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1.
Biomed Res Int ; 2020: 9680474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32596401

RESUMO

BACKGROUND: Animal tissues and tissue-derived biomaterials are widely used in the field of xenotransplantation and regenerative medicine. A potential immunogenic risk that affects the safety and effectiveness of xenografts is the presence of remnant α-Gal antigen (synthesized by GGTA1 or/and iGb3S). GGTA1 knockout mice have been developed as a suitable model for the analysis of anti-Gal antibody-mediated immunogenicity. However, we are yet to establish whether GGTA1/iGb3S double knockout (G/i DKO) mice are sensitive to Gal antigen-positive xenoimplants. METHODS: α-Gal antigen expression in the main organs of G/i DKO mice or bovine bone substitutes was detected via a standardized ELISA inhibition assay. Serum anti-α-Gal antibody titers of G/i DKO mice after immunization with rabbit red blood cells (RRBC) and implantation of raw lyophilized bone substitutes (Gal antigen content was 8.14 ± 3.17 × 1012/mg) or Guanhao Biotech bone substitutes (50% decrease in Gal antigen relative to the raw material) were assessed. The evaluation of total serum antibody, inflammatory cytokine, and splenic lymphocyte subtype populations and the histological analysis of implants and thymus were performed to systematically assess the immune response caused by bovine bone substitutes and bone substitute grafts in G/i DKO mice. RESULTS: α-Gal epitope expression was reduced by 100% in the main organs of G/i DKO mice, compared with their wild-type counterparts. Following immunization with RRBC, serum anti-Gal antibody titers of G/i DKO mice increased from 80- to 180-fold. After subcutaneous implantation of raw lyophilized bone substitutes and Guanhao Biotech bone substitutes into G/i DKO mice, specific anti-α-Gal IgG, anti-α-Gal IgM, and related inflammatory factors (IFN-γ and IL-6) were significantly increased in the raw lyophilized bone substitute group but showed limited changes in the Guanhao Biotech bone substitute group, compared with the control. CONCLUSION: G/i DKO mice are sensitive to Gal antigen-positive xenogeneic grafts and can be effectively utilized for evaluating the α-Gal-mediated immunogenic risk of xenogeneic grafts.


Assuntos
Matriz Óssea , Galactosiltransferases/genética , Xenoenxertos/imunologia , Transplante Heterólogo , Animais , Matriz Óssea/imunologia , Matriz Óssea/transplante , Substitutos Ósseos , Bovinos , Eritrócitos/imunologia , Galactosiltransferases/metabolismo , Camundongos , Camundongos Knockout , Coelhos , alfa-Galactosidase/imunologia
2.
Artif Cells Nanomed Biotechnol ; 46(sup3): S359-S369, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30207744

RESUMO

Homeotransplantation of bones for replacement therapy have been demonstrated reliably in clinical data. However, human donor bones applicable for homeotransplantation are in short supply, which facilitates the search for suitable alternatives, such as xenografts grafts. The α-Gal antigen-related immune risk of xenografts directly affects the safety and effectiveness of the biomaterials and limits their applications in the clinic. The immune risk can be prevented by depletion or breaking anti-Gal antibody prior to transplant. Therefore, how to assess the immune risk of the bone substitutes and select the reliable animal research model become extremely important. In this study, we prepared lyophilized bone substitutes (T1) and Guanghao Biotech bone substitutes (T2, animal-derived biomaterials with α-Gal antigen decreased), aimed to assess the immune risk of xenografts bone substitutes on GGTA1 knockout mice. The α-Gal antigen contents of T1 and T2 were firstly detected by ELISA method in vitro. The bone substitutes were then implanted subcutaneously into GGTA1 knockout mice for 2, 4 and 12 weeks, respectively. The total serum antibody levels, anti-α-Gal antibody levels, inflammatory cytokine and splenic lymphocyte surface molecules were detected and histology analysis of skin and thymus were performed to systematically evaluate the immune response caused by the T1 and T2 bone substitutes in mice. In vitro results showed that the amount of α-Gal epitopes in T1 bone substitutes was significantly higher than T2 bone substitutes, and the clearance rate of α-Gal antigen in T2 bone substitutes achieved about 55.6%. Results of antibody level in vivo showed that the T1 bone substitutes group possessed significantly higher total IgG, IgM, IgA and anti-α-Gal IgG levels than T2 and control group, while T2 group showed no significant changes of these indexes compared with control. In terms of inflammatory cytokines, T1 bone substitutes showed evidently higher levels of IL-4, IL-12P70 and IL-10 than T2 and control, while T2 group was comparable to control. No changes in the levels of splenic lymphocyte surface molecules were found in the three groups (T1, T2 and control group) during the experimental periods. The pathological results demonstrated that the inflammatory response in T2 group was lighter than the T1 group, which was in accordance with the inflammatory cytokines levels. The above results indicated that the process of antigen removal effectively reduced the α-Gal antigens content in T2 bone substitutes, which caused little immune response in vivo and could be used as bone healing materials. This study also demonstrated that GGTA1 knockout mice can be used as a routine tool to assess the immune risk of animal-derived biomaterials.


