| Part Ⅰ. Establishment and evaluation for titanium particle-induced murine cranium osteolysis modelObjective: Establish a murine cranium osteolysis model, simulate the pathologicalprocess of titanium(Ti) particle-induced prosthesis aseptic loosening.Methods: Twenty healthy male C57BL/6mice aged7-8weeks were randomlydivided into two groups:(A) control group;(B) Ti group. Five milligram sterile andendotoxin-free Ti particles were implanted on mice cranium surfaces in Ti group to induceosteolysis. Mice in control groups received the same operation procudures but sterile salinewas implanted as control. After two weeks, all mice were sacrificed with overdoseanesthesia and cranium specimens were collected for use. A round-shaped region ofinterest (ROI) five milimeters in diameter was established as the object for study and theintersections of coronal, sagittal sutures on cranium surfaces were set as centers of thecircles. H&E staining was used to observe osteolysis and inflammatory cells infiltrationand periosteal thickness in two groups were measured with Paint.NET software.Enzyme-linked immunosorbent assay (ELISA) detected TNF-α and IL-1β proteinconcentrations in the supernatant in which ROI were in vitro cultured. Real timequantitative reverse transcription polymerase chain reaction (QRT-PCR) analysis detectedTNF-α and IL-1β mRNAlevels.Results:No animals died during the experiment.No swelling,effusion, rash or pus were foundon the skin. Cranium specimens covered with Ti particles display rough and unevensurface. H&E staining suggested wormhole like change were observed in Ti group.A largenumber of cells infiltration in Ti group was observed and inflammatory cells were majority,fibroblasts were less.The measurement results of Paint.NET software showed periosteal thickness in Ti group increased to (0.30±0.03)mm, compared with control group(0.06±0.01)mm, the difference was statistically significant (p<0.05).ELISA resultssuggested TNF-α and IL-1β protein concentrations in Ti group were (1440.12±82.33)ng/land (872.13±42.39)ng/l respectively, compared with control group (235.01±35.41)ng/l and(140.02±21.07)ng/l,the differences were statistically significant (p<0.05). RT-PCR resultssuggested after Ti stimulation,TNF-α and IL-1β mRNAs in Ti group were7.31±0.40and9.62±0.85, compared with control group (standardization to1), the differences werestatistically significant (p<0.05).Conclusion: Ti particle-induced murine cranium osteolysis model is simple,repeatable and could more accurately mimic the pathological process of prosthesis asepticloosening. PartⅡ. Effects of icariin on titanium particles induced osteolysisObjective: To investigate the treatment effect of icariin (IC) on Ti particle-inducedosteolysis and the underlying mechanisms were preliminary explored.Methods: Eighty male healthy C57BL/6mice aged7-8weeks were randomly dividedinto four groups:(A) control group,(B) IC group,(C) Ti+placebo group and (D) Ti+ICgroup. Ti particles were implanted on mice cranium in C and D groups to creat osteolysismodel. Mice in A and B groups received only the operation procudures but no Ti particleswere implanted as control. IC was given orally to mice in B and D groups with a dose of200mg/kg/day from the day modeling. Mice in A and C groups were administrated with thesame dose of placebo(PBS). After two weeks, all mice were sacrificed with overdoseanesthesia and cranium specimens were collected for use. ROI in different groups wereevaluated with micro-CT scanning and bone resorptive lacuna numbers on the surface canbe observed on CT three dimensional reconstruction images. Parameters of ROI werecalculated with CT own software,such as bone volume（BV）,bone mineral density（BMD）,bone surface（BS）,bone surface/bone volume ratio（BS/BV) values. H&Estaining was used to observe osteolysis and inflammatory cells infiltration Periostealthickness in different groups were measured with Paint.NET software. TRAP stainingdetected osteoclasts. ELISAanalysis determined TNF-α and IL-1β protein concentrations; Immunohistochemical staining detect RANKL and RANK protein expressions;QRT-PCR analysis determined TNF-α, IL-1β, RANKL and RANK mRNAlevels.Results:1. Micro-CT three-dimensional reconstruction images suggested cranium specimensin control group showed smooth surfaces and no signs of osteolysis were observed. But inTi+placebo group