Injectable chitosan/gelatin/bioactive glass nanocomposite hydrogels for potential bone regeneration: In vitro and in vivo analyses.

The present work describes in vitro and in vivo behaviors of thermosensitive composite hydrogels based on polymers/bioactive glass nanoparticles. Assays in SBF (simulated body fluid) solution showed that loss of hydrogel mass in vitro was decreased by 4.3% when bioactive glass nanoparticles (nBG) were incorporated, and confirmed the bioactivity of nBG containing hydrogels. In vitro assays demonstrated the cytocompatibility of the hydrogels with encapsulated rat bone marrow mesenchymal stem cells (BMSC). Crystal violet assays showed a 27% increase in cell viability when these cells were seeded in hydrogels containing nBG. In vivo biocompatibility was examined by injecting hydrogels into the dorsum of Swiss rats. The results indicated that the prepared hydrogels were nontoxic upon subcutaneous injection, and could be candidates for a safe in situ gel-forming system. Injection of the hydrogels into a rat tibial defect allowed preliminary evaluation of the hydrogels' regenerative potential. Micro Computed Tomography analysis suggested that more new tissue was formed in the defects treated with the hydrogels. Taken together, our data suggest that the developed injectable composite hydrogels possess properties which make them suitable candidates for use as temporary injectable matrices for bone regeneration.

Estudo comparativo da diferenciação osteogênica das células tronco mesenquimais da medula óssea e do tecido adiposo de cães adultos / Comparative study of the osteogenic differentiation of mesenchymal stem cells from bone marrow and adipose tissue of adult dogs

O objetivo deste estudo foi comparar o potencial osteogênico das células tronco mesenquimais extraídas da medula óssea (CTM-MO) com as do tecido adiposo (CTM-AD) de cães adultos. As células foram caracterizadas fenotipicamente quanto à expressão de CD29, CD90, CD34 e CD45 e submetidas à diferenciação adipogênica e condrogênica por 21 dias e osteogênica por 7, 14 e 21 dias. Foram constituídos quatro grupos: 1) CTM-MO em meio osteogênico, 2) CTM-MO em meio basal, 3) CTM-AD em meio osteogênico e 4) CTM-AD em meio basal. Aos 7, 14 e 21 dias de diferenciação osteogênica as culturas foram submetidas às avaliações da conversão de MTT em formazan, da atividade da fosfatase alcalina (FA), da síntese de colágeno e de matriz mineralizada, avaliação do número de células por campo e foram quantificados os transcritos gênicos para osterix, sialoproteina óssea (BSP), osteonectina (ON) e osteocalcina (OC). Tanto as células extraídas da medula óssea quanto do tecido adiposo mostraram elevada expressão de marcadores para células tronco e baixa expressão de marcadores de células hematopoiéticas (menor que 2%). Além disso, foram capazes de se diferenciar em osteoblastos, condrócitos e adipócitos. As CTM-AD submetidas à diferenciação osteogênica mostraram maior conversão do MTT em formazan que as CTM-MO, sob mesmas condições aos 7 e 21 dias. O número de células por campo, a atividade da FA, a síntese de colágeno e de matriz mineralizada foram superior nas CTM-AD em diferenciação, em relação às CTM-MO sob as mesmas condições, em todos os tempos estudados. As expressões de osterix, BSP e OC foram predominantemente superiores nas CTM-MO diferenciadas, mas a expressão de ON foi superior nas CTM-AD diferenciadas aos 7, 14 e 21 dias. Conclui-se que as CTM-AD apresentam maior potencial osteogênico que as CTM-MO quando extraídas de cães adultos.(AU)

The aim of this study was to compare the osteogenic potential of mesenchymal stem cells obtained from bone marrow (BM-MSC) with those extracted from adipose tissue (AT-MSC) of adult dogs. The cells were phenotypically categorized according to the expression of CD29, CD90, CD34 and CD45, and submitted to adipogenic and chondrogenic differentiation for 21 days and osteogenic differentiation for 7, 14 and 21 days. Four groups were formed: BM-MSC in osteogenic medium (1), BM-MSC in basal medium (2), AT-MSC in osteogenic medium (3) and ATMSC in basal medium (4). On days 7, 14 and 21 of osteogenic differentiation, the cultures were submitted to evaluations of MTT conversion in formazan, of alkaline phosphatase activity (AP), of collagen and mineralized matrix synthesis, evaluation of the number of cells per field and there was quantification of the gene transcripts for osterix, bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). Both the cells obtained from bone marrow and those from adipose tissue showed high expression of stem cells markers and low expression of hematopoietic cells markers (lower than 2%). Besides, they were able to differentiate into osteoblasts, chondrocytes and adipocytes. AT-MSC submitted to osteogenic differentiation showed higher MTT conversion in formazan than BM-MSC, under the same conditions on days 7 and 21. The number of cells per field, the AP activity, the collagen and mineralized matrix synthesis were higher in AT-MSC en differentiation, in relation to BM-MSC under the same conditions in all evaluated times. Expressions of osterix, BSP and OC were predominantly higher in differentiated BMMSC, however the expression of ON was higher AT-MSC differentiated on days 7, 14 and 21. In conclusion, AT-MSC present higher osteogenic potential than BM-MSC when extracted from adult dogs.(AU)

Effect of the ionic product of bioglass 60s on osteoblastic activity in canines.

BACKGROUND: The objective of the present study was to evaluate the effect of the ionic product (IP) of BG60S on osteoblastic activity. The following media groups were created: DMEM, which is formed by osteoblasts in basal medium; IP DMEM, which is formed by osteoblasts in IP with basal medium; OST, which is formed by osteoblasts in osteogenic medium; and IP OST, which is formed by osteoblasts in IP with osteogenic medium. The osteoblasts were cultivated in an incubator at 37 °C and 5 % CO2 for 7, 14 and 21 days. After each period, the alkaline phosphatase (AP) activity, mineralised area per field and expression of osterix (OSX), bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC) were evaluated by reverse transcription (RT)-PCR. RESULTS: The IP significantly increased the AP activity in the IP DMEM group at 7 and 14 days and reduced the AP activity in the IP OST group at 14 and 21 days relative to their respective controls (DMEM and OST). The groups that received the IP displayed a significant increase in the percentage of mineralised area per field and more advance maturation of the extracellular matrix relative to those that did not receive IP. The IP significantly increased the expression of OSX, BSP and ON in osteoblast cultures maintained in IP DMEM compared with the control (DMEM) for the majority of studied periods. In osteogenic medium, IP also significantly increased OSX, BSP, ON and OC expression compared with the control (OST) for the majority of studied periods. CONCLUSIONS: The IP of BG60S alters the gene expression of canine osteoblasts, favouring the synthesis and mineralisation of the extracellular matrix.

Comparison of the osteogenic potential of mesenchymal stem cells from the bone marrow and adipose tissue of young dogs.

BACKGROUND: The aim of the present study was to compare the osteogenic potential of mesenchymal stem cells extracted from the bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) of young dogs. The following parameters were assessed: dimethyl thiazolyl diphenyl tetrazolium (MTT) conversion, alkaline phosphatase (ALP) activity, collagen and mineralised matrix synthesis, and the expressions of osterix, bone sialoprotein (BSP), and osteocalcin (OC). RESULTS: MTT conversion was greater in BM-MSCs compared to AD-MSCs after 14 and 21 days of differentiation; ALP activity was greater in differentiated AD-MSCs on day 7; collagen synthesis was greater in BM-MSCs on days 14 and 21; the percentage of mineralized area per field was greater in BM-MSCs compared to AD-MSCs; osterix expression was greater in BM-MSCs in days 14 and 21, and BSP and OC expression levels were greater in BM-MSCs at all the investigation time-points. CONCLUSIONS: It was concluded that the osteogenic potential was greater in BM-MSCs than AD-MSCs when extracted from young dogs.

Ultrastructural characteristics of fibrin clots from canine and feline platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin.

BACKGROUND: The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (µm2), the area of the fibrin fibers (µm2), and the width of the fibrin fibers (µm) were determined for the dog clots. The platelet area (µm2), the area of fibrin fibers (µm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. RESULTS: Cat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB. CONCLUSIONS: The findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.

Comparison of the effect of calcium gluconate and batroxobin on the release of transforming growth factor beta 1 in canine platelet concentrates.

BACKGROUND: The clinical use of autologous platelet concentrates (also known as platelet-rich plasma) on the field of regenerative therapy, in the last decade has been the subject of several studies especially in equine medicine and surgery. The objectives of this study was: 1) to describe and compare the cellular population in whole blood, lower fraction (A) and upper fraction (B) of platelet concentrates, 2) to measure and compare the transforming growth factor beta 1 (TGF-ß1) concentration in plasma and both platelet concentrates after be activated with calcium gluconate or batroxobin plus calcium gluconate and, 3) to determine correlations between cell counts in platelet concentrates and concentrations of TGF-ß1. Blood samples were taken from 16 dogs for complete blood count, plasma collection and platelet concentrates preparation. The platelet concentrates (PC) were arbitrarily divided into two fractions, specifically, PC-A (lower fraction) and PC-B (upper fraction). The Platelet concentrates were analyzed by hemogram. After activated with calcium gluconate or batroxobin plus calcium gluconate, TGF-ß1 concentration was determined in supernatants of platelet concentrates and plasma. RESULTS: There were differences statistically significant (P < 0.05) for the platelet count and leukocyte count and TGF-ß1 concentration between whole blood, plasma and both platelet concentrates. A significant correlation was found between the number of platelets in both platelet concentrates and TGF-ß1 concentration. Platelet collection efficiency was 46.34% and 28.16% for PC-A and PC-B, respectively. TGF-ß1 concentration efficiency for PC activated with calcium gluconate was 47.75% and 31.77%, for PC-A and PC-B, respectively. PC activated with batroxobin plus CG showed 46.87% and 32.24% for PC-A and PC-B, respectively. CONCLUSIONS: The methodology used in this study allows the concentration of a number of platelets and TGF-ß1 that might be acceptable for a biological effect for clinical or experimental use as a regenerative therapy in dogs.

In vivo evaluation of bioactive glass foams associated with platelet-rich plasma in bone defects.

The objective of this study was to evaluate the use of bioactive glass foams produced by the sol-gel process, associated or not with platelet-rich plasma (PRP), in the regeneration of bone defects. Mongrel dogs (n = 14) were divided into two groups after having their superior first premolar removed. A small piece of vestibular bone from the alveolus was intentionally removed. The area was filled with bioactive glass foam produced by the sol-gel method. Two groups were tested: group A was the glass foam; group B was the same material associated with PRP, prepared from each animal. The other side of alveolar bone was used as a control group, in which the bone defect did not receive any biomaterial. The thickness of the bone area was measured before and after the intervention. After a period of 60 days implantation, the right and left bones were measured again, and a bone biopsy on both regions was conducted for histological analysis. The findings show an increase of bone thickness for both materials implanted compared to the control group. Group B, implanted with bioactive glass foam associated with PRP, showed a thicker bone area compared to Group A. Histological results indicate bone formation for both materials used. However, the bioactive glass associated with PRP gave rise to a more mature bone formation. These results show that bioactive glass foams processed by a sol-gel method is effective in maintaining the thickness of the alveolar ridge, and the use of PRP associated with the foams improve bone formation.