Assuntos
Matriz Óssea , Transplante Ósseo , Galactosiltransferases/deficiência , Animais , Anticorpos/imunologia , Matriz Óssea/imunologia , Matriz Óssea/patologia , Matriz Óssea/transplante , Substitutos Ósseos/farmacologia , Galactosiltransferases/imunologia , Xenoenxertos , Camundongos , Camundongos Knockout , Transplante Heterólogo
3.
Sci Rep ; 8(1): 13535, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30201960

RESUMO

Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other interferon signalling pathway members as potential immunomodulatory mediators. The CD317 molecule was detected on the surface of CBF-resident cells confirming the gene expression data. Taken together, these data demonstrate that human clinically used CBFs are inherently immunomodulatory and suggest that these viable allografts may be used to deliver therapeutic immunomodulation for immune-related diseases.


Assuntos
Aloenxertos/imunologia , Matriz Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Osso Esponjoso/imunologia , Terapia de Imunossupressão/métodos , Aloenxertos/metabolismo , Aloenxertos/transplante , Antígenos CD/imunologia , Antígenos CD/metabolismo , Matriz Óssea/metabolismo , Matriz Óssea/transplante , Osso Esponjoso/metabolismo , Osso Esponjoso/transplante , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura/métodos , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Perfilação da Expressão Gênica , Humanos , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/terapia , Ativação Linfocitária , Cultura Primária de Células/métodos , Fusão Vertebral/métodos , Transplante Homólogo/métodos , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
4.
Tissue Eng Part A ; 24(5-6): 448-457, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28683667

RESUMO

Human perivascular stem/stromal cells (PSC) are a multipotent mesodermal progenitor cell population defined by their perivascular residence. PSC are most commonly derived from subcutaneous adipose tissue, and recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting-derived cell population for bone tissue engineering. Specifically, purified PSC induce greater bone formation than unpurified stroma taken from the same patient sample. In this study, we examined the differences in early innate immune response to human PSC or unpurified stroma (stromal vascular fraction [SVF]) during the in vivo process of bone formation. Briefly, SVF or PSC from the same patient sample were implanted intramuscularly in the hindlimb of severe combined immunodeficient (SCID) mice using an osteoinductive demineralized bone matrix carrier. Histological examination of early inflammatory infiltrates was examined by hematoxylin and eosin and immunohistochemical staining (Ly-6G, F4/80). Results showed significantly greater neutrophilic and macrophage infiltrates within and around SVF in comparison to PSC-laden implants. Differences in early postoperative inflammation among SVF-laden implants were associated with reduced osteogenic differentiation and bone formation. Similar findings were recapitulated with PSC implantation in immunocompetent mice. Exaggerated postoperative inflammation was associated with increased IL-1α, IL-1ß, IFN-γ, and TNF-α gene expression among SVF samples, and conversely increased IL-6 and IL-10 expression among PSC samples. These data document a robust immunomodulatory effect of implanted PSC, and an inverse correlation between host inflammatory cell infiltration and stromal progenitor cell-mediated ossification.


Assuntos
Matriz Óssea , Células Imobilizadas , Imunomodulação , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Osteogênese/imunologia , Animais , Matriz Óssea/imunologia , Matriz Óssea/transplante , Células Imobilizadas/imunologia , Células Imobilizadas/transplante , Citocinas/imunologia , Xenoenxertos , Humanos , Camundongos , Camundongos SCID
5.
Biochem Biophys Res Commun ; 456(3): 785-91, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25514036

RESUMO

Bone destruction at inflamed joints is an important complication associated with rheumatoid arthritis (RA). Interleukin-10 (IL-10) may suppress not only inflammation but also induction of osteoclasts that play key roles in the bone destruction. If IL-10-producing osteoblast-like cells are induced from patient somatic cells and transplanted back into the destructive bone lesion, such therapy may promote bone remodeling by the cooperative effects of IL-10 and osteoblasts. We transduced mouse fibroblasts with genes for IL-10 and Runx2 that is a crucial transcription factor for osteoblast differentiation. The IL-10-producing induced osteoblast-like cells (IL-10-iOBs) strongly expressed osteoblast-specific genes and massively produced bone matrix that were mineralized by calcium phosphate in vitro and in vivo. Culture supernatant of IL-10-iOBs significantly suppressed induction of osteoclast from RANKL-stimulated Raw264.7 cells as well as LPS-induced production of inflammatory cytokine by macrophages. The IL-10-iOBs may be applicable to novel cell-based therapy against bone destruction associated with RA.


