SciELO - Scientific Electronic Library Online

 
vol.25 número2A method for measuring post-extraction alveolar dimensional changes with volumetric computed tomographyPrevalence of actinic cheilitis in a population of agricultural sugarcane workers índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

  • No hay articulos citadosCitado por SciELO

Links relacionados

  • No hay articulos similaresSimilares en SciELO

Compartir


Acta Odontológica Latinoamericana

versión On-line ISSN 1852-4834

Acta odontol. latinoam. vol.25 no.2 Buenos Aires oct. 2012

 

ARTÍCULOS ORIGINALES

Histologic and histomorphometric study of bone repair under acute Trypanosoma cruzi infection in rats

 

Tammy Steimetz1, Alejandro A. Gorustovich3,4, Ana M. Collet1, Olga Sanchez Negrette2, María A. Segura2, Miguel A. Basombrío2,4, María B. Guglielmotti1,4

1 Department of Oral Pathology, School of Dentistry, University of Buenos Aires
2 Institute of Experimental Pathology, School of Health Sciences, National University of Salta
3 Institute for Interdisciplinary Studies in Engineering (IESIING). School of Engineering and Computer Science. Catholic University of Salta (FII-UCASAL). Institute of Science Technology and Engineering "Hilario Fernández Long" (INTECIN) UBA-CONICET
4 National Research Council, Argentina.

CORRESPONDENCE Dra. Tammy Steimetz M. T. de Alvear 2142, 2° A (1122AAH) Buenos Aires, Argentina tammysteimetz@gmail.com


ABSTRACT

Trypanosoma cruzi (T. cruzi) is an intracellular protozoan pathogen that causes American trypanosomiasis (Chagas disease). The aim of this study was to evaluate the histopathological effects of acute infection by T. cruzi on bone repair. Wistar rats were used throughout. The animals were assigned to two groups: Control Group (CG n =20) and Experimental Group (EG n =20). All the animals were anesthetized, at t0 the first lower right molar was extracted. The EG animals were inoculated subcutaneously at t0 with 0.1 mL of 105 trypomastigotes of the virulent strain Tulahuen of T. cruzi. The CG animals were administered an equivalent volume of saline solution subcutaneously. The animals in both groups were euthanized at 15 days post-infection and tooth extraction. The mandibles were resected, fixed in formalin solution, radiographed, decalcified and embedded in paraffin. Bucco-lingually oriented sections were obtained at the level of the mesial tooth socket of the first lower molar, and stained with hematoxylin-eosin. Total alveolar volume (TV) and bone volume (TBV/TV) in the apical third of the tooth socket were evaluated histomorphometrically. The histological analysis revealed an alteration in post-extraction bone tissue repair in animals infected by T. cruzi. A reduction in osteogenic activity was observed concomitant with a rise in quiescent and eroded bone surfaces. Histomorphometric evaluation revealed a significant reduction (19%) in total alveolar volume (TV) and bone volume (TBV/TV) (24%) in the apical third of the tooth socket in animals infected with T. cruzi in comparison to non-infected animals (p<0.05). The results obtained using this experimental model showed decreased osteogenesis in bone tissue repair under acute Trypanosoma cruzi infection in rats.

Keywords: Alveolar ridge; Wound healing; Bone; Osteogenesis; Tooth socket; Trypanosoma cruzi.

RESUMEN

Estudio histológico e histomorfométrico del efecto de la infección aguda por Trypanosoma cruzi en la reparación ósea post-exodoncia

