An Animal Experimental Study Of Steroid-induced Avascular Necrosis Of Femoral Head By Mouse Nerve Growth Factor

Objective:Steroid induced avascular necrosis of femoral head(steroid-induced avascular necrosis of femoral head,SANFH) is the mostcommon reason of the non traumatic avascular necrosis of the femoral head,the course of disease from several months to several years. It is high disabilityrate, seriously influences on quality of life in patients, and an obstinate disease.Since Pietrogrande and Mastomarine first reported that one case of thesteroid-induced avascular necrosis of femoral head caused by theglucocorticoid in1957, steroid-induced necrosis of femoral head necrosis isthe first pathogeny to the non traumatic avascular necrosis of the femoral head.Over the years both the domestic and overseas made a depth study onpathologic change and treatment of steroid-induced avascular necrosis offemoral head. Because of its pathogenesis is still unclear, currently a variety oftreatment options is given to some theory of targeted therapy, there isn'teffective treatment options could completely cured of SANFH. Nowadaysprophylactic medication to the high risk group of using hormones, and reducethe incidence of SANFH is the research hotspot in recent years. It shows thatSANFH process involves the influence of hormone on many factors, includingtoxic effects on neurons, resulting in systemic tissues (including bone tissue)nerve injury. It is true that there are innervations in the bone tissue. Theneuropeptide substance which is secreted by nerve endings could be metabolicregulation to the bone tissue. The toxic effects of hormones on the nervoussystem could affect the neuropeptide substance compound, transport andrelease, then result abnormal bone metabolism, bone resorption exceeds boneformation, bone loss, trabecular bone necrosis; the bone metabolism abnormal,bone microcirculation changes, femur head ischemia and hypoxia, abnormalbone metabolism; protected algesia weaken then cannot avoid the excessive activity of the joint, and eventually developed into SANFH. Neural tissue andbone tissue is bidirectional regulation. Neuropeptide substance secreting bynerve endings could be metabolic regulation to the bone. The nerve growthfactor-NGF secreted by bone tissue is important to peripheral sensory nerveand postganglionic sympathetic nerve on growth, nutrition and rehabilitation.It could promote the neuron survival and the axonal stimulation growth, andinduce the nerve grow into bone tissue, then through the neural secretion ofneuropeptide substance made a control on bone metabolic. Mouse nervegrowth factor for injection (mNGF): it could improve the peripheral bloodcirculation; and regulate nerve, be important to the nerve nutrition andrestoration. The study is mature, Wang Yan and other design ofsteroid-induced femoral head necrosis in animal models, application ofEscherichia coli endotoxin (Sigma Company,USA); methylprednisolonesodium succinate (Pfizer,USA) preparation pure New Zealand white rabbits ofavascular necrosis of the model; the application of the injection of mousenerve growth factor (Beijing create a feeling of God biological agents, China)in the rabbit femoral head necrosis process, in order to complete the rightfemoral head necrosis animal Preparation of the model, and to investigate theintervention of the mouse nerve growth factor on rabbit femoral head necrosis.Methods: The40New Zealand rabbits,14-16weeks of age, no limit ofmale or female,2.5±0.5kg, after Adaptive feeding2weeks, randomly dividedinto3groups. Group A as control group (n=4), group B as modeling group(n=18), group C as the treatment group (n=18). Animals of BC group given10μg/kg Escherichia coli endotoxin (LPS) by ear vein injection, after24hgiven gluteal injection of methylprednisolone sodium succinate (MPSL)20mg/kg3times, each time interval of24h; Control group wasn't treated.Group A: normal feeding; group B: after building the model then normalfeeding; group C: hormone+intramuscular injection of mouse nerve growthfactor (30units/time,1time/day, intramuscular injection, for6consecutiveweeks); each group shall be intramuscular injection of penicillin(200000U/each time,2times/week) to avoid of infection. Triglycerides and cholesterol levels were measured before treatment (0weeks) and medication4,6,8,12weeks. After treatment4,6,8,12weeks, the animals were killed inbatches for bilateral femur length and cut part of the acetabulum (pay attentionto retain the joint capsule), made X-ray and histological observation, thefemoral head was soaked in formalin specimens and fixed for24hours, by HEspecimens under light microscopy to observe the changes of trabecular bone,bone cell lacunae and marrow cavity, and fat cell morphology structure,number and changes. At last we calculate of empty osteocyte lacunaepercentage and bone necrosis rate. Select10fields at high magnification,count50lacunae per field, count the number of vacancies, get vacancies inbone lacunae percentage of average empty bone lacunae percentage (%).Incidence of osteonecrosis=any side of the femoral head osteonecrosis of theanimal number/group of animals in the total number×100%.Experimental data obtained with the mean±standard deviation (x±s),between groups comparation used single factor analysis of variance (P <0.05,difference was statistically