Assuntos
Reabsorção Óssea/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Interleucina-10/imunologia , Osteoblastos/imunologia , Osteoclastos/imunologia , Animais , Artrite Reumatoide/complicações , Matriz Óssea/imunologia , Remodelação Óssea , Reabsorção Óssea/etiologia , Calcificação Fisiológica , Fosfatos de Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/imunologia , Regulação da Expressão Gênica , Engenharia Genética , Interleucina-10/genética , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Camundongos , Osteogênese/genética , Ligante RANK/imunologia , Transdução Genética
6.
Expert Opin Biol Ther ; 10(6): 885-901, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20415596

RESUMO

IMPORTANCE OF THE FIELD: Bone is one of the most transplanted tissues worldwide. Autograft is the ideal bone graft but is not widely used because of donor site morbidity and restricted availability. Allograft is easily accessible but can transmit infections and elicit an immune response. AREAS COVERED IN THIS REVIEW: This review identifies all in vitro and in vivo evidence of immune responses following bone transplantation and highlights methods of improving host tolerance to bone allotransplantation. WHAT THE READER WILL GAIN: In humans, the presence of anti-HLA specific antibodies against freeze-dried and fresh-frozen bone allografts has been demonstrated. Fresh-frozen bone allograft can still generate immune reactions whilst freeze-dried bone allografts present with less immunogenicity but have less structural integrity. This immune response can have an adverse effect on the graft's incorporation and increase the incidence of rejection. Decreasing the immune reaction against the allograft by lowering the immunogenic load of the graft or lowering the host immune response, would result in improved bone incorporation. TAKE HOME MESSAGE: It is essential that the complex biological processes related to bone immunogenicity are understood, since this may allow the development of safer and more successful ways of controlling the outcome of bone allografting.


Assuntos
Doenças Ósseas/imunologia , Doenças Ósseas/terapia , Transplante Ósseo/imunologia , Animais , Antígenos/imunologia , Matriz Óssea/imunologia , Transplante Ósseo/efeitos adversos , Humanos , Imunossupressores/uso terapêutico , Transplante Autólogo , Transplante Homólogo
7.
J Immunol ; 178(6): 3379-89, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17339432

RESUMO

Apoptosis through Fas/Fas ligand (FasL) is an important regulator of immune system homeostasis but its role in bone homeostasis is elusive. We systematically analyzed: 1) the expression of Fas/FasL during osteoblastogenesis and osteoclastogenesis in vitro, 2) the effect of FasL on apoptosis and osteoblastic/osteoclastic differentiation, and 3) osteoblastogenesis and osteoclastogenesis in mice deficient in Fas or FasL. The expression of Fas increased with osteoblastic differentiation. Addition of FasL weakly increased the proportion of apoptotic cells in both osteoclastogenic and osteoblastogenic cultures. In a CFU assay, FasL decreased the proportion of osteoblast colonies but did not affect the total number of colonies, indicating specific inhibitory effect of Fas/FasL on osteoblastic differentiation. The effect depended on the activation of caspase 8 and was specific, as addition of FasL to osteoblastogenic cultures significantly decreased gene expression for runt-related transcription factor 2 (Runx2) required for osteoblastic differentiation. Bone marrow from mice without functional Fas or FasL had similar osteoclastogenic potential as bone marrow from wild-type mice, but generated more osteoblast colonies ex vivo. These colonies had increased expression of the osteoblast genes Runx2, osteopontin, alkaline phosphatase, bone sialoprotein, osteocalcin, and osteoprotegerin. Our results indicate that Fas/FasL system primarily controls osteoblastic differentiation by inhibiting progenitor differentiation and not by inducing apoptosis. During osteoclastogenesis, the Fas/FasL system may have a limited effect on osteoclast progenitor apoptosis. The study suggests that Fas/FasL system plays a key role in osteoblastic differentiation and provides novel insight into the interactions between the immune system and bone.


Assuntos
Apoptose/imunologia , Diferenciação Celular/imunologia , Proteína Ligante Fas/imunologia , Osteoblastos/imunologia , Osteoclastos/imunologia , Receptor fas/imunologia , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/imunologia , Apoptose/genética , Medula Óssea/imunologia , Medula Óssea/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Matriz Óssea/imunologia , Matriz Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Proteína Ligante Fas/deficiência , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Mutantes , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Receptor fas/deficiência
8.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 23(3): 551-5, 2006 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-16856387

RESUMO

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.


