The Establishment Of SCID-Hu Model And The Influence Of Peripheral Blood Mononuclar Cells On Periprosthetic Tissues

BackgroudTotal joint replacement is an effective procedure for the treatment of end-stage arthritis.It is estimated that approximately 1 million cases of total joint arthroplasty were performed each year.However,there is a relatively high incidence of aseptic loosening of the prosthetic joint component.Aseptic loosening is the most common complication of orthoapedic implants worldwide. Up to 20%of patients with total joint arthroplasty will develop radiographic evidence of aseptic loosening within 10 years.Aseptic loosening most likely results from an inflammatory response to the presence of billions of debris particulates shed from the prosthesis during normal use.20%who develop osteolysis may have a tendency to overreact to wear particles;a thorough study of these mechanisms is indicated.Several studies from our laboratory and others have defined a quantitative association between the amount of wear debris and the incidence of aseptic loosening.Phagocytosis of implant-derived wear particles by resident tissue macrophages results in cytokine secretion, inflammation,and subsequent osteoclastic bone resorption.IL-1 and TNFαare two key cytokines that play a critical role in the development of inflammatory bone resorption.In vitro studies have demonstrated the rapid induction of mRNA followed by protein expression of TNFa and IL-1 in macrophages exposed to wear debris.Using a mouse inflammation model,we have shown that wear debris provokes a significant tissue inflammation associated with significant increase of IL-1 and TNFa synthesis and release.Homeostatic bone mass is determined by the delicate balance that exists between formation and loss.,Osteoblastic cells of mesenchymal origin synthesize and deposit bone matrix and thus increase bone mass.Osteoclastic cells are large,multinucleated phagocytes of hematopoietic origin that resorb bone.Enhanced osteoclastogenesis mediated by the inflammatory cytokines is an important contributor to the development of aseptic loosening.Two important regulators of osteoclastogenesis have been recently identified.These are the receptor activator of nuclear factor kappa B(RANK) and the receptor activator of nuclear factor kappa B ligand(RANKL).RANKL, is a TNF-related cytokine that is produced by marrow stromal cells and osteoblasts.RANK,a member of TNF receptor super family,is present on osteoclasts and their precursors and initiates osteoclastogenesis after ligation with RANKLor agonistic anti-RANK antibodies.Wear of prosthetic components plays a critical role in aseptic loosening.It is generally accepted that wear debris provokes diverse biological tissue responses,including vascularized granulomatous tissue formation along the implant-to-bone interface,inflammatory cells(macrophages,lymphocytes) influx,bone resorption,and finally osteolysis and loss of prosthesis fixation.Studies have shown that the circulating peripheral blood monocytes (PBMC) are among the first cells to colonize the inflammatory site,however,the interaction and molecular mechanisms that the circulating blood monocytes home to the prosthetic joint and form the periprosthetic pseudo-membranous tissue remain not clear.In this project we mainly study the molecular and biological mechanism of osteolysi which related with PBMC.ObjectiveThis study develope a mouse-human(SCID-Hu) chimera model and the objective of this study was to test the hypothesis that the systemic hematopoitic cells(PBMC) respond to the wear debris stimulation and home to periprosthetic tissue using the novel SCID-human chimera model,and examined the trafficking of human circulating blood monocytes and its influence on the inflammation of patient periprosthetic tissues,meanwhile study the effects to block the host immune reaction of mice using ASGM1.the other objective of this study was to study the release of cytokine IL1,IL6,TNF,RANK when PBMC exposured to PMMA and Ti particles.MethodsThis study includes two parts.Part one was to establish the SCID-Hu model and examine the patient PMBC trafficking in the SCID-Hu model,and the effects of ASGM1 on the model.Part two was to study the release of cytokine IL1,IL6,TNF,RANK when PBMC exposured to PMMA and Ti particles.1.Animals and Groups Part one:Thirty-two female severe combined immunodeficient(SCID) mice that were three to four weeks old were quarantined in a pathogen-flee environment for at least one week before experimentation.the mice were divided into three groups.Group 1 was the control group,engraft human periprosthetic tissues and bone chips