The overall goal of this procedure is to modify metallic implants at multiple length scales to control cell behavior in vitro and in vivo. This is accomplished by first producing a pore size gradient within three dimensional tubular implants, Biofreeze extraction method. The second step is to develop nano fibrillar basement membrane mimics based on collagen and alginate by layer by layer methodology.
Next, an indirect method to monitor such implants following implantation using blood plasma analysis. HPLC and subsequent peptide sequencing is demonstrated ultimately an implant where different areas can accommodate different cell types as necessary. For some multicellular tissues can be obtained Though this system can provide a way to modify metallic implants.
It can also be applied to 3D cell culture model systems such as artificial tissue or disease models. We first had the idea for this method when we were trying to find a technique to control cell movement around porous titanium implants in vivo. To begin this procedure, clean the implants and design and manufacture Teflon molds as described in the written text for freezing and extraction.
Prepare the synthetic polymer PLLA solution in a binary mixture of dioxin and water by introducing the polymer powder into the organic solvent. On a magnetic Starr heat the mixture to 60 degrees Celsius in order to obtain a homogenous solution. This temperature is selected because it is the higher limit of the temperature resistance for the precision glass syringes to be used for the introduction of the solution into the implants.
Meanwhile, calculate the volume of the necessary polymer solution with respect to the porosity of the implant taking into account change in the volume of the frozen solution. Next, introduce the solution into the implants using precision glass syringes with 0.1 microliter accuracy. The lower limit for the polymer concentration is 3%for reproducible PO gradient formation, or as it becomes hard to obtain homogenous distribution in the thick samples above 6%freeze the samples either directly at minus 80 degrees Celsius or with a prior incubation period of 30 minutes at room temperature.
Freezing conditions determine partially the pore formation. Thus the freezing conditions can be adjusted according to the porosity aimed. Keep the samples overnight at minus 80 degrees Celsius.
The separation of the mold in the frozen state is a very difficult step. Care should be taken to decrease the contact of the frozen bottomer solution with the mold, with the help of a scalp, Immerse the implants in 80%pre chilled ethanol to obtain porosity gradients. Use a pre chilled scalpel to remove all the mold parts except for the mandril, for the tubular implants and all the parts except the bottom part for disc shaped implants, carry out the extraction at minus 20 degrees Celsius overnight.
After extraction, remove the remaining mold parts and air dry the implants. For characterization of the overall porosity of the structure. Mercury oximeter analysis is necessary.
In this example, mercury oximeter measurements show distinct peaks that correspond to the pores on both sides of the implant and the smaller inter dispersed pores. However, the more crucial data is the difference between the porosities of intraluminal and extraluminal surfaces, which can be analyzed by image J for pore size distribution. Following freeze fracture of the samples by observing the cross section with a scanning electron microscope.
Due to the open porous nature of the implants used and the light reflecting capacity of titanium, it is possible to do Z stacks of labeled cells within the porous implants. Cells labeled with PKH 26 or calcium AM can be used to visualize the implants with confocal laser microscopy for buildup of multi-layers. Highest reproducibility is obtained with dipping robots.
However, if a dipping robot is not available, these steps can be done manually. To prepare the collagen solution, use medical grade collagen type one and sodium alginate. The optimized concentrations are 0.5 gram per liter for each in 150 millimolar.
Sodium chloride citrate buffer at pH 3.8 dissolve the collagen solution overnight to ensure the homogeneity. The solution acidic pH of 3.8 is necessary for stable buildup of the layers as the structure is unstable. Before cross-linking.
In neutral pH design a specific holder for utilization of the implants with dipping robots used in poly electrolyte multilayer production. Deposit the layers on the surface of either titanium only implants or implants modified as described in the text. Using a dipping robot system by first immersing the implants into an alginate solution for 15 minutes, then rinse the structure with 150 millimolar sodium chloride at pH 3.8 for five minutes.