El Trypanosoma cruzi (T. cruzi) es un protozoario intracelular que causa Trypanosomoniasis Americana (Enfermedad de Chagas). El objetivo del presente trabajo fue el estudio histopatologico del efecto de la infeccion aguda por Trypanosoma cruzi sobre la reparacion del tejido oseo. Se utilizaron ratas Wistar macho que fueron asignadas a dos grupos: Grupo Control (GC n =20) y Grupo Experimental (GE n =20). Los animales de ambos grupos, bajo anestesia general intraperitoneal, fueron sometidos a t0, a exodoncia del primer molar inferior derecho, en el GE fueron inoculados,a t0 por via subcutanea en la region inguinal izquierda con 0.1 mL de 105 tripomastigotes de la cepa virulenta Tulahuen de Trypanosoma cruzi. A los animales del GC se les administro el volumen equivalente de solucion salina por via subcutanea. A los animales de ambos grupos se les practico la eutanasia a los 15 dias. Se resecaron las mandibulas, se fijaron en solucion de formol al 10%, se radiografiaron, se descalcificaron y se incluyeron en parafina. Se obtuvieron cortes orientados en sentido vestibulo-lingual a nivel del alveolo mesial del primer molar inferior derecho y se colorearon con hematoxilina–eosina para su posterior estudio histologico e histomorfometrico. Histologicamente se observo una menor actividad osteogenica a expensas de un incremento de las superficies quiescentes y de las superficies erosivas en el GE. En la evaluacion histomorfometrica se detecto disminucion estadiasticamente significativa del volumen oseo total (19%) y del volumen trabecular en el tercio apical del alveolo (24%) en el GE con respecto al GC (p<0.05). Los resultados obtenidos en este modelo experimental evidencian una disminucion de la osteogenesis en la reparacion osea en ratas con infeccion aguda por Trypanosoma cruzi.

Palabras clave: Reborde alveolar; Reparacion tisular; Hueso; Osteogenesis; Alveolo dentario; Trypanosoma cruzi.


 

INTRODUCTION

Alveolar bone is a specialized part of the mandibular and maxillary bones that forms the primary support structure for the teeth. Alveolar bone is constantly renewed by modeling and remodeling mechanisms in response to functional demands and local and systemic factors 1,2. Bone repair is a highly regulated process. All stages of the repair process are controlled by a wide variety of different growth factors and cytokines, and can be derailed by various endogenous and exogenous factors e.g. systemic infection1-6. Several species of kinetoplastid protozoa cause major human infectious diseases. Trypanosoma cruzi (T. cruzi) is an intracellular protozoan pathogen that causes American trypanosomiasis (Chagas disease), an endemic illness that affects several million people in Latin America7-9. T. cruzi is usually transmitted by infected triatomine vectors8,10,11. However, as T. cruzi develops a lifelong infection in humans, these people can serve as parasite reservoirs throughout their lifetime. Thus, the risk of congenital and/or horizontal transmission by infected blood transfusion or solid organ transplant may become a major problem in non-endemic regions, increased by the migration of people from endemic areas in South and Central America to developed countries8,9,12,13.
Trypomastigotes, the mammalian infective forms of T. cruzi, are relatively large (~20 mm in length), motile organisms that have the capacity to infect most nucleate cell types. Non-dividing trypomastigotes must establish residence within the host cell cytoplasm and differentiate into amastigotes9,10. During the acute phase of the infection, the rupture of amastigote nests provokes destruction of the host cells and triggers inflammatory processes and intense immune responses8,9,14-21. Emerging evidence shows that the immune and skeletal systems share a number of regulatory molecules including cytokines, receptors, signaling molecules and transcription factors22-25. Therefore it has been suggested that the physiology and pathology of one system may affect the other. In 2006, Morocoima A. et al. reported invasion in hyaline cartilage cells and bone cells including the marrow of laboratory mice infected with T. cruzi isolates from urban and rural areas of Venezuela26. There is no study to date on the effect of infection by T. cruzi on the bone repair response. Given that the alveolar bone healing after tooth extraction in rats provides a suitable experimental model for the study of bone formation and can be considered a sensitive indicator of bone damage under different experimental conditions27-33, the aim of this study was to assess the effects of acute infection by Trypanosoma cruzi on alveolar bone healing in rats employing histological and histomorphometric evaluation.