Assuntos
Antígenos Heterófilos/análise , Matriz Óssea/imunologia , Engenharia Tecidual , Animais , Fenômenos Biomecânicos , Feminino , Masculino , Estresse Mecânico , Suínos , Porco Miniatura , alfa-Galactosidase/análise
9.
J Mol Histol ; 36(4): 311-6, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-16200464

RESUMO

The purpose of the study was to analyze the involvement of metalloproteinase 2 (MMP-2) and macrophages in the tissue and cell response to the organic graft material produced from bovine cancellous bone. Thirty adult male white Wistar rats (Rattus norvegicus) received implants of blocks of demineralized bovine bone matrix between the fasciae of the quadriceps muscle. The specimens collected at 3, 7, 14, 21 and 28 days after implantation (n = 6/period). Sections of 6 microm thick were stained with hematoxylin and eosin and immunolabeled with anti-MMP-2 and anti-CD68 using standard avidin-biotin-peroxidase method. The tissue response to the material was initially mediated by polymorphonuclear neutrophils, evolving to a mononuclear inflammatory infiltrate with macrophages and few lymphocytes and plasma cells and presence of inflammatory multinucleated giant cells (GC) in contact with the material that exhibited signs of resorption. The number of cells immunolabeled to MMP-2 was highest at day 7 (103.2 +/- 39.1), but significantly decreased (F = 3.67; p = 0.044) until day 28 (45.9 +/- 13.1). CD68 immunostaining also significantly decreased (F = 6.75; p = 0.007) from day 7 (49.5 +/- 10.4) to day 28 (19.5 +/- 8.9). A positive and statistically significant correlation was observed between the evolutions of these two variables. The material had been almost completely resorbed at day 28. Among cells present at the granuloma, anti-MMP-2 immunostaining was predominant and more intense in macrophages, yet lightly immunolabeled multinucleated giant cells were found in close contact with the material. Thus, considering the experimental limitations of this study, we concluded that MMP-2 produced by macrophages participates in the resorption of demineralized bovine bone.


Assuntos
Técnica de Desmineralização Óssea/métodos , Matriz Óssea/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Metaloproteinase 2 da Matriz/metabolismo , Animais , Antígenos CD/imunologia , Matriz Óssea/transplante , Bovinos , Imuno-Histoquímica , Masculino , Metaloproteinase 2 da Matriz/imunologia , Porosidade , Ratos , Ratos Wistar
10.
Artigo em Chinês | MEDLINE | ID: mdl-15921329

RESUMO

OBJECTIVE: To observe the changes of immune status in recipient after implanting with xenogeneic acellular bone matrix (ACBM). METHODS: Twenty rabbits were randomly divided into 4 groups. Autograft, ACBM and bone soaked in alcohol were implanted into the 3 experimental groups separately, and No-treatment was done as control group. The CD4+, CD8+, CD25+ T lymphocytes in blood were detected by flow cytometer at 1, 2, 4 and 6 weeks after operation. After 2 and 6 weeks of implantation, the changes of bone and tissue were observed by histology. RESULTS: After 2-6 weeks, CD4+ and CD8+ T cells were significantly higher in the implanted group of bone soaked in alcohol than that in the other 3 groups (P < 0.05) and there was no statistically significant difference in the other 3 groups (P > 0.05). After 2 weeks, CD25+ T cells were significantly higher in the implanted group of bone soaked in alcohol than that in the other groups. In the 2nd week, there were inflammatory infiltration with a predominance of granulocytes. In the 6th week, there were many fibroblasts instead of granulocytes with a few lymphocytes and cartilage island formed in the implanted groups of autograft and ACBM. CONCLUSION: ACBM implanting has low influence on cellular immunity in recipient.


Assuntos
Matriz Óssea/transplante , Transplante Ósseo/métodos , Subpopulações de Linfócitos T/imunologia , Animais , Matriz Óssea/imunologia , Transplante Ósseo/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Subunidade alfa de Receptor de Interleucina-2/imunologia , Masculino , Coelhos , Distribuição Aleatória , Suínos , Fatores de Tempo , Transplante Heterólogo
11.
Scand J Immunol ; 60(5): 514-23, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15541045