Follow with immersion into a collagen solution for 15 minutes. To stabilize the basement membrane first, prepare the cross-linking solution at 100 millimolar genin. In A-D-M-S-O citrate buffer at a one to four volume volume ratio dissolve genin in the DMSO component and then at the water component to avoid clumping cross-link the samples by immersion in the cross-linking solution between 12 and 24 hours afterwards.
Rinse with copious amounts of citrate buffer at pH 3.8 After washing the steps, sterilize the samples either with UV treatment for 30 minutes or in an antibiotic antifungal bath. The main parameters that determine the quality of the basement membrane mimic are its thickness and the diameter of the fibers. Calculate the fiber diameters using atomic force microscopy or a FM images obtained in contact mode.
After drying the samples with a nitrogen flow, before imaging quantify the thickness of at least 10 fibers per image to determine the average fibrile thickness. With Image J software following drying of the collagen alginate 24 bilayers collagen multilayer films, the thickness of the films can then be determined by scratch tests. Using a FM first, use a syringe needle to scratch the film after localization of the scratch with a light microscope, obtain images with a FM on 10 by 10 square micron surfaces at the boundary of the scratch.
Calculate the heights from the profiles obtained with the A FM software, which provides the thickness of the film layer. After implanting the device into the trachea of the rabbits as described in the text, obtain blood samples from their auricular veins, centrifuge the samples at 5, 000 RPM for 20 minutes at four degrees Celsius. Use the supernatant obtained for analysis after extracting the rabbit plasma with 0.1%of trior acetic acid.
Perform reverse phase HPLC purification of the plasma protein content by purifying the extract using a nucleus cell reverse phase 305 C 18 column. Record the absorbance at 214 and 280 nanometers using the solvent system and flow rate found in the text protocol. Collect the peak fractions before concentrating them through evaporation.
Using speed vacuum application, it is important to stop the speed vacuum before complete dryness. Correlate the peaks obtained at different time points over the course of the implantation period. Use the purified peptides that are showing consistent trends during the course of implantation for identification by automatic Edmond sequencing.
Load the sample to poly brain treated glass fiber filters determine the end terminal sequence of the purified peptides by automatic Edmond degradation using a pro precise micro sequencer. The next step is the identification of phenyl th andone amino acids by chromatography on a C 18 column. After the sequence is obtained, it can be identified by blast software using Swiss Prote database.
By changing the concentration of the PLLA solution, it is possible to control the size of the pores on the extra luminal side of the implants. Pore size and shape was significantly affected by the presence of titanium implants. Pore sizes ranged from 40 to 100 micrometers with utilization of lower concentrations resulting in smaller pores, whereas the intraluminal side pore size was governed by the restricted extraction and was around nine microns, which is less than the average size of fibroblasts.
By adding an incubation step at room temperature, double porous structures where the pore walls of the bigger pores have their own porosity can be obtained. This feature is important for thick implants as it would facilitate the gas and nutrient movement. After the pore gradient is formed, it is possible to add the collagen alginate film layer on top of the structure.
This film layer is stable on top of the PLLA foam and can also be maintained on the surface in the absence of the foam nanoscale collagen fibers form. As the film layer grows, the growth of the film is exponential. Thus, a thick film of several hundred nanometers can be obtained.
Porous titanium implants integrate with the host tissue and are completely filled between four and six weeks in vivo, as shown in the explanted implant cross-sections. The tissue within the pores is a mature connective tissue with a good level of vascularization. During this period, HPLC analysis showed distinct peaks that fluctuate during the time course of implantation.
The peak fractions of interest were sequenced and determined to be alpha and beta hemoglobin half chains, which had shown a similar trend with C-reactive protein readings. Once master, this technique can be done in one day if it is performed properly. After watching this video, you should have a good understanding of how to produce 3D multiscale PO gradients for modification of metallic implants and for cell culture experiments.
Freeze extraction and layer by the film formation are simple metals to develop your own modified implants or microenvironments for cells.
