Residual stress remains in bone tissues after press-fit-fixation of a joint prosthesis, recently employed for joint arthroplasty. The response of bone tissues to the residual stress is, however, unknown because it is not physiological. This unnatural stimulus may have adverse effects on bone tissues, including causing thigh pain or bone resorption. In the present study, we designed an experimental method to apply a stationary load from inside an animal femur using a loop spring of titanium alloy with super elasticity. The femoral response was assessed based on the migration of the wire into bone twelve weeks after implantation. As the results, wire migration was noted in 10 of 11 cases. We developed a method using a loop spring made of super elastic titanium alloy, which can maintain sufficient stress in a rat femur for a prolonged period. This titanium alloy, which contains 43.94% titanium and 56.06% nickel, was supplied as a wire (WDL1, Actment Co., Ltd., Kasukabe, Japan). In the present study, an experimental method was designed to apply a stationary load from inside a rat femur by inserting a loop spring made of super elastic wire.Background
Methods
It is generally accepted that strong hammering is necessary for the press fit fixation of a joint prosthesis. In this regard, large stress must remain within bone tissues for a long period. This residual stress is, however, some different from the feasible mechanical stimuli for bone tissues because that is stationary, continuous and directed from within outward unlike physiological conditions. The response on this residual stress, which may induce the disorder of the fixation of implant, has not been discussed, yet. In the present study, we designed an experimental method to exert a stationary load from inside of a femur of a rat by inserting a loop spring made from a super elastic wire of titanium alloy. Response of the femur was assessed by bone morphology mainly about the migration of the wire into the bone twelve weeks after the implantation. We developed a method using a loop spring made of super elastic wire of titanium alloy, which can maintain sufficient magnitude of stress in a rat femur during the experimental period. The loop spring was fabricated with a wire of 0.4 mm diameter before the quenching process. Eleven Wistar rats of ten weeks old were used for the experiments. The loop spring was inserted the right femur, as shown in Figure 1. The left femur was remained intact. The compressive load was added from within outward of bone marrow when the spring was compressed with the insertion into a bone marrow of a rat femur, as shown in Figure 2. The average contact stress was calculated by dividing the elastic force by the spring and bone contact area. The contact stress was distributed from 62 to 94 MPa, which are sufficiently lower than the yield stress of cortical bone [1]. The assessment of bone morphology around the implanted loop spring was performed by micro-CT imaging after the twelve weeks of cage activity.INTRODUCTION
MATERIALS AND METHODS
Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Human ACL cells were harvested from ligament samples donated by patients who underwent total knee arthroplasties with informed consents. Interface cells were isolated from the 5-mm-end of ACL. Midsubstance cells were cultured from the middle part of ACL. The cells were seeded onto stretch chambers (2Ä−2 cm, 50,000 cells/chamber) and uni-axial cyclic mechanical stretch (0.5 Hz, 7%) was applied for 2 h using a ST140. RNA samples were reverse-transcripted and quantitative real-time RT-PCR analysis were performed. To inhibit the function of integrin alphaVbeta3 subunit or alpha5 in stretching experiments, anti-human integrin alphaVbeta3 and alpha5 functional blocking antibodies (alphaVbeta3: 20 mg/ml, alpha5: 4 mg/ml) were used. To investigate the cellular attachments responding to mechanical stretch, we observed the distribution of integrins and stress fibers in both ACL cells. The shape of midsubstance cells showed spindle and fibroblastic cellular morphologies. On the other hand, the interface cells displayed chondroblastic appearances such as small and triangular morphologies. The expressions of COL1A1, COL2A1, and COL3A1 genes were detected in the tissue RNAs of interface zones. However, these expressions were decreased in cultured interface cells. In midsubstance cells, the expression of COL1A1 gene was equally detected in both tissues and cultured cells. COL3A1 gene expression was maintained in cultured midsubstance cells. These results indicated that the phenotypes of both ACL cells were changed by cultured conditions, especially in the interface cells. After mechanical stretch, the COL1A1 expression of midsubstance and interface cells were stimulated up to 6 and 14-fold levels of each non-stretched control, respectively. The COL3A1 expressions were also enhanced up to 1.8-fold level of controls by stretching treatment in both cells. Integrin alphaVbeta3 was shifted to the peripheral edge of cells by stretching treatment. In addition, mechanical stretch changed the integrin alphaVbeta3-dependent stress fiber formation in both ACL cells. The functional blocking of integrin alphaVbeta3 inhibited stretch-activated COL1A1 and COL3A1 expressions. However, the functional blocking of integrin alpha5 did not suppress the stretch-induced COL1A1 and COL3A1 expressions in both ACL cells. Cultured interface cells loose their phenotypes in collagen gene expressions. However, mechanical stretch reproduces the expression of COL1A1 and COL3A1 genes in cultured ACL cells. The present study demonstrated that stretch-activated collagen gene expressions depend on the integrin alphaVbeta3-mediated cellular adhesions.