RESUMO

In osteoarthritis (OA), cartilage and bone fragments have been described within the synovial tissue which are surrounded by synovial cells (i.e. detritus synovitis). These cells appear to attach actively to the cartilage and bone fragments. In rheumatoid arthritis (RA), on the other hand, synovial fibroblasts (SF) have also been shown to be localized at sites of invasion into cartilage and bone and to degrade extracellular matrix (ECM) by secreting proteolytic enzymes. One prerequisite for exerting their aggressive properties is the attachment to cartilage and bone ECM. This attachment appears to be mediated by the expression of different adhesion molecules for which corresponding binding sites on ECM components are known. As it has not been addressed to which ECM proteins SF adhere and with which affinity this process takes place, we investigated the adherence of SF from patients with OA and RA to different cartilage and bone matrix proteins. Synovial tissue samples were obtained during synovectomy or arthroplastic surgery and used for isolating and culturing SF. Synovial cells attaching to cartilage/bone fragments were characterized using immunohistochemistry. The adherence of SF to ECM proteins was examined using an adhesion assay with the following proteins coated on 96-well plates: aggrecan (AGG), bone sialoprotein (BSP), cartilage oligomeric matrix protein (COMP), collagen type I, II and VI, proline arginine-rich, end leucine-rich repeat protein (PRELP), osteopontin (OPN) and recombinant chondroadherin (CHAD). Bovine serum albumin was used as negative control. In addition, adhering fibroblasts were photographed using a phase-contrast microscope. As compared with RA-SF, significantly higher numbers of OA-SF adhering to collagen type II, OPN and CHAD could be detected (P < 0.05). In contrast, RA-SF showed increased attachment to collagen type II, OPN and BSP. Adhesion to AGG, COMP and PRELP appeared not to be significantly increased and differed widely among the SF samples, and, apart from one exception (BSP), OA-SF adhered in higher numbers to the matrix proteins than did RA-SF. Using immunohistochemistry, synovial cells attached to cartilage/bone fragments could be shown to predominantly express CD68 (>/=50%). The CD68-negative population was of the fibroblast phenotype (AS02 positive). The study demonstrates that the binding pattern of OA-SF and RA-SF to ECM proteins differs considerably and therefore provides novel insights into the difficult pathophysiology of OA and RA. In general, it appeared that SF adhere primarily to ECM proteins that contain known binding sites for adhesion molecules (e.g. integrins: collagen/integrin alpha(2)beta(1)) and that higher numbers of OA-SF adhered to the cartilage and bone matrix proteins than did RA-SF.


Assuntos
Artrite Reumatoide/imunologia , Matriz Óssea/imunologia , Cartilagem/imunologia , Osteoartrite/imunologia , Membrana Sinovial/imunologia , Adesão Celular/imunologia , Fibroblastos/imunologia , Humanos , Imuno-Histoquímica , Osteoartrite/etiologia
12.
J Bone Miner Res ; 15(9): 1825-34, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10977002

RESUMO

The aim of the present study was to investigate a systemic induction of bone formation in rats by immunosuppression with FK506 (1 mg/kg body weight intraperitoneally [ip]) in a model of osteoinduction of isogeneic and xenogeneic demineralized bone matrix (DBM) for a period of 28 days. In particular, alterations of in vitro cytokine synthesis and changes of lymphocyte subsets were studied. DBM was implanted intramuscularly in the abdominal wall of Lewis rats (seven per group). Blood was sampled on days -7, 0, 7, and 28 for determination of in vitro tumor necrosis factor a (TNF-alpha) synthesis and lymphocyte subsets by flow cytometry (CD3+, CD4+, CD8+, CD45+, ED9+, and Ia+ antibodies). Ossicles of de novo formed bone and the tibias were removed on day 28 after double tetracycline labeling for histomorphometric analysis. Immunosuppression with FK506 significantly decreased lipopolysaccharide (LPS)-stimulated in vitro cytokine synthesis after 7 days and 28 days (p < 0.05). Compared with control animals FK506 treatment significantly increased the volume of induced bone in isogeneic (2.1 +/- 0.3 mm3 vs. 10.8 +/- 0.9 mm3) and xenogeneic (O mm3 vs. 4.7 +/- 0.8 mm3) DBM. Bone histomorphometry of the tibias revealed that immunosuppression increased both bone formation and bone resorption, accompanied by a significant reduction in the relative trabecular area (Tb.Ar). FK506 caused a decrease in the counts of CD8+ T cells probably because of destruction or dislocation of these cells. This suggests that the amount of CD8+ cells and the degree of T cell activation in terms of mean fluorescence intensity (MFI) may be associated with bone metabolism. In support of this, statistical analysis revealed a significant positive correlation between parameters of bone formation as well as bone resorption and the CD4+/CD8+ ratio. There was a significant negative correlation between parameters of remodeling of the metaphysis of the tibia and induced bone volume (BV), respectively, and MFI values of CD3+/Ia+ cells. These findings suggest an important role of T lymphocytes in bone formation and bone resorption in vivo. FK506 caused a marked increase of bone formation in DBM. However, the conclusion that immunosuppression increases fracture healing warrants further investigation.


Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Matriz Óssea/efeitos dos fármacos , Matriz Óssea/transplante , Calcificação Fisiológica , Imunossupressores/farmacologia , Tacrolimo/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Antígenos CD/análise , Peso Corporal , Desenvolvimento Ósseo/imunologia , Matriz Óssea/imunologia , Matriz Óssea/metabolismo , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/imunologia , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/imunologia , Diferenciação Celular/efeitos dos fármacos , Citocinas/biossíntese , Imunossupressores/sangue , Imunossupressores/uso terapêutico , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Coelhos , Ratos , Ratos Endogâmicos , Tacrolimo/sangue , Tacrolimo/uso terapêutico , Tíbia/efeitos dos fármacos , Tíbia/fisiologia , Transplante Heterólogo/imunologia , Transplante Isogênico/imunologia
13.
J Bone Miner Res ; 14(1): 47-56, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9893065

RESUMO

Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO-1 antigen exclusively (STRO-1+/ALP-), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone-related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO-1-/ALP+ and STRO-1-/ALP- phenotypes appeared to represent fully differentiated osteoblasts, while the STRO-1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO-1/ALP NHBC subsets were also found to express the DNA-binding transcription factor CBFA-1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA-1 in each of the STRO-1/ALP subsets, with the exception of the proposed preosteoblastic STRO-1+/ALP- subpopulation. Furthermore, upon re-culture of the four different STRO-1/ALP subsets only the STRO-1+/ALP- subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO-1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.


Assuntos
Fosfatase Alcalina/imunologia , Antígenos de Superfície/biossíntese , Desenvolvimento Ósseo/fisiologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Biomarcadores , Densidade Óssea/fisiologia , Matriz Óssea/citologia , Matriz Óssea/crescimento & desenvolvimento , Matriz Óssea/imunologia , Linhagem da Célula , Células Cultivadas , Humanos , Pessoa de Meia-Idade , Osteogênese/fisiologia , Fenótipo , Valores de Referência , Fatores de Transcrição/biossíntese
14.
Clin Orthop Relat Res ; (345): 229-38, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9418645

RESUMO

The effects of cyclosporin A on the occurrence of neuroendocrine peptides in bone induced by demineralized allogeneic and xenogeneic bone matrix were studied in rats. Cyclosporin A enhanced bone induction in demineralized allogeneic bone matrix implants by 40% to 50% at 4 weeks, whereas there was no difference to the control group at 8 weeks. In demineralized xenogeneic bone matrix implants there was virtually no cartilage or bone formation at 4 weeks, but some bone and cartilage formation was seen at 8 weeks. In both cyclosporin A treated groups the net bone formation in demineralized xenogeneic bone matrix implants was increased four to five times at 4 weeks. Cyclosporin A treatment did not alter the temporal occurrence or distribution of neuropeptide containing nerve fibers in the bone induced by allogeneic bone matrix. Fibers containing substance P, calcitonin gene related peptide, neuropeptide Y, vasoactive intestinal peptide, and tyrosine hydroxylase were detected in the ossicles of cyclosporin A treated and control rats. In the xenogeneic bone matrix of the control group, no immunoreactive nerve fibers could be detected at 4 weeks, but at 8 weeks all five neuropeptides were detected. However, after cyclosporin A treatment immunoreactive nerve fibers could be seen at 4 weeks in the demineralized xenogeneic bone matrix implants. Thus, immunologic properties of the inductive matrix affect the yield of mineralized bone and the degree of innervation. Cyclosporin A decreases the immune response and enhances the formation of bone and the number of transmitter identified nerves in demineralized xenogeneic bone matrix induced ossicles.


Assuntos
Matriz Óssea/transplante , Osso e Ossos/química , Ciclosporina/farmacologia , Terapia de Imunossupressão , Imunossupressores/farmacologia , Neuropeptídeos/análise , Ossificação Heterotópica/metabolismo , Animais , Matriz Óssea/imunologia , Matriz Óssea/inervação , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/imunologia , Osso e Ossos/inervação , Peptídeo Relacionado com Gene de Calcitonina/análise , Cartilagem/metabolismo , Técnica de Descalcificação , Masculino , Fibras Nervosas/metabolismo , Fibras Nervosas/ultraestrutura , Neuropeptídeo Y/análise , Ossificação Heterotópica/induzido quimicamente , Osteogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Substância P/análise , Fatores de Tempo , Imunologia de Transplantes , Transplante Heterólogo , Transplante Homólogo , Tirosina 3-Mono-Oxigenase/análise , Peptídeo Intestinal Vasoativo/análise
15.
Spine (Phila Pa 1976) ; 20(22): 2373-8, 1995 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-8578386