MATERIALS AND METHODS

Animals
Forty male Wistar rats (International Laboratory Code Registry: Hsd:Wi-ffyb), 21-25 days old, were used throughout. The animals were not given a special diet. They were fed rat chow and given water ad libitum, housed in steel cages and maintained on a 12:12 hour light-dark cycle. All animal experiments were carried out according to the guidelines of the National Institutes of Health for the care and use of laboratory animals (NIH Publication No 85- 23, Rev. 1985). The protocol was examined and approved by the institutional ethics committee at the School of Dentistry, University of Buenos Aires.

Experimental Procedure
Surgical procedure

The animals were assigned to two groups: Control Group (CG n =20) and Experimental Group (EG n =20). All the animals were anesthetized by intraperitoneal administration of a 4:1 solution of ketamine/xylazine (ketamine chlorhydrate, 50 mg/mL, Ketamina 50R Holliday-Scott, Buenos Aires, Argentina) and xylazine, 20 mg/mL (RompunR Bayer, Buenos Aires, Argentina) at a dose of 0.15 mL per 100 g body weight. At t0 the first lower right molar was extracted according to the technique described by Guglielmotti et al.27

Experimental infection
The EG animals were inoculated subcutaneously (s.c.) in the left inguinal region at t0, under ether anesthesia, with 0.1 mL of 105 trypomastigotes of the virulent strain Tulahuen of Trypanosoma cruzi kindly provided by the Institute of Experimental Pathology, School of Health Sciences, National University of Salta. The CG animals were administered an equivalent volume of saline solution subcutaneously.

Parasitaemia
Evaluation was performed by direct detection of parasitaemia (fresh blood observation) in blood samples extracted from the tail vein under anesthesia (10 μL with a heparinized capillary tube) at 7 and 15 days after the initial infection. The samples were placed between a glass slide and coverslip and examined by light microscopy. The number of parasites in 100 fields was counted with a x40 objective.

Haematological parameters
The values of haematocrit (Htc) and haemoglobinemia (Hb) were determined at baseline (t0) and 7 and 15 days post-initial infection. The animals in both groups were euthanized 15 days post-infection and tooth extraction. The mandibles were resected, fixed in 10% formalin solution and radiographed.

Histological processing
The mandibles were decalcified in 5% formic acid, embedded in paraffin, and semi-serially sectioned, at the level of the mesial tooth socket of the first lower right molar, in a frontal plane (bucco-lingual direction) at 10 mm thickness and stained with hematoxylin-eosin.

Histomorphometric
Evaluation
Total Alveolar Volume
Total alveolar volume (TV) was considered as the bone tissue and its marrow spaces situated above line a drawn tangential to the upper cortical border of the mandibular canal and perpendicular to the external surface of the buccal plate30.

Bone volume in the apical third of the tooth socket
Bone volume (TBV/TV) was considered as the ratio between the trabecular volume (TBV) and the total volume (TV), measured in the apical third of the socket as previously reported 30. The following parameters were determined in the apical third of the tooth socket: percentage of osteoblast surface (Ob.S), eroded surface (ES), and quiescent surface (QS). Osteoblast surfaces are covered with osteoid seams and mature osteoblasts. Eroded surfaces are scalloped with Howship's lacunae with or without osteoclasts. Quiescent surfaces are covered with bone lining cells.34 Histomorphometric determinations were performed on sections using a light microscope (Zeiss Axioscop 2 MOP, Carl Zeiss, Jena, Germany), on line with an image analysis system (Kontron KS300 v. 2, Kontron Elektronik, Munich, Germany).

Statistical Analysis Student's t-test was used for statistical analysis of the data (p<0.05). Data are presented as means ±SD.

RESULTS

No immediate or mediate post-operative complications were observed.

Parasitaemia
Parasitaemia (5 ±2 /100 fields) was detected in the EG at 7 days post-infection. A tendency to negative parasitaemia values was observed at 15 days postinitial infection (1 ±1 /100 fields).