significant), the non-normal distribution andheterogeneity of variance used non-parametric tests analytical (α=0.05). Allthe experimental data used SPSS17.0software package for statistical analysis.Results:①T he general observation results, blank control group (group A):animal responses normally, the femoral head specimens also showed noabnormalities. Model group (group B): after given an injection of LPS, allanimals showed varying degrees of depression, loss of appetite, after8weeks,experimental animal appeared limp, and reduced activity. After modeling for12weeks, these phenomena increase and the femoral head articular surfaceroughness. During the experiment we found3cases femoral cartilage surfacecollapse. The others appeared limp change, and Activity was significantlyreduced. Three rabbit died in the experiment. The treatment group (group C):initially experimental animal spirit is dispirited, after8weeks; the femoralhead cartilage surface is smooth, normal color, profiles of articular cartilageare uneven thickness, subchondral without cystic change, no destruction ofbone. After modeling for12weeks, the femoral head cartilage surface color is dark, slightly off-white, portion of the femoral head can be seen in the fattydegeneration, bone crisp, partially within the femoral head were necrosistissue. During experiment there wasn't femoral head cartilage surface collapse,1cases in the group died.②Light microscope observation, the control group(group A): trabecular bone cells arranged in rows, clearly visible bone cell,normal configuration, distribution uniformity, uniformity saw empty bonelacuna cell; bone marrow adipose cell were normal morphology, moderatecontent. Model group (groupB): when12weeks HE staining appeared femoralhead bone marrow cavity fat cell proliferation, partial fat cell loss of nuclei,fragmentation, sections of bone marrow cell lyses and necrosis, inflammatorycell infiltration; part appear cystic degeneration regions of fibrosis; trabecularbone became thinning, degeneration and necrosis. Bone cell edge deeplystained, partial bone lacuna bone cells disappeared, empty osteocyte lacunaeincreased evidently. Osteoblasts were uncommon, new bone cells were less.The treatment group (group C): after modeling12weeks: intra-bone marrowbone trabecular structure became slightly sparse; trabecular bone surface hadnew bone tissue formation. Subchondral bone plate were continuous, therewasn't obvious large bone necrosis and bone necrosis repaired.③The modelgroup (group B) empty osteocyte lacunae percentage and bone necrosis ratecompared with those in blank control group increased evidently (P<0.05). Itshows that the model of preparation of femoral head necrosis was successful.The treatment group (group C) empty osteocyte lacunae percentage and bonenecrosis rate compared with those in blank control group increased evidently(P<0.05), and compared with the model group empty osteocyte lacunaepercentage decreased evidently (P<0.05), bone necrosis rate was no evidentlydifference (P>0.05). It shows mouse nerve growth factor for steroid-inducedavascular necrosis of the femoral head has a certain therapeutic effect, butnecrosis of the femoral head is not completely repair.④Triglycerides measure:Experiments in12weeks the pure hormone group (B) compared with blankcontrol group (A) triglyceride level increased evidently(P<0.05). Thetreatment group (C) compared with the blank control group (A) also increased significantly (P <0.05), B group and C group were no significant difference,without statistical significance.⑤Cholesterol measure: experiment in12weeks, the pure hormone group (B) compared with a blank control group (A)cholesterol levels were significantly increased (P<0.05). The treatment group(C) compared with the blank control group (A) also increased significantly (P<0.05), B group and C group were no significant difference, without statisticalsignificance.⑥X-ray comparison: pure hormone group (B) in a rabbit modelmade after8weeks, it shows that the bilateral femoral head area were mildosteoporosis, the cortex were thin, trabecular bone were fuzzy, when12weeksit appeared different degree of osteoporosis, bone trabecular structural disorder,confusion, and appearance of cartilage point radiolucent shadow, lighttransmission increased and cartilage surface does not appear collapse, anormal joint space.The treatment group (C) from the8week after radiographicchanges is not obvious; at12weeks of trabecular bone vague, without obvioussclerosis band, no joint surface subsidence.Conclusion:Endotoxin (LPS) combined with methylprednisolonesuccessfully causing the models of osteonecrosis of the femoral head, animalmortality is low, and the incidence of avascular necrosis of the femoral head ishigh.Mouse nerve growth factor could effectively intervene in femoral headnecrosis disease progression, treated early femoral head necrosis, increasedosteoblast number, reduced empty osteocyte lacunae and osteoclast numberand reduced the extent of osteonecrosis of the femoral head. Its mechanism ofaction may influence to improve the peripheral microcirculation and hormoneon nervous system toxicity, improved peripheral secretion of neuropeptidesubstance, thereby improving bone metabolism balance, thereby reducingosteoporosis to delay the occurrence and progression of the disease for thepurpose of SANFH.