RESUMO

STUDY DESIGN: Herniated cervical disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced. OBJECTIVES: Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in cervical intervertebral disc degeneration and in the pathophysiology of cervical radiculopathy. SUMMARY OF BACKGROUND DATA: Neck pain with or without radiculopathy is a common clinical problem, but the etiology of neck pain and the exact pathophysiology of radiculopathy remain uncertain. We have previously reported the production of various biochemical agents by herniated lumbar disc specimens in vitro. Because of a lack of such studies in the literature with respect to the cervical spine, the purpose of this study was to determine whether similar biochemical agents of inflammation and tissue degradation were being produced by herniated cervical disc specimens. METHODS: Eighteen herniated cervical discs were obtained from 15 patients undergoing anterior disc surgery. The specimens were cultured and incubated for 72 hours, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, six cervical discs specimens were obtained from three patients undergoing anterior surgery for traumatic burst fractures, and similar biochemical analyses were performed. RESULTS: The culture media from the herniated cervical disc specimens showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated disc specimens compared with the control discs. Interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of the herniated or control discs. CONCLUSIONS: Herniated cervical disc specimens were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These results were similar to those obtained in herniated lumbar disc specimens that we have previously reported. These products may be intimately involved in the biochemistry of disc degeneration and the pathophysiology of radiculopathy.


Assuntos
Matriz Óssea/metabolismo , Vértebras Cervicais/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Adulto , Matriz Óssea/enzimologia , Matriz Óssea/imunologia , Estudos de Casos e Controles , Vértebras Cervicais/enzimologia , Vértebras Cervicais/imunologia , Técnicas de Cultura , Dinoprostona/biossíntese , Feminino , Humanos , Interleucina-6/biossíntese , Deslocamento do Disco Intervertebral/enzimologia , Deslocamento do Disco Intervertebral/imunologia , Masculino , Metaloendopeptidases/biossíntese , Pessoa de Meia-Idade , Óxido Nítrico/biossíntese
16.
Histochemistry ; 101(5): 347-54, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7928418

RESUMO

The morphology of osteoclasts, primary cells that resorb bone, is well documented; however, the precise details of their terminal differentiation remains obscure. To date, the only morphological criterion for identifying activated functional osteoclasts has been the presence of ruffled borders. We have developed a rat bone marrow culture system in which osteoclast-like cells formed. These cells fulfilled most of the criteria of osteoclasts, and when they were reseeded on calcified tissue, formed numerous resorption lacunae in vitro. To find an immunological marker for functional osteoclasts, we have used these cells in a functional state as antigens for the preparation of monoclonal antibodies (mAb) that reacted with rat osteoclasts; we obtained mAb Ch1 and Ch2. Interestingly, these mAbs reacted with the marginal portion of authentic osteoclasts, where they attached to the bone surface on frozen sections. The reactivity of Ch1 to rat osteoclasts was more restricted than that of Ch2: Ch1 reacted with few tartrate-resistant acid phosphatase (TRAP)-positive cells on a culture plate. These TRAP-positive cells (including mono- and multinucleated cells) were, however, converted to Ch1-positive cells when they were reseeded on calcified tissues. These findings suggested that the antigen recognized by the Ch1 antibody was induced by some factors of matrix proteins released from calcified tissues.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Matriz Óssea/imunologia , Osteoclastos/imunologia , Fosfatase Ácida/imunologia , Fosfatase Ácida/metabolismo , Animais , Sequência de Bases , Células da Medula Óssea , Matriz Óssea/citologia , Fusão Celular , Células Cultivadas , Feminino , Hibridomas/imunologia , Hibridomas/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley
17.
Clin Orthop Relat Res ; (293): 360-5, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8339503

RESUMO

Human bone matrix gelatin (hBMG) was implanted into the quadriceps muscles of mice to determine its osteoinductive activity and immune responses. The host mouse muscle assay reacted positively to the hBMG, providing an experimental basis for clinical application. At two weeks after implantation, cartilage was produced in the mouse muscle, and new bone and bone marrow were formed at three to four weeks after implantation. Human bone matrix gelatin, which was thus shown to have osteoinductive activity, was subsequently implanted in 24 patients. Generally, the bone defects were filled with new bone tissue with increased density within two to four months after operation. Nonunion and delayed unions were healed at two to six months after surgery.