Haematological Parameters T
he EG exhibited an increase (10%) in haematocrit at 7 days post-infection compared to baseline values (p<0.05; Table 1). An increase above control values was observed in haematocrit (5%) and haemoglobinemia (33%) at 15 days post-infection (p<0.05) (Table 1).

Table 1: Haematological parameters.

Radiographic Study
The post-extraction tooth sockets of control and experimental animals were filled with radiopaque tissue.

Histological Study
Active osteogenesis evidenced by neoformed trabeculae filling almost the entire tooth socket was observed with light microscopy in control animals
15 days post-extraction (Fig. 1A). The woven bone tissue was lined with cuboidal osteoblasts. Full epithelialization of the alveolar ridge was observed.


Fig. 1:
Microphotograph of the bucco-lingual section of the mesial tooth socket of the first lower right molar 15 days post-extraction. (A) Note that the socket is almost completely filled with woven bone in a CG sample (hematoxylin and eosin; original magnification x100). (B) EG sample exhibiting a reduction in total alveolar volume and in bone volume in the apical third of the tooth socket (hematoxylin and eosin; original magnification x100).

The tooth socket of experimental animals was filled with woven bone tissue. Trabeculae lined with cuboidal osteoblasts and a predominance of bone lining cells, eroded surfaces and osteoclasts were observed (Fig. 1B). Noticeable presence of eosinophils, granuloma-like structures, with foam cells (macrophages) containing amastigotes were detected between the trabeculae (Fig. 2 A and B). No difference in the healing of soft tissues lining the alveolar ridge was observed compared to control.


Fig. 2:
(A) Note the granuloma-like structures between the trabeculae (arrows) (hematoxylin and eosin; original magnification x400) and (B) the presence of eosinophils (arrowhead) and foam cells containing amastigotes (arrow) (hematoxylin and eosin; original magnification x1000) in EG samples.

Histomorphometric evaluation
Total Alveolar Volume (TV, in mm2) The EG exhibited a reduction in total alveolar volume (1.4 x 106 ±2 x 105) as compared to CG (1.7 x 106 ±2 x 105). Statistically significant differences were observed between the groups (p< 0.05).

Bone Volume in the Apical Third (TBV/TV, in %)
The EG exhibited reduced bone volume in the apical third of the tooth socket (44 ±10) as compared to control values (58 ±7). Statistically significant differences were found between the groups (p< 0.05). The EG showed a statistically significant reduction (69%) (p<0.05) in the percentage of osteoblast surfaces concomitant with an increase in eroded and quiescent surfaces (Fig. 3).


Fig. 3:
Histomorphometric evaluation showed a significant reduction in the percentage of osteoblast surfaces (Ob.S) and a significant increase in eroded (ES) and quiescent surfaces (QS) in experimental animals as compared to controls. Values are means ±SD (*p < 0.05).

