【Abstract】 Objective To measure the changes of bone mineral density and bone micro-architecture of thefemoral head that harvested from the three-foot bearing ethanol destroyed canine model for osteonecrosis of femoral head, and discuss the influences of local injection of ethanol and biomechanical loading to the structural properties of the femoral head. Methods Twenty-four Beagles were divided randomly into four-foot bearing canines and three-foot bearing canines. One fore-l imb was fixed randomly in three-foot bearing canines. Osteonecrosis was induced in all experimental animals by local injection of 5 mL pure ethanol into one side of the femoral head. The hind l imbs injected with NS were acted as control group, that of three-foot canines injected with ethanol were acted as three-foot canine group, and that of four-foot canines injected with ethanol were acted as four-foot canine group. The contralateral femoral head was injected into equal amount of NS. Animals were sacrificed at the time intervals of 1, 3, 6, and 12 weeks after the injection of ethanol. Quantitative microcomputedtomography was used to characterize changes in bone micro-architecture and bone mineral density of femoralhead. Results The clear three-dimensional model of trabecular bone of necrotic femoral head were obtained. There were no significant differences among 3 groups according to the time l ine by 1 week after ethanol injection(P gt; 0.05). At 3 weeks after injection of ethanol, in three-foot canine group and four-foot canine group, the volume of BMC, BMD, BVF, and BS/BV increased gradually as the distance to the drill ing canal increased. There were significant differences between 3 regions (P lt; 0.05). At 6 weeks, in three-foot canine group and four-foot canine group, the volume of BMC, BMD,BVF, and Tb.N of region I and II decreased significantly compared with region III (P lt; 0.05). At 12 weeks, there are no differences among 3 groups (P gt; 0.05). There were significant decreases in BMD values, BVF, BS/BV, Tb.N, Tb.Sp and Tb.Th after the injection of pure ethanol. And, the changes were more and more obvious by the time l ine. These changes were differentiable at 3 weeks after injection of ethanol, and became obvious at 6 weeks. These changes were more obvious at the part that near the injection canal. The changes in threefoot canine group were more obvious than that in four-foot canine group. Conclusion Resorption of necrotic compact bone trabecular may weaken the structural properties of the femoral head. Moreover, remodel ing and repairing process of necrotic bone trabecular may be hampered by constant biomechanical loading that presented in three-foot bearing canines, and thereby further weaken the structural properties of the femoral head. Biomechanical loading may be one of the critical reasons that lead to the collapse of femoral head.
ObjectiveTo analyze the correlation between the trabecular microstructure and the clinical imaging parameters in the fracture region of osteoporotic hip so as to provide a simple method to evaluate the trabecular microstructure by a non-invasive way. MethodsBetween June 2012 and January 2013, 16 elderly patients with femoral neck fracture underwent hip arthroplasty were selected as the trial group; 5 young patients with pelvic fracture were selected as the control group. The hip CT examination was done, and cancellous bone volume/marrow cavity volume (CV/MV) was analyzed with Mimics 10.01 software in the control group. The CT scan and bone mineral density (BMD) measurement were performed on normal hips of the trial group, and cuboid specimens were gained from the femoral necks at the place of the tensional trabeculae to evaluate the trabecular microstructure parameters by Micro-CT, including bone volume fraction (BV/TV), trabecular number (Tb. N), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th), connect density (Conn.D), and structure model index (SMI). The correlation between imaging parameters and microstructure parameters was analyzed. ResultsIn the trial group, the BMD value was 0.491-0.698 g/cm2 (mean, 0.601 g/cm2); according to World Health Organization (WHO) standard, 10 cases were diagnosed as having osteoporosis, and 6 cases as having osteopenia. The CV/MV of the trial group (0.670 1±0.102 0) was significantly lower than that of the control group (0.885 0±0.089 1) (t=-4.567, P=0.000). In the trial group, CV/MV had correlation with BV/TV, Tb.Th, and SMI (P<0.05); however, CV/MV had no correlation with Tb.N, Tb.Sp, or Conn.D (P>0.05). BV/TV had correlation with Tb.Th, Tb.N, Tb.Sp, and SMI (P<0.05), but it had no correlation with Conn.D (P=0.075). There was no correlation between BMD and microstructure parameters (P>0.05). ConclusionCV/MV obviously decreases in the osteoporotic hip, and there is a correlation between CV/MV and the microstructure parameters of BV/TV, Tb.Th, and SMI, to some extent, which can reflect the variety of the microstructure of the trabeculae. There is no correlation between BMD of femoral neck and microstructure parameters.
