Experimental model of heterotopic ossification in Wistar rats

Heterotopic ossification (HO) is a metaplastic biological process in which there is newly formed bone in soft tissues adjacent to large joints, resulting in joint mobility deficit. In order to determine which treatment techniques are more appropriate for such condition, experimental models of induced heterotopic bone formation have been proposed using heterologous demineralized bone matrix implants and bone morphogenetic protein and other tissues. The objective of the present experimental study was to identify a reliable protocol to induce HO in Wistar rats, based on autologous bone marrow (BM) implantation, comparing 3 different BM volumes and based on literature evidence of this HO induction model in larger laboratory animals. Twelve male Wistar albino rats weighing 350/390 g were used. The animals were anesthetized for blood sampling before HO induction in order to quantify serum alkaline phosphatase (ALP). HO was induced by BM implantation in both quadriceps muscles of these animals, experimental group (EG). Thirty-five days after the induction, another blood sample was collected for ALP determination. The results showed a weight gain in the EG and no significant difference in ALP levels when comparing the periods before and after induction. Qualitative histological analysis confirmed the occurrence of heterotopic ossification in all 12 EG rats. In conclusion, the HO induction model was effective when 0.35 mL autologous BM was applied to the quadriceps of Wistar rats.

Caractérisation du motif acidique de la sous-unité p28 de l’IL-27 et étude des propriétés partagées entre cette protéine, le CNTF, CLC/CLF et l’interleukine-6

L’interleukine 6 (IL-6) est une cytokine qui joue un rôle essentiel dans l’inflammation. Son récepteur (IL-6R) est composé de la chaîne non signalétique IL-6Rα et de la chaîne transductrice du signal gp130, commune aux cytokines de la famille IL-6. La liaison de l’IL-6 à son récepteur permet l’activation de plusieurs voies de signalisation, notamment des voies Jak/STAT1 et préférentiellement Jak/STAT3. De façon complémentaire, nous avons démontré que l’IL-6 est capable d’activer la voie Jak/STAT5 dans les lymphocytes T CD4. L’activation de cette voie de signalisation pourrait être impliquée dans le rétrocontrôle des effets pro-inflammatoires de l’IL-6 sur les cellules T CD4. Le facteur neurotrophique ciliaire (CNTF) et la « cardiotrophin-like cytokine/cytokine-like factor 1 » (CLC/CLF) sont deux cytokines de la famille de l’IL-6 qui signalent à travers un récepteur commun, le récepteur au CNTF (CNTFR), composé du CNTFRα, « leukaemia inhibitory factor receptor β » (LIFRβ) et gp130. Toutes deux exercent des actions au niveau du système immunitaire, or la chaîne CNTFRα de leur récepteur n’y est pas exprimée. Il a été montré que le CNTFR humain peut également activer un récepteur formé des sous-unités IL-6Rα, LIFRβ et gp130. Nous avons comparé les effets du CNTF et du CLC/CLF de souris sur des transfectants exprimant LIFRβ et gp130 et les chaines α connues de la famille IL-6 (IL-6Rα, IL-11Rβ et CNTFRα). Nos résultats indiquent que le CNTF de souris, comme le CNTF humain est capable d’activer un récepteur formé de l’IL-6Rα, LIFRβ et gp130. Toutefois cette propriété n’est pas partagée par CLC/CLF et le récepteur impliqué dans les effets de cette cytokine sur le système immunitaire reste donc à identifier. L’IL-27 appartient à la famille de l’IL-6 composée d’une sous-unité cytokinique, p28, associée à un récepteur soluble « l’Epstein-Barr virus-induced gene 3» (EBI3). La sous-unité p28 peut s’associer avec le récepteur soluble CLF pour former une cytokine capable d’activer les lymphocytes T. Dans le but de caractériser cette cytokine, nous avons montré que p28/CLF agit aussi sur les lymphocytes B et permet leur différenciation en plasmocytes. Le partage de l’IL-6R par l’IL-6 et p28/CLF semble être à l’origine de la similarité des effets de ces deux cytokines. De plus, nous avons observé des effets semblables à ceux de l’IL-6 suite à l’association de la sous-unité p28 seule avec la chaîne IL-6Rα. En effet, afin de mieux caractériser la cytokine p28/CLF, nous avons étudié les effets dus au recrutement de la chaîne IL-6Rα par la sous-unité p28. Les cytokines de la famille de l’IL-6 sont composées de quatre hélices α disposées de façon anti-parallèle deux à deux. La sous-unité p28 possède, au niveau d’une boucle reliant deux hélices α, un motif de plusieurs acides glutamiques consécutifs (motif polyE) qui n’est retrouvé dans aucune autre cytokine de cette famille. Nous avons démontré que ce motif est impliqué dans la liaison de cette sous-unité avec l’hydroxyapatite et l’os. Cette caractéristique de p28 pourrait permettre un ciblage de l’IL-27 (p28/EBI3) et de p28/CLF préférentiellement vers la niche endostéale des cellules souches et des cellules immunitaires.