DISCUSSION

Morocoima A. et al. were the first to report the presence of T. cruzi stages in bone in a murine experimental parasitism model 26. We describe in the experimental model used, for the first time, the bone tissue repair response to in vivo acute T. cruzi infection. Our results show that acute infection by the virulent strain Tulahuen of T. cruzi affects bone tissue repair in rats. The histological and histomorphometric analyses showed a decrease in osteogenic acitivity concomitant with an increase in quiescent and eroded bone surfaces. These alterations resulted in a reduction in alveolar total volume and bone volume in the apical third of the tooth socket in animals infected with T. cruzi. In a previous study by our laboratory we described the chronology of socket healing after tooth extraction in the rat employing radiographic, histologic, and histomorphometric techniques27. The newly formed bone after tooth extraction undergoes intramembranous ossification35-37. Our previous studies using histomorphometric methods showed that under normal conditions maximum bone formation occurs on the fourteenth day after tooth extraction27-33.
Various studies have demonstrated that experimental infection of rats with T. cruzi reproduces several aspects of the clinicopathological features of human chagasic infection17,18,20,38,39. In these studies, inoculation at weaning with living T. cruzi in rats resulted in a self-resolving acute infection characterized by marked parasitaemia and production of specific antibodies17,18,20,21. Briefly, parasites were evident microscopically by day 7 post-infection and declined gradually, as the adaptive immune response developed. In our study the parasitaemia reached its peak on day 7 post-infection. At this experimental time, an increase in haematocrit value was observed. Recently, Berra et al.40 demonstrated increased plasma viscosity in experimentally T. cruzi – infected rats that was correlated with high blood parasite levels at 7 days post-infection. Marcondes et al.41 reported that acute T. cruzi infection in mice results in alterations of the haematopoietic system associated with bone marrow suppression. The mechanisms involved in myelosuppression are not clear. The blood and bone marrow alterations may result from suppression of precursor cells via secreted cytokines or parasite or cell-dependent cytotoxicity. Host resistance to T. cruzi infection, both in humans and in experimental models, induces cells from the monocyte/macrophage lineage and other nonimmune cells to produce high levels of proinflammatory cytokines15,16,19. Various inhibitory cytokines exert profound inhibitory signals at critical stages of erythropoiesis, the production of erythropoietin and the maturation and differentiation of colony forming units-erythroid (CFU-E)41. These findings would explain the haematologic response observed 15 days post-infection in the present study.
Inflammatory reactions are among the first host responses to infection with T. cruzi15,16,19. Most inflammatory cells can interact with different life cycle stages of T. cruzi, causing parasite destruction extracellularly by antibody-dependent, cell-mediated cytotoxicity and intracellularly without antibody
requirement15,16,18. In the present study we observed the presence of foam cells (macrophages) containing amastigotes in the granulation tissue and eosinophils in bone marrow in agreement with the results informed by Morocoima , et al.26 Rowland and Sibley-Phillips reported an increase in femoral bone marrow eosinophil levels during T. cruzi infection in mice with a peak coincident with that of parasitaemia42.
Oba et al. showed that the eosinophil chemotactic factor- L (ECF-L), a previously described chemotactic factor for eosinophils, acts at the later stages of osteoclast formation43. In addition, cytokines play a critical role in the regulation of osteoclast differentiation and activation of initiation of bone resorption44,45. In the present study we demonstrated that acute infection by T. cruzi induces an increase in areas of bone resorption and quiescent bone and a concomitant reduction in bone formation surfaces. Recently, Unnikrishnan and Burleigh46 reported that T. cruzi elicits the selective repression of basal connective tissue growth factor (CTGF) expression in fibroblasts, a TGF-b dependent cytokine normally up-regulated in tissue repair processes. CTGF is a member of the CCN (cyr61, ctgf, nov) proteins, which are an important family of matricellular regulatory factors involved in internal and external cell signaling. This family participates in angiogenesis, chondrogenesis, and osteogenesis, and they are probably involved in the control of cell proliferation and differentiation47. Kanyama et al. demonstrated that CTGF was expressed at an early stage of the rat tooth extraction wound healing process, and stated that CTGF may play an important role in angiogenesis and granulation tissue formation specifically at the early healing stage after tooth extraction to initiate alveolar bone repair48. In addition, Safadi et al. demonstrated that CTGF plays a role in osteoblast differentiation and function in vitro and elicits an osteogenic response in vivo49.
Unnikrishnan and Burleigh indicated that the mechanisms of T. cruzi – mediated repression of CTGF are complex and involve targeted inhibition of the TGF-b-induced host signaling pathway followed by down-regulation of the extracellular matrix proteins, fibronectin, and collagen I a1 expression.46 These mechanisms could be responsible for the deleterious effects of acute T. cruzi infection on bone tissue repair observed herein. The results of our study reveal that acute infection by the virulent strain Tulahuen of Trypanosoma cruzi affects the bone tissue repair process in the post-extraction tooth socket in rats quantitatively and qualitatively.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the technical assistance of Federico Ramos (Institute of Experimental Pathology, School of Health Sciences, National University of Salta). This study was supported by grants 1295 of the National University of Salta and O020 of the University of Buenos Aires.