ObjectiveTo introduce a new bone defect model based on the anatomical measurement of radius and ulna in rabbits for offering a standard model for further tissue engineering research. MethodsFifteen healthy 4-month-old New Zealand rabbits were selected for anatomic measurement and radiological measurement of the radius and ulna. Another 30 healthy 4-month-old New Zealand rabbits were randomly divided into groups A, B, and C (n=10). The radius bone defect was created bilaterally in 3 groups. In group A, the periosteum and interosseous membranes were fully removed with jig-saw by approach between extensor carpi radialis muscle and musculus extensor digitorum. The periosteum and interosseous membranes were fully removed in group B, and only periosteum was removed in group C with electric-saw by approach between extensor carpi radialis muscle and flexor digitorum profundus based on anatomical analysis results of ulnar and radial measurement. The gross observation, X-ray, micro-CT three-dimensional reconstruction, bone mineral density (BMD), and bone mineral content (BMC) were observed and recorded at immediate and 15 weeks after operation. HE staining and Masson staining were performed to observe bone formation in the defect areas. ResultsBlood vessel injury (1 rabbit), tendon injury (2 rabbits), postoperative hematoma (1 rabbit), and infection (1 rabbit) occurred in group A, postoperative infection (1 rabbit) in group C, and no postoperative complications in group B; the complication rate of group A (50%) was significantly higher than that of groups B (0%) and C (10%) (P<0.05). The radiological examination showed bone defects were fully repaired in groups A and B at 15 weeks, but bridging callus formation was observed in group C. There was no significant difference in BMC and BMD among 3 groups (P>0.05). HE staining and Masson staining results showed bone formation in group A, with structure disturbance and sclerosis. New bone formed in groups B and C, cartilage cells were observed in the center of bone cells. ConclusionThe radius bone defect model established by approach between extensor carpi radialis muscle and flexor digitorum profundus is an ideal model because of better exposures, less intra-operative blood loss, less complications. Interosseous membranes play a role in bone tissue repair process, and the mechanism needs further study.
ObjectiveTo explore the degradation of AZ31 magnesium alloy and poly (lactic-co-glycolic acid) (PLGA) in the femoral condyle, and then evaluate the laws of degradation of AZ31 magnesium alloy by Micro-CT images and data. MethodsForty 3-month-old male New Zealand white rabbits (weighing, 2.5 kg) were randomly divided into 4 groups, 10 rabbits each group. Forty micro-arc-oxidized AZ31 magnesium alloy pins and 40 PLGA pins were implanted into the right and left femoral condyle, respectively. Micro-CT images and data analysis were used to evaluate the degradation at 4, 8, 12, and 16 weeks after operation (n=10). Degradation was evaluated by weight difference between pre-and post-implantation. The inflammatory response was observed around the implants by HE staining. The weight loss of magnesium alloy and Micro-CT results were compared. ResultsThe Micro-CT images showed that PLGA pins had gray low signal, which was similar to the soft tissue around. At 4 weeks after operation, no signs of degradation were observed, and there were little corrosion pitting on the magnesium alloy. At 8 weeks, corrosion pitting gradually expanded, the boundary between the longitudinal axis and the cross section became blurred; at 16 weeks, corrosion pitting became bigger, and the boundary was discontinuous. Micro-CT quantitative analysis showed that the volume fraction of magnesium pins decreased slowly at 4 and 8 weeks; it was significantly lower at 12 and 16 weeks than 4 and 8 weeks (P < 0.05). The magnesium cylinder mineral density continuously decreased during the study period, it had a rapidly speed from 12 to 16 weeks (P < 0.05). However, the magnesium CT image density showed a slight change (P>0.05). The surface-to-volume ratio of the pins constantly increased, and the ratio was significantly larger at 12 and 16 weeks than 4 and 8 weeks, and at 