Preliminary histological study on bone- ingrowth Characteristics of titanium metal of classical pore structure in the greater tuberosity of proximal humerus in rabbits

Abstract

Background The postoperative nonunion of greater tuberosity is the most common complication after the treatment of complex proximal humeral fracture with traditional shoulder prosthesis replacement, which severely affects shoulder joint function and reduces operative effect. Currently, the porous-coated prosthesis applied in clinic has several defects, such as low porosity, interfacial shear force between coating and prosthetic frame space, galvanic effect, etc., which can affect bone ingrowth and reduce biological fixation strength. However, the titanium alloy biological implant with classical regular hexahedral pore structure made by electron beam melting can overcome the shortcomings of traditional coated prosthesis and further improve the effect of bone ingrowth theoretically.In this study, the New Zealand rabbit was used for experiment. The classic pore structure, namely the titanium alloy biological implant with regular hexahedral pore structure, was implanted into greater tuberosity. With the help of histological research, the purpose of this study includes the confirmations of whether the titanium alloy of classical pore structure has the potential of bone ingrowth over time; what are the histological characteristics over time. This study can provide theoretical basis and evidential support for the application of titanium alloy biological implant in the field of shoulder surgery and the design of artificial shoulder prosthesis for enhancing the healing of greater tuberosity. Methods (1) Design: animal modeling observation. Time and location: it was completed in the animal laboratory of our hospital from May 2015 to November 2016. Materials: 15 adult male New Zealand rabbits of 4 months old (random allocation with three rabbits in each group) . The body weight was 2.41-2.70 kg, and the rabbits were raised in single cage and fed with standard diet. All rabbits were observed preoperatively for 1 week to confirm healthy. The disposal of animal during the experimental process is in conformity with the standard of medical ethics. (2) Methods. Establishment of rabbit proximal humeral biological implant model: The rabbit was given the intramuscular injection of mianmixin II (0.2 ml/kg) for general anesthesia. Afterward, the rabbit was placed in left lateral decubitus. Skin preparation, cleaning, regular disinfection and draping were carried out preoperatively. A 2-cm longitudinal incision was made in the shoulder, and the deltoid muscle was bluntly split to expose greater tuberosity. A 2.0 Kirschner wire was drilled through the center of humeral shaft at the insertion of greater tuberosity, which had an angle of 120° with the long axis of distal humerus. The titanium alloy screw with classic pore structure was inserted through bone tunnel to establish the model of proximal humeral titanium alloy biological implant. Without active hemorrhage, the wound was closed layer by layer. (3) Management after modeling: the rabbits were awakened naturally and fed regularly. They were kept in cage (65 cm × 40 cm × 40 cm) for activity restriction throughout the experiment, and the affected limb was immobilized. Penicillin (4 × 105 U) was injected intramuscularly to prevent infection for the first 3 days after modeling, and the samples were collected randomly and respectively at the 3rd, 6th and 12th week. The bone sample from humeral head to its distal end of 3 cm was kept for use, and all the muscle and soft tissue were removed. The bone sample was fixed with neutral buffered formalin for 48 hours and then dehydrated with gradient alcohol of 70%, 80% and 90% for 7 days and 100% for twice and 2 days per time. Afterward, the samples were infiltrated with Technovit7200VLC UV-curable resin for 1 month to complete photocurable embedding. The slices were made by EXAKT 400 CS/AW micro grinding system, which were cut parallel to humeral shaft with anatomical axis as the center. With about 1 mm set aside in both lateral and medial sides, the bone tissue was cut into slices of approximately 2 mm in thickness for toluidine blue staining. Under optical microscope (Nikon Eclipse 90i; Nikon Instruments, Inc., Melville, NY, USA) , the image acquisition was performed by 4 times magnification. The measurement of bone in growth area was processed and analyzed by Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA) . A rectangle that contained the length a of the screw and its width b was made in the sagittal section of the slice, and the total area S0 = a × b. The rectangular area S1 with screws removed was calculated as well as the toluidine blue stained area Six, and the definition of bone in growth area S% = Sx / S1 ×100%. All the histological measurements were performed by the same operator. (4) Major observation indexes. ① Qualitative observation: general situation of bone ingrowth of biological implant with time. ② Quantitative measurement: changes of bone in growth area percentage with time. (5) Statistical analysis.SPSS 20.0 software (SPSS/PC Inc., Chicago, IL) was used for statistical analysis. The data was expressed as mean value ± standard division, and a P value < 0.05 was regarded as statistically significant. The ANOVA variance analysis was performed to evaluate the difference of bone in growth area percentages in the 3 experimental groups at 3-time points. Results (1) Quantitative analysis of experimental animals.All rabbits were in good health after operation, and they all began to take food independently on the same day of modeling. No wound infection or unpredicted death occurred during observation. In the process of modeling, the local anatomical structure of rabbit shoulder joint was similar to that of human being’s, and the anatomical position of surgical approach was relatively constant. The establishment of biological implant model of greater tuberosity was highly repeatable. (2) General change of biological implant bone ingrowth with time. Under the microscopic view of histological staining, the number of chondrocyte in screw gap showed enhancing trend with expanding range of distribution based on the 3-time points of 3rd, 6th and 12th months. (3) Changes of bone ingrowth percentage with time.The histological data showed that the difference of bone ingrowth percentage among 3 groups was statistically significant, and the percentage of bone in growth area of 12th week increased remarkably compared to those of 3rd week (P 0.05) .Meanwhile, we made the histogram and line chart of bone in growth area percentage of 3-time points. The percentage of bone in growth area and its corresponding time point were used for function fitting, and it was discovered that the bone ingrowth and time were in logarithmic relationship within 3 months. S% = 13.706ln (T) + 5.3095; R2 = 0.9868. Conclusion In this study, the histological sections confirmed that the bone in growth area of titanium alloy biological implant with classic pore structure in rabbit proximal humerus increases over time. Within 12 weeks of modeling, the bone in growth area and time were in exponential relationship, and the growth rate slowed down gradually. The results of this study suggest that it is necessary to strictly follow the rehabilitation protocol of active functional exercise after shoulder arthroplasty to reduce the occurrence of postoperative complication in clinic. Key words: Biological titanium implant; Pore structure; Regular hexahedron; Proximal humerus; Bone ingrowth