Assuntos
Matriz Óssea/transplante , Neoplasias Ósseas/cirurgia , Fraturas Ósseas/cirurgia , Fraturas não Consolidadas/cirurgia , Tumores de Células Gigantes/cirurgia , Osseointegração , Adulto , Animais , Matriz Óssea/imunologia , Neoplasias Ósseas/fisiopatologia , Feminino , Fraturas Ósseas/fisiopatologia , Fraturas não Consolidadas/fisiopatologia , Tumores de Células Gigantes/fisiopatologia , Humanos , Masculino , Camundongos , Músculos , Transplante Heterotópico
18.
Matrix ; 11(6): 404-11, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1787832

RESUMO

Subcutaneous implantation of xenogeneic demineralized bone matrix does not initiate endochondral bone differentiation. Dissociative extraction in 4 M guanidine-HCl or 6 M urea has shown that the apparent species-specificity of intact bone matrix resides in its insoluble immunogenic component, since there is homology in solubilized osteogenic proteins amongst mammals. To further investigate the species-specificity and cross-species reactivity of bone matrix components, baboon and human demineralized bone matrix (DBM) and bovine osteogenin, purified greater than 50,000-fold and with an apparent molecular mass of 28-42 kilodaltons, were implanted in the subcutaneous space of athymic and euthymic rats and into the rectus abdominis of 16 baboons (Papio ursinus). Baboon DBM was also implanted in athymic and euthymic mice. Alkaline phosphatase activity and histology of implants harvested at day 11 and 30 showed that baboon and human DBM induced endochondral bone differentiation both in athymic rats and baboons. Bovine osteogenin in conjunction with baboon insoluble collagenous matrix induced extensive bone differentiation in athymic rats and baboons. Baboon and human DBM did not induce bone differentiation in euthymic rats and, in athymic mice, baboon DBM failed to induce bone differentiation, determining instead the recruitment of multinucleated giant cells. The results indicate that in rodents bone differentiation induced by intact bone matrix is species specific and that T-cell functions are not a requirement for bone induction, although immunologically competent rats block bone differentiation from xenogeneic matrix. Bone differentiation induced by human DBM in baboons suggests that intact bone matrices may not be species-specific amongst primates.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Matriz Óssea/transplante , Proteínas Morfogenéticas Ósseas , Osteogênese/efeitos dos fármacos , Proteínas/farmacologia , Animais , Matriz Óssea/imunologia , Proteína Morfogenética Óssea 3 , Bovinos , Humanos , Camundongos , Camundongos Nus/imunologia , Papio/imunologia , Ratos , Ratos Endogâmicos F344/imunologia , Ratos Nus/imunologia , Especificidade da Espécie , Linfócitos T Citotóxicos/imunologia , Transplante Heterólogo
19.
Calcif Tissue Int ; 43(4): 263-7, 1988 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3145133

RESUMO

Decalcified and nondecalcified sections of fetal bovine tibia were stained immunohistochemically with a monoclonal antibody against dentin phosphophoryn. In the epiphyseal portion of the long bone, osteoblasts, osteocytes and the bone matrix were stained, but chondrocytes and the cartilage matrix were not. Similar staining was observed in the epiphyseal and diaphyseal portions of bones. These findings suggest that a protein(s) with the same epitope as phosphophoryn may be synthesized and secreted by osteoblasts at the beginning of ossification and may be involved in mineralization of bone tissue. On Western blots of proteins extracted from fetal bovine bone, the antibody reacted with two bands of molecular weights of about 71,000 and 63,000. These proteins and antibody(s) to the proteins may be useful for detection of the phenotype of osteogenesis.


Assuntos
Anticorpos Monoclonais/imunologia , Osso e Ossos/imunologia , Osteogênese , Fosfoproteínas/imunologia , Proteínas/imunologia , Animais , Reações Antígeno-Anticorpo , Matriz Óssea/imunologia , Osso e Ossos/análise , Bovinos , Immunoblotting , Osteoblastos/imunologia , Osteócitos/imunologia , Fenótipo
20.
Biochem Biophys Res Commun ; 151(1): 382-7, 1988 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-3162363

RESUMO

Novel proteins synthesize predominantly in bone have been identified by antibody screening of bone cell cDNA expression libraries. Two unique cDNAs were identified whose structures do not match any known nucleic acid or protein sequence in the NIH computer bank. The first cDNA clone, BP-I, encoded a mRNA of 2300 bases in size which was expressed at high levels in 17/2.8 rat osteosarcoma cells, rat calvarial bone cells and placenta. A second clone, BP-II, encoded a mRNA of 1500 bases which was expressed at high levels in 17/2.8 osteosarcoma cells and in salivary gland. Expression of both mRNAs in osteosarcoma cells was modulated by the calciotropic hormone, vitamin D. Southern blot analyses indicated that the two cDNAs represented distinct, single copy genes in the rat genome. These novel gene products may serve as potential new markers to study bone turnover in metabolic bone disease.


Assuntos
Osso e Ossos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Matriz Óssea/imunologia , Neoplasias Ósseas , Osso e Ossos/citologia , Células Cultivadas , DNA/genética , Eletroforese em Gel de Ágar , Genes , Soros Imunes/imunologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Osteossarcoma , Placenta/citologia , Proteínas/análise , Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Células Tumorais Cultivadas , Vitamina D/fisiologia
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