16 weeks than 12 weeks (P < 0.05). There was more and more corrosion pitting on the surface with time, which resulted in a decrease in the radius that mean trabecular thickness gradually decreased, showing significant difference between different time points after 8 weeks (P < 0.05). The weight loss detection showed that the degradation of magnesium pin and PLGA gradually increased with time (P < 0.05), and the degradation rate of magnesium pin was significantly lower than that of PLGA at 8-12 weeks (P < 0.05), but the degradation rate of magnesium pin was higher than that of PLGA at 16 weeks. At each time point, the weight loss of magnesium alloy was similar to that by Micro-CT, but mass fraction was lower than volume fraction and had significant differences at 8, 12, and 16 weeks (P < 0.05). HE staining revealed that slight inflammatory response was observed around the magnesium pins at 4 weeks, and inflammatory reaction gradually reduced with time and disappeared at 16 weeks, but no inflammatory reaction was seen around PLGA. ConclusionMicro-CT has the advantages of non-trauma, in vivo detection, quantitative analysis, and precise data in evaluating the degradation of AZ31 magnesium alloy. Regarding the degradation of the magnesium alloy and PLGA in vivo, the degradation rate is slow in the early stage, and then increases with time. The degradation of PLGA is faster and earlier but it is then overtaken by AZ31 magnesium alloy at 16 weeks. During the degradation, the density of the magnesium has almost no change. The biomaterials can not firmly attach to the surrounding tissues due to inadequate holding forces.
Objective To investigate the effect of domestic porous tantalum encapsulated with pedicled fascial flap on repairing of segmental bone defect in rabbits’ radius. Methods A total of 60 New Zealand white rabbits (aged 6- 8 months and weighing 2.5-3.0 kg) were randomly divided into the experimental group and control group (30 rabbits each group). A 1.5 cm segmental bone defect in right radius was established as the animal model. The porous tantalums encapsulated with pedicled fascial flaps (30 mm×20 mm) were implanted in the created bone defect in the experimental group, and the porous tantalums were only implanted in the control group. X-ray films were observed at the day after operation and at 4, 8, and 16 weeks after operation. Specimens were taken out at 4, 8, and 16 weeks after operation for HE staining and toluidine blue staining observation. The maximum load force and bending strength were detected by three point bending biomechanical test, and the Micro-CT analysis and quantitative analysis of the new bone volume fraction (BV/TV) were performed at 16 weeks after operation to compare the bone defect repair abilityin vivo in 2 groups. Results All incisions healed by first intention without wound infection. At 4, 8, and 16 weeks after operation, the X-ray films showed that the implants were well maintained without apparent displacement. As followed with time, the combination between the implants and host bone became more and more closely, and the fracture line gradually disappeared. HE staining and toluidine blue staining showed that new bone mass and maturity gradually increased at the interface and inside materials in 2 groups, and the new bone gradually growed from the interface to internal pore. At 16 weeks after operation, the three point bending biomechanical test showed that the maximum load force and bending strength in the experimental were (96.54±7.21) N and (91.26±1.76) MPa respectively, showing significant differences when compared with the control group [(82.65±5.65) N and (78.53±1.16) MPa respectively] (t=3.715, P=0.004; t=14.801, P=0.000). And Micro-CT analysis exhibited that there were a large amount of new bone at the interface and the surface of implant materials and inside the materials. The new bone BV/TV in the experimental group (32.63%±3.56%) was significantly higher than that in control group (25.07%±4.34%) (t=3.299, P=0.008). Conclusion Domestic porous tantalum encapsulated with pedicled fascial flap can increase local blood supply, strengthen material bone conduction ability, and promote the segmental bone defect repair.
