The overall goal of this procedure is to assess the anchorage and stability of newly formed reparative bone to candidate implant surfaces. This is accomplished by first creating two groups of custom titanium implants. One group treated with a standard grit blast treatment to create an underlying micro topography and a second group with the same grit blasting treatment, as well as superimposed calcium phosphate nano crystals.
The second step is to place implants into the distal femur of male wistar rats for nine days, at which point fem are collected. Next, the fem are cleaned and the surrounding bone is trimmed to the width of the implant, leaving mechanical testing specimens with the cortical arch on either side of the implant. The final step is to put the test specimens in a flowable dental composite using a custom breakaway mold, which isolates 0.5 millimeters of peri-implant bone and to conduct mechanical testing on the specimens.
Ultimately, mechanical testing results provide an assessment of the ability of the candidate implant surface to act as an anchorage surface for newly formed reparative bone. This method can be employed to demonstrate the importance of different scale ranges of implant surface topography on the various mechanisms of bone anchorage from bone bonding at the nanometer scale to bone ingrowth at the course micron scale. The technique is particularly suited to measuring the effects of candidate surface designs, such as anchored surfaces for newly formed reparative bone.
Since it limits the mechanical testing region to within half a millimeter of the implant surface. This method can provide insight into the effects on bone formation as a function of implant surface design, and can also be applied to other metal, ceramic and polymeric implant materials. To begin this procedure, fabricate rectangular shaped titanium implants from commercially pure titanium, then drill a hole centrally down the long axis and prepare their surfaces for implantation as described in the accompanying text protocol, assign implants one to the left femur and one to the right by partial randomization.
This ensures that one of each implant type ends up in the same animal and allows for optimization of the statistical analysis. As with all animal procedures, obtain protocol approval from the local animal care committee before beginning any experiments for this procedure. Obtain young wistar rats that will weigh between 200 and 250 grams at the time of surgery.
Sedate one rat at a time using inhalation anesthesia for induction. Administer 4%isof fluorine in oxygen at a flow rate of one liter per minute. Then use 2%isof fluorine in a mixture of one liter per minute of nitrous oxide and 0.6 liters per minute of oxygen.
To maintain sedation, conduct a standard toe pinch test to ensure effective sedation before continuing with the procedure. Once fully sedated, apply artificial tear ointment to both eyes and administer preoperative analgesic through a subcutaneous injection of 0.01 to 0.05 milligrams per kilogram buprenorphine. Next, shave the anterolateral aspect of each hind leg and then disinfect the surgical area by alternating three times with 10%Betadine and 70%ethanol.
Place the rat on a warm water circulation pad to prevent hypothermia during the procedure using a number 15 surgical scalpel. Make an incision through the skin and along the lateral aspect of the thigh to expose the muscle. Then expose the distal femur using blunt dissection to deflect the muscle bodies in a minimally invasive manner.
Next, scrape away the thin layer of periosteum, which overlays the femur using a perote elevator to fully expose the cortical bone for drilling. Take care not to damage the growth plate or articular cartilage of the knee joint during blunt dissection or removal of periosteum. Once the femur has been cleaned and inspected, rotate the femur laterally to expose the anterior aspect of the distal femur.
Also ensure that there is plenty of sterile saline available to irrigate the tissue during drilling to avoid overheating of the tissue Before drilling, measure the location for two holes, 2.5 millimeters apart along the midline of the femur using a caliper. Then attach a 1.3 millimeter dental burr to a dental handpiece and create the holes by drilling through the anterior cortex. Next, attach a twist 1.3 millimeter dental burr to the handpiece and extend the holes through the opposing cortex of the femur resulting in bicortical parallel holes.
Finally, switch out the twist burr for a custom side cutting burr and use it to join the holes in a proximal distal direction, forming the site for the implant. Once the defect is complete, pass a biodegradable suture through the opening using the attached needle and return it around the outer femoral cortex. Then thread the implant over the free end of the suture and guide it into the defect where it should be pressure fitted.
Tie the suture around the lateral aspect of the femur to provide implant stability during postoperative recovery and the early stages of healing. Use the remaining suture to close the muscle tissue, then repose the cutaneous tissue using surgical staples. Finally, administer a postoperative analgesic through a subcutaneous injection.
Inspect surgical sites for signs of infection and monitor the animals daily. Exclude animals that do not fully recover ambulation or those found to have femoral fractures at sacrifice Upon sacrifice, detach the femur and clean the bone of the surrounding soft tissue. Immediately store it in a 15%sucrose buffer solution to maintain tissue hydration.
In preparation for mechanical testing, draw a thin black line in permanent marker on the lateral arch for identification purposes. Next, trim the bone to the width of the implants using a cylindrical diamond burr attached to a high speed system. The final test specimens consist of two arches of bone attached to each face of the implant.
Use a mechanical testing apparatus equipped with a load cell for all testing and set the operating speed to 30 millimeters per minute. Assign a testing value of zero newton's for arches that fall off during preparation or transportation to test specimens. First, place the specimen into a custom breakaway mold and stabilize it by placing the stabilizing plate on the posterior side of the mold.
Then surround the cortical arch in flowable dental composite and cure the composite for 60 seconds using a sapphire plasma arc high intensity curing light. This design allows for isolation of a 0.5 millimeter region of peri-implant bone for consistent testing. After curing open the mold and remove the hardened specimen block.
Next, fix a prefabricated replica of the test specimen in a vice and center the unit on the base of the instron. Then anchor the sample to the system by placing it into the vice thread, the 10 pound nylon fishing line through the implant and attach the loose ends to the moving crosshead For consistency, always label and test the lateral side first. Next, move the crosshead away from the sample at 30 millimeters per minute as before until failure.
Once the bone pulls away from the implant surface, repeat this process with the medial arch and then observe the implant surfaces using a dissecting microscope. Shown here are SEM images of the grit blasted implant surfaces presented with and without the addition of calcium phosphate nano crystals. The modified nano surfaces look similar to the control surfaces at 10, 000 x, but when viewed at 100, 000 x, the superimposed nano topography created by the calcium phosphate nano crystals becomes quite obvious.
Representative forced displacement curves are shown here. The red lines represent the micro topographically complex surfaces, while the black line represents implants with the superimposed calcium phosphate nano crystals. The surface with the added calcium phosphate submicron topographical features had significantly higher disruption forces than that with an unmodified micro topography.
In analyzing the fracture planes following mechanical testing, 92%of those with the bone encased in flowable dental composite fractured within the targeted implant region. While attempting this procedure, it is important to pay strict attention when potting the test specimens due to their miniature size and weight. They're difficult to accurately place, so does necessary to properly align everything before applying the dental composite.
Following this procedure, advanced imaging methods such as back scattered electron imaging in a scanning electron microscope can be performed in order to answer additional questions about the bone implant interaction at a significantly higher magnification.
