each 1:2 dilution (ΔCt) versus the logarithm (base 2) of the dilution factor. Calculate the amplification efficiency as the slope of the best linear fit of the efficiency curve (see Note 25). To compare the expression levels of one gene between different samples, the expression level must first be normalized to the expression level of β-actin or GAPDH within each sample according to the following equation: (1 + E HKG ) = (1 + EGOI ) Ct Ct HKG RGOI GOI where RGOI is the expression of the gene of interest (GOI) normalized to that of the housekeeping gene (HKG), EHKG and EGOI are the amplification efficiencies of the HKG and GOI target sequences, respectively, and CtHKG and CtGOI are the Ct values for the HKG and GOI, respectively.
Sterilize by autoclaving and store at 4 °C. Tissue dissection solution: 1 % (v/v) antibiotic solution in PBS. Type II collagenase stock solution: for cartilage tissue digestion. Dissolve type II clostridial collagenase (Sigma-Aldrich) in DMEM base medium at a concentration equivalent to 3000 U/mL for fetal cartilage, or 10 mg/mL (approx. 4300 U/mL) for adult cartilage (see Note 2). 2-μm single-use syringe filter. Aliquot and store at −20 °C for up to 1 year (see Note 3). 1 mL of antibiotic solution, and 1 mL of Type II collagenase stock solution made up to 10 mL using DMEM base medium.
Cartilage Tissue Engineering: Methods and Protocols by Pauline M. Doran