This study has been reviewed and approved by the Institutional Review Board (IRB) of the Third Hospital of Hebei Medical University and that it conformed to the provisions of the Declaration of Helsinki. The experimental flowchart of this biomechanical study was summarized in Fig. 1.
Specimen preparation
Fourteen formalin-soaked specimens were all adult male donated bodies( Provided by the department of human anatomy, Hebei Medical University). The body height of the specimen was 172 cm on average (range, from 163 to 182 cm). The age when donated was 55 years on average (range, from 40 to 65 years old). Samples from those who had rheumatism, tuberculosis, or tumors were excluded. The specimens were then examined with digital radiography and excluded if they were identified to have osteoporosis, pathological or anatomical deformity, irregular joint surface, or other imaging abnormalities. All samples were required to have intact ligaments and tendons around knee joint and unbroken joint capsule. The muscular tissue of each specimen was removed. Then, the upper part of the femur and the distal part of the tibial and fibula were dissected. The remaining femur and tibial and fibula were both about 25 cm in length. The specimens were maintained in standby packages to prevent dehydration, and kept at -20°C for cryopreservation.
Establishment of femoral fracture malunion model
The cadaver specimens were thawed at room temperature for 12 hours before the experiment. The middle and lower femoral fracture models were created, and fixed using locking plate and screws at various residual valgus deformity (3 degrees, 7 degrees, 10 degrees), neutral position (0 degrees, anatomically reduced), or varus deformity (3 degrees, 7 degrees, 10 degrees), respectively. A horizontal incision about 3-4cm in length was made at the level of the joint space on each side. The subcutaneous fat was separated, the bursa was cut, and the joint space was exposed. The anterior and posterior cruciate ligaments and the medial and lateral menisci were preserved so as not to affect the normal distribution of the contact stress of knee joint.
Specimen assembled to biomechanical testing machine
The specimen was firstly erected, and the femur end was fixed perpendicularly to the homemade clamp with the use of denture base resin and solution (Type II self-setting dental powder and tray water). After the dental powder and tray water solidified, the tibial end of the specimen was fixed in the same way. Then the two-ends of the clamp were assembled to the biomechanical testing machine (Electroforce 3520-AT, Bose company, USA), and the fixed position of the femoral and tibia stumps was adjusted to make the lower limb mechanical axis close to the position when standing naturally (Fig. 2).
Stress measurement and data collection
An ultra-low pressure sensitive film (0.5 to 2.5 MPa) was used to measure the contact pressure on the medial and lateral plateaus of the femur. The pressure sensitive film is trimmed into a suitable shape and sealed in a polyethylene film bag. The total thickness of the pressure sensitive film and the polyethylene film bag is controlled to 250 μm. The fixed specimen was loaded with a tension of 200 N to pull the knee joint, and the pressure sensitive films were carefully placed under the knee meniscus separately through the medial and lateral incisions (Fig. 3). The incisions were then sutured tightly to close the joint capsule. After stabilization, the specimen was pressurized to 200N at 10 N/s to eliminate creep. A vertical load was applied on the specimen at a rate of 10 N/s to 400 N for 2 min. Then, the pressure sensitive film was carefully removed from the knee joint. As the pressure-sensitive film material varies with the humidity and temperature, the color development is also different. Therefore, humidifiers and air conditioners are used in the experiment to keep the indoor temperature at 25~30℃ and the relative humidity at 35% RH~ 80% RH.
The stress values of the ultra-low pressure sensitive films were read with the use of FPD-305E densitometer and FPD-306E pressure transducer (Fuji Company, Japan). The contact area of each pressure sensitive film was divided into four quadrants (front outer, front inner, rear inner and rear outer). Five points were selected in each quadrant for stress value reading. A total of 20 values in each film were recorded, and the average was calculated for final analysis.
Statistical analysis
SPSS 21.0 software (SPSS, Chicago, IL, USA) was applied for statistical analysis. The variables in this study include stress values and angles of varus or valgus deformity. The normality of the stress data is verified using the Shapiro-Wilk test, and the variance consistency is verified using the Levene test. Normally distributed measurement data was recorded as mean ± standard deviation. The stress values on the medial and lateral plateau of the femur at different positions were calculated and recorded, respectively. The analysis of variance (ANOVA) for random block groups was used to compare the stress values on the medial or lateral plateau at different angles of femur varus or valgus deformity under vertical load. The Student-Newman-Keuls (SNK) test was applied to make pairwise comparisons between the multiple sample measurements. Differences in the stress data between medial and lateral plateaus were tested using the paired samples t test. A P-value less than 0.05 indicated a statistically significant difference.