REFERENCES

1. Principles in Bone Biology. Bilezikian J, Raisz L, Rodan G. 2nd Edition, 2002. Academic Press.         [ Links ]

2. Pathologic Basis of Disease. Robbins and Cotran. Tissue renewal and repair: regeneration, healing, and fibrosis. Kumar V, Abbas AK, Fausto N. 7th Edition, 2005. Elsevier Saunders.         [ Links ]

3. Thomas T. New actors in bone remodelling: a role for the immune system. Bull Acad Natl Med. 2010;194:1493-1503.         [ Links ]

4. Arnott JA, Lambi AG, Mundy C, Hendesi H, Pixley RA, Owen TA, Safadi FF, Popoff SN. The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis. Crit Rev Eukaryot Gene Expr. 2011;21:43-69.         [ Links ]

5. Schett G. Effects of inflammatory and anti-inflammatory cytokines on the bone. Eur J Clin Invest. 2011;41:1361-1366.         [ Links ]

6. Bone Regeneration and Repair. Biology and Clinical Applications. Lieberman JR, Friedlaender GE. 2005. Humana press.         [ Links ]

7. El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN, Ghedin E, Worthey EA, Delcher AL, Blandin G, Westenberger SJ, Caler E, Cerqueira GC, Branche C, Haas B, Anupama A, Arner E, Aslund L, Attipoe P, Bontempi E, Bringaud F, Burton P, Cadag E, Campbell DA, Carrington M, Crabtree J, Darban H, da Silveira JF, de Jong P, Edwards K, Englund PT, Fazelina G, Feldblyum T, Ferella M, Frasch AC, Gull K, Horn D, Hou L, Huang Y, Kindlund E, Klingbeil M, Kluge S, Koo H, Lacerda D, Levin MJ, Lorenzi H, Louie T, Machado CR, McCulloch R, McKenna A, Mizuno Y, Mottram JC, Nelson S, Ochaya S, Osoegawa K, Pai G, Parsons M, Pentony M, Pettersson U, Pop M, Ramirez JL, Rinta J, Robertson L, Salzberg SL, Sanchez DO, Seyler A, Sharma R, Shetty J, Simpson AJ, Sisk E, Tammi MT, Tarleton R, Teixeira S, Van Aken S, Vogt C, Ward PN, Wickstead B, Wortman J, White O, Fraser CM, Stuart KD, Andersson B. The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 2005;15;309:409-415.         [ Links ]

8. Barrett MP, Burchmore RJS, Stich A, Lazzari JO, Frasch AC, Cazzulo JJ, Krishna S. The trypanosomiases. Lancet 2003;362:1469-1480.         [ Links ]

9. Manson's Tropical Disease. Cook GC, Zumla A American trypanosomiasis (Chagas disease). Miles MA. 21st Edition, 2003, Elsevier Science.         [ Links ]

10. Tyler KM, Engman DM. The life cycle of Trypanosoma cruzi revisited. Int J Parasitol 2001;31:472-481.         [ Links ]

11. Diosque P, Padilla AM, Cimino RO, Cardozo RM, Negrette OS, Marco JD, Zacca R, Meza C, Juarez A, Rojo H, Rey R, Corrales RM,Nasser JR, Basombrio MA. Chagas disease in rural areas of Chaco Province, Argentina: epidemiologic survey in humans, reservoirs, and vectors. Am J Trop Med Hyg 2004;71:590-593.         [ Links ]

12. Assal A, Corbi C. Chagas disease and blood transfusion: an emerging issue in non-endemic countries. Transfus Clin Biol 2011;18:286-291.         [ Links ]

