Reparo de defeitos ósseos críticos usando hidroxiapatita sintética ou bovina com ou sem fração mononuclear da medula óssea: análise histomorfométrica e imuno-histoquímica / Repair of critical size bone defects using synthetic or bovine hydroxyapatite with or without bone marrow mononuclear fraction: histomorphometric and immunohistochemical analysis

O presente trabalho teve como objetivo avaliar a regeneração óssea de defeitos ósseos críticos na calvária de ratos com a utilização de enxerto xenógeno Bio-Oss Small ( Bio-Oss® Geistlich Pharma AG, Wolhusen, Suíça) e hidroxiapatita sintética, (Alobone poros Osseocon Biomateriais Ltda., Rio de Janeiro/RJ, Brasil ) puros e enriquecidos com a fração mononuclear medular de célula tronco. Material e métodos: quarenta ratos adultos jovens machos, Sprague Dawley, com doze semanas distribuídos em cinco grupos com oito animais, sendo assim denominados: grupo controle, grupo 1, grupo 2, grupo 3, e grupo 4. Além dos quarenta ratos adultos jovens, mais oito ratos adultos jovens machos, Sprague Dawley, com doze semanas foram utilizados como doadores de medula óssea. Os cinco grupos foram submetidos a defeitos ósseos críticos com trefina de 8 (oito) milímetros de diâmetro, na região temporoparietal. No grupo controle não teve a colocação de biomaterial, no grupo 1 os defeitos críticos foram preenchidos por hidroxiapatita sintética ,0.10 gramas, (Alobone poros Osseocon Biomateriais Ltda., Rio de Janeiro/RJ, Brasil), no grupo 2 foram preenchidos com hidroxiapatita bovina,0,10 gramas, Bio-Oss Small (Bio-Oss® Geistlich Pharma AG, Wolhusen, Suíça), no grupo 3 os defeitos críticos foram preenchidos por hidroxiapatita sintética,0,10 gramas, (Alobone poros Osseocon Biomateriais Ltda, Rio de Janeiro/RJ, Brasil) enriquecida com 300 µl da fração mononuclear medular de células tronco na concentração de 1 x 10 6, obtida dos animais doadores de medula, e no grupo 4 os defeitos críticos foram preenchidos por hidroxiapatita bovina, 0,10 gramas, Bio-Oss Small (Bio-Oss® Geistlich Pharma AG, Wolhusen, Suíça) também enriquecida com 300 µl da fração mononuclear medular de células tronco na concentração de 1 x 10 6, obtida dos animais doadores de medula Após oito semanas, os cinco grupos tiveram todos os animais eutanasiados, por intermédio de overdose anestésica de Pentobarbital (180 mg/ kg intraperitoneal). Os grupos, foram submetidos a análises histomorfométrica, preparados corados com tricrômico de Masson, e técnicas imuno-histoquímica de VEGF e Osteopontina. Não existiu diferença estatisticamente significativa entre os grupos 1, 2, 3 e 4, porém existiu entre o grupo controle e os demais grupos que receberam tratamento para regeneração dos defeitos ósseos críticos.