ObjectiveTo investigate the pathological and microstructural features of the osteonecrosis samples from subjects with steroid-induced or alcohol-induced osteonecrosis of the femoral head (ONFH).MethodsThirty femoral head bone samples were collected from ONFH patients who underwent total hip arthroplasty between August 2015 and April 2016. There were 22 males and 8 females. The etiology of ONFH was alcohol-induced in 15 patients and steroid-induced in 15 patients. No significant difference of Association Research Circulation Osseous (ARCO) stage was found between alcohol-induced and steroid-induced ONFH (Z=2.143, P=0.143). The femoral head bone samples in different areas (necrosis, sclerosis, and normal) from involved subjects was taken, and gross observation, HE staining were carried out (the rate of empty lacunaes was calculated). The intact femoral head was scanned by Micro-CT and the parameters of bone microstructure were analyzed quantitatively. The parameters included bone volume to total volume (BV/TV), bone surface area to bone volume ratio (BS/BV), bone mineral density (BMD), bone mineral content (BMC), structural model index (SMI), trabecular plate number (Tb. N), trabecular plate thickness (Tb. Th), and trabecular spacing (Tb. Sp).ResultsAs observed in hard tissue slicing of both groups, the integrity of trabecular bone was destructed and cystic lesions left by the bone resorption was replaced by granulation tissues. Significant revascularization was found in granulation tissues of steroid-induced ONFH, but not in the alcohol-induced one. HE staining showed that the bone marrow structure was disordered in both group, as well as bone marrow necrosis and empty bone lacunaes noticed. The structure and integrity of trabecular bone of steroid-induced ONFH was far more severe whereas that of alcohol-induced one were thicker and better. The rate of empty bone lacunae in necrosis area of steroid-induced group was significantly higher than that of alcohol-induced one (P<0.05), but no significant difference was found in sclerotic and normal areas between 2 groups (P>0.05). Micro-CT showed that necrotic and sclerotic areas of both groups were low bone density. Bone structure in the former area was mostly heterogeneous. Further blood-rich granulation tissues formation was observed in the same places of hard tissue slicing, while the sclerotic one wasn’t. The results of quantitative bone structure analysis showed that BV/TV, BMD, BMC, Tb.N, and Tb.Th of the necrotic and sclerotic areas of steroid-induced ONFH were significantly lower than those of alcohol-induced one (P<0.01), BS/BV, SMI, and Tb.Sp of steroid-induced ONFH were significantly higher than those of alcohol-induced one (P<0.01). No significant difference among the indexes above was found in the normal areas of both groups (P>0.05).ConclusionThe integrity of trabecular bone was destroyed in necrotic area of steroid-induced or alcohol-induced ONFH. However, they performed different features of osteonecrosis and contrasted with each other. The steroid-induced ONFH was characterized by multiple " osteolytic bone destruction”, while the alcohol-induced one was manifested by some kinds of " coagulative destruction”.