13. Schwartz BS, Paster M, Ison MG, Chin-Hong PV. Organ donor screening practices for Trypanosoma cruzi infection among US Organ Procurement Organizations. Am J Transplant 2011;11:848-851.         [ Links ]

14. Burleigh BA, Woolsey AM. Cell signaling and Trypanosoma cruzi invasion. Cell Microbiol 2002;4:701-711.         [ Links ]

15. Hall BS, Pereira MA. Dual role for transforming growth factor beta-dependent signaling in Trypanosoma cruzi infection of mammalian cells. Infect Immun 2000; 68:2077- 2081.         [ Links ]

16. Fabrino DL, Leon LL, Parreira GG, Genestra M, Almeida PE, Melo RC Peripheral blood monocytes show morphological pattern of activation and decreased nitric oxide production during acute Chagas' disease in rats. Nitric Oxide 2004;11:166-174.         [ Links ]

17. Revelli S, Davila H, Ferro ME, Romero-Piffiguer M, Musso O, Valenti J, Bernabo J, Falcoff E, Wietzerbin J, Bottasso O. Acute and chronic experimental Trypanosoma cruzi infection in the rat. Response to systemic treatment with recombinant rat interferon-gamma. Microbiol Immunol 1995; 39:275-281.         [ Links ]

18. Une C, Andersson J, Orn A. Role of IFN-alpha/beta and IL- 12 in the activation of natural killer cells and interferongamma production during experimental infection with Trypanosoma cruzi. Clin Exp Immunol 2003;134:195-201.         [ Links ]

19. Kierszenbaum F, Villalta F, Tai PC. Role of inflammatory cells in Chagas' disease. III. Kinetics of human eosinophil activation upon interaction with parasites (Trypanosoma cruzi). J Immunol 1986;136:662-666.         [ Links ]

20. Marcipar IS, Risso MG, Silber AM, Revelli S, Marcipar AJ. Antibody maturation in Trypanosoma cruzi-infected rats. Clin Diagn Lab Immunol 2001;8:802-805.         [ Links ]

21. Pascutti MF, Bottasso OA, Hourquescos MC, Wietzerbin J, Revelli S. Age-related increase in resistance to acute Trypanosoma cruzi infection in rats is associated with an appropriate antibody response. Scand J Immunol 2003; 58:173-179.         [ Links ]

22. Arron JR, Choi Y. Bone versus immune system. Nature 2000;408:535-536.         [ Links ]

23. Rho J, Takami M, Choi Y. Osteoimmunology: interactions of the immune and skeletal systems. Mol Cells 2004;17:1-9.         [ Links ]

24. Walsh MC,Kim N, Kadono Y, RhoJ, Lee SY, Lorenzo J, Choi Y. Osteoimmunology: Interplay Between the Immune System and Bone Metabolism. Annu. Rev. Immunol 2006; 24: 33-63.         [ Links ]

25. Lee SH, Kim TS, Cho Y, Lorenzo J. Osteoimmunology: cytokines and the skeletal system. BMB reports 2008;4: 495-510.         [ Links ]

26. Morocoima A, Rodriguez M, Herrera L, Urdaneta-Morales S. Trypanosoma cruzi: experimental parasitism of bone and cartilage. Parasitol Res. 2006;99:663-668.         [ Links ]

27. Guglielmotti MB, Cabrini RL. Alveolar wound healing and ridge remodeling after tooth extraction in the rat: a histologic, radiographic and histometric study. J Oral Maxillofac Surg 1985;43:359-364.         [ Links ]

28. Guglielmotti MB, Ubios AM, Cabrini RL 1986 Alveolar wound healing after X-irradiation: a histologic, radiographic and histometric study. J Oral Maxillofac Surg 44: 972-976.         [ Links ]

29. Guglielmotti MB, Ubios AM, Cabrini RL. Alveolar wound healing under uranyl nitrate intoxication. J Oral Pathol 1985;14:565-572.         [ Links ]