The present study aimed to evaluate the bone regeneration of critical bone defects in the calvaria of rats using xenogenous grafts, Bio-Oss Small (Bio-Oss® Geistlich Pharma AG, Wolhusen, Switzerland) and synthetic hypooxypatite, (Alobone poros Osseocon Biomateriais Ltda., Rio de Janeiro / RJ, Brazil) pure or enriched with the medullary mononuclear fraction of stem cells, in the material and methods: forty young adult male rats, Sprague Dawley, were used, with twelve weeks distributed in five groups with eight animals, being thus named: control group, group 1, group 2, group 3, and group 4. In addition to the forty young adult rats, eight more animals were used as bone marrow donors. The Five groups were submitted to critical size bone defects with 8 (eight) millimeter diameter trephine, in the temporoparietal region. In the control group there was no biomaterial placement, in group 1 the critical size bone defects were filled with synthetic hydroxyapatite, 0,10 grams, (Alobone poros Osseocon Biomateriais Ltda., Rio de Janeiro / RJ, Brazil), in group 2 they were filled with bovine hydroxyapatite,0,10gramas, Bio-Oss Small (Bio-Oss® Geistlich Pharma AG, Wolhusen, Switzerland), in group 3 the critical size bone defects were filled with synthetic hydroxyapatite,0,10 grams, (Alobone poros Osseocon Biomateriais Ltda., Rio de Janeiro / RJ, Brazil) enriched with 300 µl of BMMF containing 1x106 cells, obtained from bone marrow donors animals, and in group 4 the critical size bone defects were filled with bovine hydroxyapatite, 0,10 grams Bio-Oss Small (Bio-Oss® Geistlich Pharma AG, Wolhusen, Switzerland) enriched with 300 µl of BMMF containing 1x106 cells, obtained from bone marrow donors animals. After eight weeks, all five of animals were euthanized by Pentobarbital anesthetic overdose (180 mg / kg intraperitoneal). The groups were submitted to histomorphometric analysis, using Masson's trichrome Stain, and immunohistochemical techniques of VEGF and Osteopontin. There was no statistically significant difference among groups 1, 2, 3 and 4, but it did exist with the control group and the other groups that received treatment for the regeneration of critical bone defects.

Safety of Autologous Umbilical Cord Blood Therapy for Acquired Sensorineural Hearing Loss in Children

BACKGROUND AND OBJECTIVES: Sensorineural hearing loss (SNHL) in children is associated with neurocognitive morbidity. The cause of SNHL is a loss of hair cells in the organ of Corti. There are currently no reparative treatments for SNHL. Numerous studies suggest that cord blood mononuclear cells (human umbilical cord blood, hUCB) allow at least partial restoration of SNHL by enabling repair of a damaged organ of Corti. Our objective is to determine if hUCB is a safe treatment for moderate to severe acquired SNHL in children. SUBJECTS AND METHODS: Eleven children aged 6 months to 6 years with moderate to severe acquired SNHL were treated with intravenous autologous hUCB. The cell dose ranged from 8 to 30 million cells/kg body weight. Safety was assessed by measuring systemic hemodynamics during hUCB infusion. Infusion-related toxicity was evaluated by measuring neurologic, hepatic, renal and pulmonary function before and after infusion. Auditory function, auditory verbal language assessments and MRI with diffusion tensor imaging (DTI) were obtained before and after treatment. RESULTS: All patients survived, and there were no adverse events. No infusionrelated changes in hemodynamics occurred. No infusion-related toxicity was recorded. Five subjects experienced a reduction in auditory brainstem response (ABR) thresholds. Four of those 5 subjects also experienced an improvement in cochlear nerve latencies. Comparison of MRI with DTI sequences obtained before and after treatment revealed increased fractional anisotropy in the primary auditory cortex in three of five subjects with reduced ABR thresholds. Statistically significant (p < 0.05) reductions in ABR thresholds were identified. CONCLUSIONS: TIntravenous hUCB is feasible and safe in children with SNHL.

Uso de células mononucleares da medula óssea no tratamento de tendinites induzidas experimentalmente em equinos / The use of mononuclear cells from bone marrow on the treatment of experimentally induced tendinitis in horses

Foram induzidas lesões no tendão flexor digital superficial (TFDS) de ambos os membros anteriores de seis equinos, seguidas de implante autólogo de células da fração mononuclear de medula óssea em apenas um membro de cada animal. Os animais foram avaliados por parâmetros clínicos, ultrassonográficos, histopatológico e imunoistoquímico. Paralelamente, realizou-se o cultivo de novas amostras para a caracterização das células utilizando-se marcadores CD34 e CD45 por meio da citometria de fluxo, confirmando a presença de células mesenquimais na fração mononuclear. A caracterização das fibras colágenas tipo I e tipo III no tecido neoformado mostrou melhora na qualidade da cicatrização tendínea dos membros tratados. A terapia com implante autólogo das células da fração mononuclear melhorou a organização tecidual e a sua qualidade, apresentando maior expressão significativa para colágeno tipo I.

