The overall goal of this experiment is to identify sequences that potentiate mesenchymal cell differentiation and promote bone repair. The initial step is to isolate by biop panning the phage that binds to bone. Proceed to identify the peptide sequences that bind to mesenchymal cells, and also determine the subsequent changes in gene expression.
Perform experiments to determine how the peptide sequences can be delivered to bone for regenerative purposes. As a final step, examine bone repair histologically taken together the results can offer important insights into mechanisms of cell differentiation in vitro and bone repair in vivo. The advantages of using the biop panning technique with a phage display library are that we were able to identify peptides that target bone and can be used to stimulate the differentiation of mesenchymal cells and potentiate bone repair.
Ultimately, these peptide sequences may be useful in therapeutic modalities for bone repair and for the treatment of bone loss such as osteopenia and osteoporosis. Based on a study where ULA and Pascal showed that biop panning can be used to isolate peptide sequences that target blood vessels, we opted to adapt this method to query mechanisms in bone. For the in vivo biop panning procedure, perfused 10 times 10 to the 10th plaque performing units of the N-E-B-P-H-D 12 phage display peptide library through the heart of a 10 week old valve C mouse.
After approximately five minutes, euthanize the mouse, expose and dissect the femurs, cut off the ends and remove the bone marrow. Rinse the left femur both inside and outside with PBS 10 times. Then add the bone to ER 2 5 3 7 bacteria.
In order to remove non bound phage, use 2%dried skim milk in a syringe with a 21 gauge needle to rinse the intermediary canal of the right femur 10 times. Now elute the bound phage from the intermediary canal with hydrochloric acid glycine buffer. pH 2.2 neutralized with one mo la triss.
To amplify the phage, add EIT to ER 2 5 3 7 bacteria and culture for 4.5 hours. Carefully transfer the supernatants into fresh tubes and precipitate the phage three times by the addition of polyethylene glycol. Sodium chloride solution, incubate the tubes overnight at four degrees Celsius.
Finally, resuspend the pellet in TBS with 0.02%Sodium azide. Plaque the phage onto plates to determine the plaque forming units of the amplified EITs. Repeat the in vivo bio panning procedure for both the eluded and non eluted phage four times.
Amplify pooled phage in bacterial cultures. Purify and quantify the DNA sequence, the final phage DNA using 98 primer provided with the phage display kit. Importantly, calculate the frequency of phage DNA sequence representation.
Now synthesize peptides incorporating a glycine glycine glycine sine lysine sequence seed and amplify D one cells. A cloned mouse multipotential bone marrow stromal cell line that was developed in our lab at 90%confluence. Trypsin is the cells and centrifuge to pellet and count for peptide staining plate cells on gelatin coated chamber well slides and culture for 24 hours.
Wash the cells with PBS and fix with 4%paraldehyde for 30 minutes. Now perform blocking incubations with glycine followed by BSA Aden and BBSA. Biotin blocking solutions.
Add 100 micromolar of biotinylated L seven and R one peptides. Proceed to wash the cells three times with PBS for five minutes each. Add a one to 1000 dilution of LOR strippin cover and foil and incubate in a dark drawer for 30 minutes.
Wash the cells and mount cover slips with vector shield Mounting media. Now analyze the slides under a fluorescent microscope 24 hours before surgery. Soak the gel foam cylinders.
Transfer the gel foam cylinders with 20 micromolar peptide either L seven or R one or PBS buffer without peptide to sterile 12. Well culture dishes and refrigerate overnight at four degrees Celsius at the time of surgery. Place the gel phone composite on ice until use.
Using a pneumatic burr under saline irrigation, create a large oblong uni cortical defect of three millimeters width by eight millimeters length. Treat the bone defect with prepared gel foams by inserting all implanted scaffolds using gentle tapping. Euthanize the rats and collect tibia at defined time points carefully check for the bone defect and fix the harvested tibia in 10%neutral buffered formalin for paraffin sectioning.
Finally stain all the tissue sections with hematin eosin to label the osteo matrix. Fluorescein isothiocyanate labeled Aden permits microscopic detection of the binding of peptides on cells grown in vitro. Here, the peptides bind to mesenchymal cells in culture to determine the osteogenic effect of peptides on cells.
In vitro mesenchymal cells in culture can be treated with test peptides to determine changes in cell morphology. For instance, at day four, the cells start to aggregate and the aggregates enlarged with time to form nodules. Interestingly, bone section stained with h and e autofluorescence under UV light.
This greater contrast between bone and non bone tissues facilitates analyses quantify the presence of bone in the sections to determine bone repair in the defects treated with gelfoam plus peptide. By comparison to gelfoam alone here, defects that were filled with plus peptide showed the largest amount of cortical repair with time While attempting this procedure. It's important to be extremely methodical in your data entry because of the large number of specimens that will invariably accumulate over the time until you have selected the final group of peptides.
The development of in vivo biop panning as a technique to identify peptides that target bone means that we now have potential drugs that could be used as a therapy for bone repair and for bone loss. One of the mechanisms that underlies bone repair is also involved in bone loss. Understanding these mechanisms in more detail and having tools that could be used for the treatment of disorders such as delayed fractures and osteoporosis will help improve the therapies that can be devised for healthcare.