30. Guglielmotti MB, Ubios AM, Cabrini RL. Morphometric study of the effect of a low dose of uranium in bone healing. Acta Stereol 1987;6:357-366.         [ Links ]

31. Ubios AM, Guglielmotti MB Cabrini RL. Effect of diphosphonate against the inhibition of bone formation by Xradiation. J Oral Pathol 1986;15:500-505.         [ Links ]

32. Ubios AM, Guglielmotti MB, Cabrini RL. Ethane-1- hydroxy-1,1-diphosphonate (EHDP) counteracts the inhibitory effects of uranyl nitrate on bone formation. Arch Env Health 1990;45:374-373.         [ Links ]

33. Ubios AM, Jares Furno G, Guglielmotti MB. Effect of calcitonin on alveolar wound healing. J Oral Path Med 1991; 20:322-324.         [ Links ]

34. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987;2:595-610.         [ Links ]

35. Hsieh YD, Devlin H, Roberts C. Early alveolar ridge osteogenesis following tooth extraction in the rat. Arch Oral Biol 1994;39:425-428.         [ Links ]

36. Devlin H. Early bone healing events following rat molar tooth extraction. Cells Tissues Organs 2000;167:33-37.         [ Links ]

37. Elsubeihi ES, Heersche JN. Quantitative assessment of postextraction healing and alveolar ridge remodelling of the mandible in female rats. Arch Oral Biol 2004;49:401-412.         [ Links ]

38. Mahler E, Hoebeke J, Levin MJ. Structural and functional complexity of the humoral response against the Trypanosoma cruzi ribosomal P2 beta protein in patients with chronic Chagas' heart disease. Clin Exp Immunol 2004;136: 527-534.         [ Links ]

39. Savio-Galimberti E, Dos Santos Costa P, Campos De Carvalho AC, Ponce-Hornos JE. Mechanical and energetic effects of chronic chagasic patients' antibodies on rat myocardium. Am J Physiol Heart Circ Physiol 2004;287: H1239-H1245.         [ Links ]

40. Berra HH, Piaggio E, Revelli SS, Luquita A. Blood viscosity changes in experimentally Trypanosoma cruzi-infected rats. Clin Hemorheol Microcirc 2005;32:175-182.         [ Links ]

41. Marcondes MC, Borelli P, Yoshida N, Russo M. Acute Trypanosoma cruzi infection is associated with anemia, thrombocytopenia, leukopenia, and bone marrow hypoplasia: reversal by nifurtimox treatment. Microbes Infect 2000; 2:347-352.         [ Links ]

42. Rowland EC, Sibley-Phillips S. Bone marrow eosinophil levels in Trypanosoma cruzi infected mice. J Parasitol 1984; 70:819-820.         [ Links ]

43. Oba Y, Chung HY, Choi SJ, Roodman GD. Eosinophil chemotactic factor-L (ECF-L): a novel osteoclast stimulating factor. J Bone Miner Res 2003; 18:1332-1341.         [ Links ]

44. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003; 423:337-342.         [ Links ]

45. Takayanagi H. Mechanistic insight into osteoclast differentiation in osteoimmunology. J Mol Med 2005; 83:170-179.         [ Links ]

46. Unnikrishnan M, Burleigh BA. Inhibition of host connective tissue growth factor expression: a novel Trypanosoma cruzi-mediated response. FASEB J 2004; 18:1625-1635.         [ Links ]

47. Perbal B. CCN proteins: multifunctional signaling regulators. Lancet 2004; 363:62-64.         [ Links ]

48. Kanyama M, Kuboki T, Akiyama K, Nawachi K, Miyauchi FM, Yatani H. Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction. Arch Oral Biol 2003; 48: 723-730.         [ Links ]

49. Safadi FF, Xu J, Smock SL, Kanaan RA, Selim AH, Odgren PR. Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo. J Cell Physiol 2003; 196:51-62.         [ Links ]

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons