This method presents a way to create scaffolds derived from native tissue which can be used for cartilage regeneration. The main advantage of this technique is that components of native cartilage are preserved in the scaffolds that can promote cartilage formation and regeneration in vitro and in vivo. Although these method uses cartilage from equine stifle joints, cartilage from any other joints can be used as well.
To begin, obtain an intact cadaveric joint for cartilage isolation and remove the skin excess fat and muscle tissue as described in the accompanying text protocol. Next, define the location where the joint articulates by performing extension inflection of the joint. Make a horizontal incision to reach the joint cavity.
Next, make a vertical incision to open up the joint area. The joint cavity is filled with synovial fluid which will likely drip from the cavity when performing of correct incision. Continue to open up the joint completely by removing excessive fat, muscles and tendons, and by cutting through the tendons that keep the joint together.
Carefully inspect the cartilage for any macroscopic damage. If the articular cartilage does not have a glossier smooth appearance or if evidence, blistering, clefts or defects are present, discard the cartilage and start again. Use a sterile scalpel to remove the cartilage from the bone.
Cut all the way down to the subchondral bone to remove the deep zone cartilage. Collect the removed cartilage slices in 50 milliliter tubes containing previously prepared cartilage washing solution. While being processed, regularly drip cartilage washing solution on the cartilage to prevent the cartilage from drying out.
After collecting the cartilage from all of the areas of interest, snap freeze the cartilage slices in liquid nitrogen for five minutes. Then transfer the cartilage slices into 50 milliliter tubes and immediately placed the frozen slices in a freeze dryer. Lyophilize the cartilage slices for 24 hours with the freeze dryer.
When finished, store the freeze dried cartilage slices in a dry place at room temperature. For manual processing, pre chill a mortar and pestle with liquid nitrogen, then place the lyophilized cartilage slices in the mortar and submerge them and liquid nitrogen. Directly grind the samples by hand for approximately 45 minutes until they are sufficiently pulverized.
Alternatively, to grind the samples automatically using a milling machine, begin by pre cooling the grinding compartment of the milling machine by adding liquid nitrogen. Then add the lyophilized cartilage slices and mill the sample at its preset speed for a few seconds up to a minute. Make sure that all particles are ground and that no particles stay in the bottom of the grinding compartment.
Finally, follow along with the decellularization technique described in the accompanying text protocol prior to forming scaffolds. Transfer the cartilage particles with a small label to a cylindrical plastic mold. Press all of the air bubbles out to avoid cavities in the scaffold and fill the mold completely.
To avoid cavities in the scaffoldS, ensure that air bubbles are pressed out while filling the mold with cartilage particles. After formation, freeze the mold for 10 minutes at minus 20 degrees Celsius. Then lyophilize the cartilage scaffolds within the mold for 24 hours in a freeze dryer.
After lyophilization, carefully remove the scaffold from the mold. Place the scaffold under a 365 nanometer wavelength UV light and cross link it overnight. Form scaffold discs by cutting sterilized scaffolds into three millimeter thick slices.
Then transfer the scaffolds into separate wellS of a six well plate. Add one milliliter of Chondrocyte Expansion Medium on top of the scaffolds and allow them to rehydrate for 30 minutes. While the scaffolds rehydrate, prepare the cells and pipette 50 microliter of the prepared cell suspension on top of the pre soaked scaffold.
Then incubate the scaffold for one hour at 37 degrees Celsius. When using chondrocytes, make sure they have not been expanded past the first passage in order to minimize the number of the differentiated cells. After one hour, return the plate to the culture hood and carefully turn the scaffold over.
Pipette the remaining 50 microliters of cell suspension on the scaffold and incubate for another hour at 37 degrees Celsius. After incubation, add three milliliters of medium to the wells with scaffolds. Avoid pipetting the medium directly on the scaffolds and handle the culture plate gently to avoid detachment of the cells.
Return the plate to the incubator and culture cell ceded scaffolds at 37 degrees Celsius. A combination of historlogical staining and DNA quantification have been used to show that the methods presented in this article result in sufficient cartilage scaffold decellularization. The resulting scaffolds were found to contain no cells along with undetectable levels of DNA.
They also lack glycosaminoglycans and are rich in collagen II.Here, neo matrix formation is observed on scaffold ceded with mesenchymal stem cells that were cultured for four weeks. The formation of glycosaminoglycans is abundant after four weeks. After attempting this procedure, it is important to examine whether decellularization has been successful, before using it in any application.
Following this procedure, additional biochemical analysis like Western blot analyzer can be performed in order to answer specific questions about the presence of other components such as growth factors. This technique paved the way for researchers in the field of tissue engineering to explore strategies for cartilage regeneration using decellularized tissue from allogeneic and syngeneic sources. Precautions such as wearing a lab coat and gloves should always be taken.