without PBMC transfusion.Group 2 was the PBMC group;engraft human periprosthetic tissues and bone chips,PBMC.transfusion three day later.Group 3 was the PBMC+ ASGM1 group;engraft human periprosthetic tissues and bone chips,PBMC transfusion,and injection of ASGM-1.Part two:Thirty-six female SCID mice were divided into three groups.every group had 12 mice.Group 1 was the control group,transfusing PBMC without particle stimulation.Group 2 was the PMMA group;transfusing PBMC with PMMA particle stimulation.Group 3 was the Ti group;transfusing PBMC with Ti particle stimulation.2.PBMC Labeling and Transplantation We first in vitro labeled the PBMC with a florescent dye.The autologous human peripheral blood 25 ml was mixed with histopaque-1077 and PBS before centrifugation.A whit layer of PBMC was formed and collected.After further purification with the Histopaque-1077 gradient centrifugation,PBMC was resuspended in PRMI1600 medium and labeled with PKH2 green fluorescent dye at 2 um per 107 cells.Efficiency of labeling was examined daily under a fluorescent microscope.At three days after cell labeling, 5×106 cells per mouse were intraperitoneally injected into the SCIC-Hu mice.Total of 24 mice received the cell transfusion.3.Establishment of the Mouse-Human Chimera Model Human periprosthetic tissue and adjacent bone at obvious osteolystic site were obtained from patient undergoing revision surgery due to aseptic knee or hip prosthetic component loosening,the periprosthetic tissue was diced into tissue cubes of 2 mm~3 and mixed with 1x1x2 mm~3 chips of human cancellous bone tissue to surgically embed into the left quadriceps and the paravertebral muscles of SCID mouse under strict sterile conditions.Similar pieces of the patient tissues were snap-frozen and stored at -80℃as pre-implantation controls.Since periprosthetic tissue usually appeared heterogeneous in composition,attention was paid to avoid obvious fibrotic or fatty tissue.It is a general phenomenon that the inflammatory granulomatous tissues selected for implantation contain numerous wear debris particles and inflammatory cells.4.Anti-asialo GM1 Treatment Group 1 was the control group,engraft human periprosthetic tissues and bone chips without PBMC transfusion.Group 2 was the PBMC group;engraft human periprosthetic tissues and bone chips,PBMC transfusion three day later.Group 3 was the PBMC + ASGM1 group;engraft human periprosthetic tissues and bone chips,PBMC transfusion,and were treated with anti-asialo GM1 rabbit sera(ASGM1) while the rest of the mice were given PBS as controls.20μl of ASGM1 was intraperitoneally injected into SCID mice 4 hours before human tissue transplantation and the same amount was repeated again 7 days later.The part two hasn't this procedure.5.Histological and Immunohistochemical(IHC) Analyses The frozen xenograts and mice tissues(spleen,liver,lung,kidney) were cut into 6μm sections and observe the fluorescyte under the fluorescence microscope.The decalcificated tissues made slides and stained with HE methods.Observed and counted the PBMC in xenograft.Immunohistochemical stains were carried out to evaluate hematopoitic cell markers(CD45-R,CD14and CD68),pro-inflammatory cytokines(IL-1,and TNF) and mediators of osteoclastogenesis(RANKL) in transplanted tissues.Antibody again mouse-origin CD68 was also employed to reveal the mouse macrophage contamination.Digital images of IHC stained sections were captured and analyzed using the Image-Pro Plus software package. The level of the positive stains and localization was evaluated in six different fields, and expressed as integrated optical density(IOD).6.RT-PCR for Gene Expression The expression of proinflammatory cytokines IL-1,TNF,IL-6,RANK,CPK,and CTR was determined by real-time PCR using the ABI Prism 7700 sequence detector.7.ELISA The level of IL-1,IL-6,TNF was evaluated by using ELISA method.8.MicroCT for BMD Scan the SCID mice the first day and fourteen day after operation using Microct.And calculate the BMD.Results1.Acceptance of Xenografts in SCID Mice The mice tolerated the surgical procedure well and the embedded human periprosthetic tissue survived in SCID mouse hosts.No infection or tissue rejection reactions were appreciable at the time of tissue harvest.Histology assessment on H&E stained d that Wear debris particles were found ubiquitous in implanted tissues,surrounded by extensive cellular infiltrates.2.Human PBMC Labeled with PKH2 Green Fluorescent Dye At 24 hours after in vitro labeling with PKH2 green fluorescent dye,100%of human PBMCs in the culture were luminated under fluorescent microscope,and the membrane labeling last up to 6 weeks in