The present study was developed inducing a lesion in the SDFT of both thoracic limbs of six horses followed by autologous implantation of mononuclear cells from bone marrow in only one affected limb of each horse. The horses were evaluated through clinical and ultrasonography exams, and through histopathology and immunohistochemistry patterns. Concomitantly, new samples were cultivated and characterized using CD34 and CD45 markers, proving the presence of mesenchymal cells in the mononuclear fraction. The characterization of collagen fibers type I and type III in the new tissue has showed an improvement in tendon healing in treated limbs. The therapy with autologous implant of the mononuclear fraction has improved tissue organization and its quality, having a significanlyt higher expression of collagen type I.

A safety and feasibility study of cell therapy in dilated cardiomyopathy

The aim of this study was to determine if bone marrow mononuclear cell (BMMC) transplantation is safe for moderate to severe idiopathic dilated cardiomyopathy (IDC). Clinical trials have shown that this procedure is safe and effective for ischemic patients, but little information is available regarding non-ischemic patients. Twenty-four patients with IDC, optimized therapy, age 46 ± 11.6 years, 17 males, NYHA classes II-IV, and left ventricular ejection fraction <35 percent were enrolled in the study. Clinical evaluation at baseline and 6 months after stem cell therapy to assess heart function included echocardiogram, magnetic resonance imaging, cardiopulmonary test, Minnesota Quality of Life Questionnaire, and NYHA classification. After cell transplantation 1 patient showed a transient increase in enzyme levels and 2 patients presented arrhythmias that were reversed within 72 h. Four patients died during follow-up, between 6 and 12 weeks after therapy. Clinical evaluation showed improvement in most patients as reflected by statistically significant decreases in Minnesota Quality of Life Questionnaire (63 ± 17.9 baseline vs 28.8 ± 16.75 at 6 months) and in class III-IV NYHA patients (18/24 baseline vs 2/20 at 6 months). Cardiopulmonary exercise tests demonstrated increased peak oxygen consumption (12.2 ± 2.4 at baseline vs 15.8 ± 7.1 mL·kg-1·min-1 at 6 months) and walked distance (377.2 ± 85.4 vs 444.1 ± 77.9 m at 6 months) in the 6-min walk test, which was not accompanied by increased left ventricular ejection fraction. Our findings indicate that BMMC therapy in IDC patients with severe ventricular dysfunction is feasible and that larger, randomized and placebo-controlled trials are warranted.

Decanoato de nandrolona no hemograma e nas células mononucleares da medula óssea de ratos Wistar hígidos / Nandrolone decanoate in the hemogram and bone marrow mononuclear cells of healthy Wistar rats

O decanoato de nandrolona (DN), um estimulante do sistema hematopoético, caracteriza-se por ser um medicamento acessível aos proprietários de animais com escassos recursos econômicos. Assim, este estudo objetivou avaliar o efeito de diferentes doses do DN no hemograma e na quantificação e a viabilidade das células mononucleares (CM) da medula óssea (MO), juntamente com a marcação das células hematopoéticas CD34+ de ratos Wistar saudáveis. Para isso, 48 animais foram separados em seis tratamentos, de forma aleatória, com oito animais cada. Os grupos foram constituídos por: G1 - controle (solução fisiológica); G2 - controle diluente (somente veículo oleoso de origem vegetal); G3 - 0,42mg kg-1 de DN; G4 - 1,8mg kg-1 de DN; G5 - 4,6mg kg-1 de DN; e G6 - 10,0mg kg-1 de DN. O fármaco foi aplicado semanalmente por três semanas. Os parâmetros hematológicos e medulares avaliados não tiveram diferença significativa entre os grupos, o que pode ter sido influenciado pela condição da MO ou pelo intervalo entre as doses. De acordo com os resultados obtidos e nas condições em que esta pesquisa foi desenvolvida, pode-se concluir que o DN não altera o hemograma, a quantificação e a viabilidade das CM e a marcação de CD34+ em ratos wistar saudáveis.

Nandrolone Decanoate (ND), a hematopoietic system stimulant, is characterized as an accessible medicament for low-income pet owners. The aim of this research is to study the effect of different ND doses in the blood cytological parameters and the quantification and viability of the bone marrow (BM) mononuclear cells (MC), together with the labeling of CD34+ hematopoietic stem cells of healthy Wistar rats. Forty eight animals were randomly separated into six different groups of treatment, each composed of eight animals. These groups were divided in: G1 - control group (physiologic solution); G2 - diluent control (only vegetal oily vehicle); G3 - ND 0.42mg kg-1; G4 - ND 1.8mg kg-1; G5 - ND 4.6mg kg-1 and G6 - ND 10.0mg kg-1. The drug was weekly applied for three weeks. The hematologic and medullar analyzed parameters showed no significant difference between the groups, which may have been influenced by the BM conditions or by the applications frequency. According to the results obtained and according to the conditions under which this research was developed, it can be concluded that ND did not affect the blood cytological parameters, quantification and viability of MC and CD34+ labeling in healthy Wistar rats.

Transplante autólogo de células mononucleares da medula óssea em úlcera de córnea experimental em cães / Bone marrow mononuclear cells autotransplant in experimental corneal ulcer in dogs

As células mononucleares (CM) da medula óssea (MO) despertam grande interesse nas pesquisas sobre regeneração tecidual. O limbo é a fonte de células-tronco (CT) para repor ceratócitos lesados e uma disfunção destas é denominada deficiência límbica. Essa condição é desenvolvida por diversas afecções, sendo que a queimadura por base é a mais comum. A fim de confirmar a presença das CM da MO transplantadas, a ocorrência de quimiotaxia destas e comparar histopatologicamente os grupos tratado e controle, utilizou-se um modelo experimental de úlcera de córnea associado ao autotransplante de CM. Para tanto, 16 cães machos ou fêmeas, sem raça definida, foram submetidos à úlcera experimental de córnea com papel filtro embebido em hidróxido de sódio (NaOH). Após as lesões, os animais foram submetidos a transplante subconjuntival de CM da MO, previamente marcadas com nanocristais. A avaliação pós-operatória foi realizada por imunofluorescência no sexto dia após o transplante e por histopatologia passados 15 dias do procedimento, quando foi possível notar que as CM fixaram-se na região lesionada, não sofreram quimiotaxia e, apesar de diminuírem a inflamação, não auxiliaram o processo de cicatrização corneana a curto prazo. Assim, sugerem-se estudos adicionais no transplante de CM da MO na cicatrização da córnea.

Bone marrow (BM) mononuclear cells (MC) are a great subject in tissual regeneration. The main stem cell source to the eye is the limbus. Theses cells replace injured corneal cells, however, if the limbal stem cells are not functional, a limbal deficiency with concomitant conjunctivalization takes place. This pathological condition can be caused for several reasons, in which alkali burns are the most common. To conduct a research about transplanted BM MC presence, the cells homing and to histopathologically compare the treated and sham group, an experimental corneal ulcer model associated with MC autotransplant was used. Sixteen, male or female, stray dogs suffered experimental corneal ulceration with sodium hydroxide soaked filter discs. After the lesions, animals were submitted to subconjunctival autotransplant of previously marked BM MC. The evaluation was made by immunofluorescence on the sixth day after lesions creation and histopathology was conducted 15 days after the same procedure, when it was possible to observe that the MC grafted in the injured area, the cells did not execute the homing process and, despite the inflammatory decrease, they did not help the corneal epithelial healing process in a short term evaluation. Thus, future studies about MC transplantations in corneal ulcers are indicated.