In Vivo Comparison of Hard Tissue Regeneration with Human Mesenchymal Stem Cells Processed with Either the FICOLL Method or the BMAC Method

Objective: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. Background: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. Methods: Mononuclear cells, including MSCs, were concentrated with either the synthetic poylsaccharid (FICOLL) method (classic open system-control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system-test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. Results: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. Conclusion: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Função osteoindutora de fragmentos ósseos conservados em glicerina a 98%: estudo experimental em ratos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

EFFECT OF A PERSISTENT INFLAMMATORY PROCESS ON EXPERIMENTAL HETEROTOPIC OSSIFICATION - THE INFLUENCE OF MACROPHAGES

1. We investigated the effect of a persistent carrageenin- or nystatin-induced inflammatory reaction on heterotopic ossification produced by the subcutaneous implant of a demineralized bone matrix in female Swiss mice (25 to 35 g).2. Subcutaneous carrageenin injection (0.3 ml of a 2% solution in saline) into mice induced an inflammatory reaction characterized by a mature granuloma predominantly of macrophages containing particles of the irritant in their cytoplasm and which remained unchanged until the end of the experiment (40th day).3. Subcutaneous nystatin inoculation (30,000 IU in 0.3 ml saline) induced an inflammatory reaction consisting initially of macrophages (4th day) but later turning into an epithelioid granuloma (7th day) consisting predominantly of epithelioid cells and which was present up to the 2 lst day when it was gradually replaced by adipocytes up to the 30th day.4. An intramuscular implant of demineralized bone matrix (DBM, approximately 10 mg) induced the formation of cartilage and bone tissue and of hemopoietic bone marrow (heterotopic ossification) in 100% of the control animals (N = 5). An intramuscular DBM implant in animals that received carrageenin (N = 19) or nystatin (N = 21) induced heterotopic ossification in 100 and 57% (P<0.01)) of the animals, respectively.5. The response to a dorsal subcutaneous DBM implant was essentially negative in control animals (N = 5), whereas implants performed near the site injected with carrageenin (N = 28) or nystatin (N = 31) produced a response in 71 (P <0.01) and 36 % (P<0.01) of the animals, respectively. A DBM implant into the contralateral (control) dorsal subcutaneous tissue of the same animals that received carrageenin (N = 25) or nystatin (N = 29) resulted in heterotopic ossification in 64 (P<0.01) and 7% of the animals, respectively.6. The results suggest that the macrophages present in the mature granuloma induced by carrageenin somehow favored the development of metaplastic plates after subcutaneous DBM implant and that this effect may be systemic since the same response was observed in contralateral subcutaneous tissue.

The effects of low laser irradiation on angiogenesis in injured rat tibiae

The influence of He-Ne laser radiation on the formation of new blood vessels in the bone marrow compartment of a regenerating area of the mid-cortical diaphysis of the tibiae of young adult rats was studied. A small hole was surgically made with a dentistry burr in the tibia and the injured area received a daily laser therapy over 7 or 14 days transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 Jcm-2 were applied during the period of the tibia wound healing investigated. Light microscopic examination of histological sections of the injured area and quantification of the newly-formed blood vessels were undertaken. Low-level energy treatment accelerated the deposition of bone matrix and histological characteristics compatible with an active recovery of the injured tissue. He-Ne laser therapy significantly increased the number of blood vessels after 7 days irradiation at an energy density of 94.5 Jcm-2, but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage. These effects were attributed to laser treatment, since no significant increase in blood vessel number was detected between 8 and 15 non-irradiated control tibiae. Molecular mechanisms involved in low-level laser therapy of angiogenesis in post-traumatic bone regeneration needs further investigation.

Interaction between mesenchymal stem cells and Ti-30Ta alloy after surface treatment

In this study, in vitro cytocompatibility was investigated in the Ti-30Ta alloy after two kinds of surfaces treatments: alkaline and biomimetic treatment. Each condition was evaluated by scanning electron microscopy/energy-dispersive X-ray spectroscopy. Cellular adhesion, viability, protein expression, morphology, and differentiation were evaluated with Bone marrow stromal cells (MSCs) to investigate the short and long-term cellular response by fluorescence microscope imaging and colorimetric assays techniques. Two treatments exhibited similar results with respect to total protein content and enzyme activity as compared with alloy without treatment. However, it was observed improved of the biomineralization, bone matrix formation, enzyme activity, and MSCs functionality after biomimetic treatment. These results indicate that the biomimetic surface treatment has a high potential for enhanced osseointegration. © 2013 Wiley Periodicals, Inc.

Aplicação do copolímero PLA/PGA adicionado ao fosfato de cálcio ao redor de implantes osseointegráveis instalados sem estabilidade primária em tíbia de coelhos: estudo biomecânico, histométrico e imunoistoquímico

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Processo de reparo ósseo de cavidades cirúrgicas preenchidas ou não com osso autógeno e recobertas por membrana de matriz óssea homógena em diabéticos: análise histomorfométrica em tíbia de ratos

Pós-graduação em Odontologia - FOA

Estudo do fenótipo osteoblástico em células-tronco mesenquimais de ratos normotensos e espontaneamente hipertensos na presença ou não de doença periodontal

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo do comportamento biológico da matriz óssea homógena desmineralizada conservada em meio químico: estudo em defeitos ósseos experimentais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ação de biomateriais e laser de baixa intensidade na reparação tecidual óssea: estudo histológico em ratos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A influência da Diabetes Mellitus no reparo de enxertos ósseos autógenos associados ou não à membrana colágena reabsorvível: estudo histomorfométrico em ratos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influência da ovariectomia no processo de reparo de enxerto ósseo autógeno em bloco na mandíbula: estudo histológico em ratas adultas

Pós-graduação em Odontologia - FOA