culture.3.Distribution of Transfused PBMCs in the SCUD-HU Model Transplanted human periprosthetic tissues and many host tissue/organs were harvested and frozen sectioned at 2 weeks after transfusion of PKH2-1abeled patient PBMCs into the SCID-HU model.A fluorescent microscope was employed to examine the presence of fluorescent cells.Remarkable amount of fluorescent cells were identified in the transplanted human periprosthetic tissues.Although sporadic labeled-cells were also detected in host spleen tissue;examinations on kidneys, liver,and lungs resulted negative.Cellular compositions in xenograft tissues with PBMC transfers were scientifically higher than that in the tissues without systemic cell transfer(p＜0.05).Notably however,transplanted periprosthetic tissues in mice receiving ASGM1 treatment contained less cell infiltration comparison with the tissue in mice treated with PBS.Transplanted periprosthetic tissues in mice receiving PBMC using PMMA stimulating contained more cell infiltration comparison with PBS stimulating.4.Immunohistochemical Assessments The xenografts retrieved from the hosts were immunostained with anti-human and anti-mouse CD68,respectively.While human-CD68 positive cells were composed of the majority cell population within the xenografts from all the groups,host-originated macrophages(mouse CD68+ cells) infiltrated into the xenografts.However,ASGM1 treatment effectively eliminated the mouse CD68 positive cell contamination in the xenografts,cytokine stains showed that the IOD of CD68,RANKL,TNF was higher in two test group than that in control group(p＜0.05).IOD of IL1,TNF was significantly higher in Ti stimulating group than that in non particle group(p＜0.05).And IOD of IL1 was significantly higher in PMMA stimulating group than that in non particle group(p＜0.05).5.RT-PCR results Gene expressions of common proinflammatory cytokines and the osteoclast markers in the harvested xenogrfts were examined by real-time PCR. Notably,there were significantly higher mRNA expressions of IL-1,TNF,and RANK in the samples with PBMC transfusion compared to the non-transfusion controls and the periprosthetic tissue prior to transplantation to mouse hosts.There were significantly higher mRNA expressions of IL-1,TNF,and RANK in the samples with PMMA stimulating group compared to the non-particle controls.6.ELISA results The proinflammatory cytokines in the harvested xenograft were examined by ELISA.There was significantly higher level of IL-1,TNF in the samples with PBMC transfusion compared to the non-transfusion controls(P＜0.05), and IL6 level was also high in sample of PBMC+ASGM1(p＜0.05).There was significantly higher level of IL-1,IL6,TNF in the samples using PMMA particle stimulating and Ti stimulating compared to the non-particle controls (p＜0.05).7.MicroCT results All the mice were scaned with microCT.The results of part one in these studies showed that BMD at havesting time was lower than that of at the operating time.there were significantly lower level of BMD in the samples with PBMC group and PBMC+ASGM-1 group compared to the non-transfusion controls.Part two results:there were significantly lower level of BMD in the samples using PMMA stimulation group(p＜0.05).there were significantly lower level of BMD in the samples using PMMA stimulation group(p＜0.05).ConclusionsSCID mice can accept prosthetic tissues and bone chips.SCID-Hu chimera model is a good model to study the mechenic of particulate wear debris related osteolysis.The data suggests that circulating PBMC tends to home the implanted periprosthetic tissues and contributes the local inflammation.The host nonspecific immune cells appear to participate in some level of cell component within xenografts.Pretreatment of ASGM1 antibody to deplete the host macrophages is necessary in the establishment of the SCID-human mouse model. PMMA and Ti particles can stimulate the expression of cytokines,such as RANK,IL1,IL6,TNF.And promote PBMC home to peristhetic tissue. Also activate the function of osteoclast.Different particles have different bioactivation.This may be useful for selection of the prosthesis.Concomitantly,peripheral white blood cells were introduced to establish an in situ setting of human failing prosthetic joint with systemic hematopoietic environment.This would make a more realistically simulated condition of aseptic loosening process.Using this successful model would investigate the formation of human periprosthetic tissue and the mechnism of wear debris, which can provide insights on prevention or treatment of osteoclastogenesis and osteolysis.It also provides evidence for prosthetic components selecting.