ObjectiveTo explore the osteogenesis effect of advanced-platelet-rich fibrin (A-PRF) and β-tricalcium phosphate (β-TCP) composite. MethodsThirty-two healthy female New Zealand rabbits were randomly selected. A-PRF was prepared by collecting blood from middle auricular artery. Rabbits were randomly divided into 6 groups: groups A, B, C, D, and E (6 rabbits in each group) and group F (2 rabbits). Bone defects (6 mm in diameter, 8 mm in depth) were drilled into femur condyle of each rabbit’s both back legs. Then A-PRF and β-TCP composites manufactured by different proportion were planted into bone defects of group A (1∶1), group B (2∶1), group C (4∶1), group D (1∶2) and group E (1∶4) (V/V). The bone defect was not repaired in group F. The specimens were collected at 8 and at 12 weeks after operation. Then gross observation, X-ray examination, Micro-CT examination, and biomechanical test were performed. The bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp), compressive strength, and modulus of elasticity were calculated. ResultsThe gross observation and X-ray examination showed that the osteogenesis effect at 12 weeks was better than that at 8 weeks. At the same time point, the repair of bone defect and the formation of new bone in group B were better than those in other groups. Micro-CT examination showed that the trabeculae of new bone in group B were the most and the trabeculae arranged closely at 8 and 12 weeks. Besides there were significant differences in BV/TV, Tb.N, and Tb.Sp between group B and the other groups (P<0.05). There were significant differences in Tb.N and Tb.Th in group B, BV/TV and Tb.Sp in group C, Tb.Sp in group D between 8 weeks and 12 weeks (P<0.05). Biomechanical tests showed that the compression strength and elastic modulus of group B were the highest, and the compression strength and elastic modulus of group C were the lowest at 8 and at 12 weeks, showing significant differences (P<0.05). There were significant differences in compression strength and elastic modulus of each group between 8 weeks and 12 weeks (P<0.05). ConclusionThe A-PRF and β-TCP composite can repair femoral condylar defects in rabbits, and the osteogenesis is better in proportion of 2∶1.
ObjectiveTo explore the effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits.MethodsFifty mature New Zealand rabbits (weighing, 2.5-3.0 kg) were randomly divided into control group (n=10), model group (n=20), and experimental group (n=20). The rabbits of model and experimental groups were injected with lipopolysaccharide and methylprednisolone to establish the animal model of early steroid-induced osteonecrosis of the femoral head. The rabbits of experimental group were feeded with icariin solution once a day for 6 weeks since the first injection of methylprednisolone, whereas the rabbits of control and model groups were given normal saline at the same time points. The left femoral heads were removed after 6 weeks and gross morphological features were evaluated. Micro-CT scan was performed to analyze the trabecular microstructure with the following parameters: trabecular bone volume to total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.Sp). The Micro-CT scan was also converted to three-dimensional reconstruction images for observation. HE staining was applied to observe the trabecular structure and morphological changes of osteocytes and marrow adipocytes. It was also used to determine whether the samples of femoral heads occurred osteonecrosis based on the criteria for pathological diagnosis, and calculate the rate of empty lacunae.ResultsSeven rabbits died during the study, and 9, 16, and 18 rabbits in the control, model, and experimental groups, respectively, enrolled the final analysis. Compared with control group, the femoral head collapse and trabecular breaks were more obvious, and the trabeculae were sparse with irregular arrangement in the model group according to the results of gross observation, Micro-CT scan, and three-dimensional reconstruction images. But in the experimental group, the surface of femoral head was slight shrinking without obvious collapse, and the degeneration of trabecular structure was mild. According to bone microstructures analysis, the Tb.N, Tb.Tn, and BV/TV of femoral head in model and experimental groups were lower than those in control group, while the Tb.Sp in the model and experimental groups were significantly higher. The Tb.N, Tb.Tn, and BV/TV of femoral head in experimental group were higher than those in model group, while the Tb.Sp in the experimental group was significantly lower. The differences between groups were all significant (P<0.05). In the model group, HE staining showed that the number of osteocytes reduced, the number of empty lacunae increased, and the marrow adipocytes piled up in the space between femoral trabeculae, some even mashed together like a cyst. In the experimental group, the trabecular structure was still relatively complete compared with model group, no obvious apoptosis of osteocytes was observed, the size and number of adipocytes were basically normal. None of the animals in control group occurred osteonecrosis of the femoral head based on the criteria for pathological diagnosis, and the incidence of osteonecrosis were 81.3% (13/16) in the model group and 66.7% (12/18) in the experimental group, and the difference was not significant (P=0.448). The rate of empty lacunae of osteonecrotic femoral heads in the model group was 33.1%±1.4%, which was higher than that in experimental group (18.9%±0.8%) and in control group (12.7%±1.5%), and the differences between groups were significant (P<0.05).ConclusionThe icariin has a protective effect on the early steroid-induced osteonecrosis of the femoral head in rabbits, which can decrease osteocytes apoptosis, improve the bone microstructure, and delay such disease processes.
ObjectiveTo investigate the effect of natural hirudin on revascularization of ischemic skin flap in rats using Micro-CT and three-dimensional (3D) reconstruction.MethodsThirty-two Sprague Dawley rats were prepared a ischemic skin flap (8.0 cm×1.8 cm) model on the back and randomly divided into hirudin group and control group (16 rats in each group). At immediate and within 3 days after operation, the rats were treated with hypodermic injection of natural hirudin 0.3 mL (including natural hirudin 6 ATU) every day in hirudin group and the equal amount of normal saline in control group. At 6 days after operation, the survival rate of skin flap was evaluated, histological changes were observed by HE staining, and the volemia, length of blood vessels, and number of blood vessels were analyzed with Micro-CT 3D reconstruction.ResultsBoth groups of rats survived to the end of the experiment without infection. Different degrees of necrosis occurred in the distal part of the skin flaps in both groups at 6 days after operation, but the flap survival rate of the hirudin group (72.11%±8.97%) was significantly higher than that of control group (58.94%±4.02%) (t=3.280, P=0.008). Histological observation showed that the histological hierarchy of the hirudin group was clearer than that of the control group, with more microangiogenesis and less inflammatory response and inflammatory cell infiltration. Micro-CT 3D reconstruction showed that the flap vessels in the hirudin group were more and denser, and the volemia, length of blood vessels, and number of blood vessels were significantly higher than those in the control group (P<0.05).ConclusionNatural hirudin can reduce the inflammation of tissue, promote the regeneration and recanalization of blood vessels in ischemic skin flap, so as to improve the survival rate of the flap.
Objective To analyze the similarities and differences of bone microstructure and apoptosis phenotype of lateral column, middle column, and medial column in type L2 and L3 osteonecrosis of the femoral head (ONFH) specimens classified by China-Japan Friendship Hospital (CJFH) classification, so as to carry out a quantitative study of ONFH “three-columns structure theory” and to provide research support for the treatment of ONFH by rotational osteotomy through the base of femoral neck. MethodsDiscarded femoral head specimens from 16 patients (16 hips) with CJFH type L2 and L3 ONFH undergone total hip arthroplasty between April 2020 and February 2021 were selected as the research objects. First, the “three-column structure” of the femoral head was three-dimensionally segmented by Micro-CT, and the bone volume to total volume (BV/TV), bone surface area to bone volume ratio (BS/BV), trabecular spacing/separation (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number (Tb.N) in the lateral column, middle column, and medial column were analyzed to compare the similarities and differences of parameters related to bone mass and trabecular structure among the three columns. Then, the specimens were cut with a oscillating saw and made into paraffin sections for HE staining and immunohistochemical staining of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax). The differences of apoptosis phenotype between the three columns of ONFH samples of CJFH type L2 and L3 were evaluated by comparing the rate of empty lacunae and the rate of positive cells of immunohistochemical staining. ResultsThere were significant differences in BV/TV, Tb.Th, and Tb.N among the three columns of CJFH type L2 ONFH femoral head (P<0.05), with the largest in the medial column and the smallest in the lateral column; BS/BV and Tb.Sp of the lateral column were significantly greater than those of the medial column and middle column (P<0.05), no significant difference was found between the middle column and medial column (P>0.05). There was no significant difference in the bone parameters between the three columns of the CJFH type L3 ONFH femoral head (P>0.05). There was no significant difference in the rate of empty lacunae between the three columns of the CJFH type L2 and L3 ONFH femoral head (P>0.05). Immunohistochemical staining showed that a large number of tissue sections were detached, and only a small amount of non-specific staining was found in the sections without detachment, so the positive cell rate could not be calculated. Conclusion The middle and medial columns of the CJFH type L2 ONFH has better trabecular structures than the lateral column, and there is no significant difference in trabecular structures among the three columns of the CJFH type L3 ONFH.