the bone erosion was serious and bone resorption lacunae clouds oncranium surface. In Ti+IC group, however,when mice were orally administrated with ICfor two weeks, the resorption lacunae numbers reduced significantly compared withTi+placebo group.3D quantitative analysis suggested BV,BMD,BS and BS/BV values in control groupwere(3.79±0.23)mm3,(0.71±0.05)mg/mm2,(35.71±1.98)mm2,(9.41±1.06)1/mmrespectively. BV and BMD values in Ti+placebo group decreased to(2.03±0.12)mm3,(0.58±0.03)mg/mm2respectively, but BS,BS/BV values increased to (45.73±3.84)mm2,(22.50±2.31)1/mm. Compared with control group, the differences were statisticallysignificant（P<0.05）. In response to IC treatment, BV and BMD values in Ti+IC groupincreased to (3.51±0.23)mm3,(0.69±0.04)mg/mm2respectively and BS,BS/BV valuesdecreased to(37.21±1.98)mm2,(10.57±1.29)1/mm, compared with Ti+placebo group, thedifferences were statistically significant (p<0.05).2. H&E staining suggested little cells infiltrate in control group, among them,fibroblasts were majority and inflammatory cells were less. Measurement results ofPaint.NET software showed periosteal thickness in control group, IC group, Ti+placebogroup and Ti+IC group were (0.07±0.01)mm，(0.06±0.01)mm，(0.28±0.04)mm，(0.12±0.02) mm respectively. The differences between control and Ti+placebo group,Ti+placebo and Ti+IC group were statistically significant（P<0.05）.3. TRAP staining suggested lots of dark brown TRAP positive cells accumulating inTi+placebo group. But few TRAP-positive cells were observed in control and IC group.TRAP-positive cells counting results as follows, control group:5.0±1.3, IC group werenot found, Ti+placebo group:36.0±4.8, Ti+IC group:11.0±2.3. The differences betweencontrol and Ti+placebo group, Ti+placebo and Ti+IC group were statisticallysignificant(p<0.05). IC obviously suppressed Ti particle-induced osteoclasts formation invivo. 4. ELISA analysis suggested TNF-α and IL-1β protein concentrations in thesupernatant of control group were (237.13±37.36)ng/l and (133.09±19.64)ng/l respectively.The two concentrations in Ti+placebo group increased to (1447.52±78.23)ng/l and(889.14±45.94)ng/l,compared with control group, the differences were statisticallysignificant (p<0.05). TNF-α and IL-1β concentrations in Ti+IC group decreased to(568.86±42.32)ng/l and (279.39±19.44)ng/l respectively. Compared with Ti+placebo group,the differences were statistically significant(p<0.05).5. RANKL and RANK protein immunohistological staining suggested color positivesites are intensive and distributed widespread in Ti+placebo group compared with controlgroup. However, color positive sites are sparse and distributed narrow in Ti+IC group.This suggest Ti particle induced up-regulation of RANKL and RANK proteins. Bysuppressing RANKL and RANK expressions, IC effectively inhibit particle inducedosteolysis.6. QRT-PCR assay results suggested if mRNA levels of TNF-α and IL-1β in controlgroup were set as1, mRNA levels of the two in IC group were0.81±0.13,0.83±0.12respectively, in Ti+placebo group were7.60±0.60,9.80±0.94; in Ti+IC group were2.40±0.18,2.10±0.23. Compared with control group, TNF-α and IL-1β mRNA levels in Ti+placebo group up-regulated significantly and the differences were statisticallysignificant(p<0.05). However,orally administration of IC result in obvious down-regulationof these two mRNA levels in Ti+IC group.Compared with Ti+placebo group, thedifferences were statistically significant(p<0.05).These results suggest the therapeuticeffect of IC may be associated with down-regulation of TNF-α and IL-1β mRNAlevels.RANKL and RANK mRNA levels in Ti+placebo group were9.02±1.05,11.01±0.97,compared with control group, the differences were statistically significant(p<0.05).Withthe stimulation of Ti particles, RANKL and RANK mRNA levels in Ti+placebo groupup-regulate obviously. In contrast,RANKL and RANK mRNA levels in Ti+IC groupdecreased to2.50±0.36,3.03±0.31in response to IC treatment,compared with Ti+placebogroup, the differences were statistically significant(p<0.05).Conclusion: By suppressing inflammation reaction and down-regulatingRANKL/RANK expressions, IC significantly inhibit Ti particle-induced osteolysis, orallyadministration with IC may become a effective method for prevention and treatment ofperiprosthetic osteolysis. |
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