Long-term rabbits bone response to titanium implants in the presence of inorganic bovine-derived graft

This study evaluated bone responses to titanium implants in the presence of an inorganic graft material. The bilateral mandible incisors of 24 rabbits were surgically extracted and one of the exposed sockets, chosen at random, was filled with an inorganic xenogenic bone graft (Gen-ox (R)), whereas the remaining socket was left to heal naturally and served as a control. After 60 days, titanium implants were inserted in the specific areas, and on days 0, 30, 60, and 180 after the implant insertions, six animals of each group were killed. Digital periapical radiography of implant region was obtained and vertical bone height (VBH) and bone density (BD) were evaluated by digital analysis system. In the undecalcified tissue cuts, bone-to-implant contact (BIC) and bone area (BA) within the limits of the implant threads were evaluated and compared statistically by means of two-way ANOVA and Tukey's test (rho < 0.05). No significant differences were detected in VBH and BA, either between groups or between different experimental intervals. The BD was significantly higher in the experimental group than in the control group in all the intervals tested, but there were no significant differences by interval. The BIC was statistically lower in the control group on day 0; however, a significant increase was observed on days 60 and 180 (rho < 0.05). The use of an inorganic xenograft prior to insertion of a titanium implant did not interfere with the course of osseointegration.

Bone repair and augmentation using block of sintered bovine-derived anorganic bone graft in cranial bone defect model

To histomorphometrically investigate the repair of critical size defects (CSDs) and bone augmentation in cranial walls using block of sintered bovine-derived anorganic bone (sBDAB) graft. Forty guinea-pigs were divided into test (n=20) and CSD control (n=20) groups. In each animal, a full-thickness bone defect with 9.5 mm diameter was made in the frontal bone. The defects were filled with an sBDAB block soaked in blood in the test group and with blood clot in the CSD control group. The skulls were collected at 0 h (n=2) and 30, 90 and 180 days (n=6/group and period) postoperatively. The volume density and total volume of newly formed bone, sBDAB, blood vessels and connective tissue, vertical thickness of removed bone plug, sBDAB block and graft area were evaluated. The vertical thickness of the adapted sBDAB block was 3.8 times higher than that of the removed bone plug and did not show significant difference between periods, filling in average 29.8% of the total graft region. The sBDAB block exhibited complete osseointegration with the borders of the defect at 90 days. At 90 and 180 days, the vertical thickness of the graft was 279% in the average, and the total volume of bone augmentation was, respectively, 78.8% and 148.5% higher compared with the removed bone plug. The defects of the CDS control group showed limited osteogenesis and filling by connective tissue plus tegument. The sBDAB block can be used to promote repair of CSDs and bone augmentation in the craniomaxillofacial region, due to its good osteoconductive and slow resorptive properties. To cite this article:Cestari TM, Granjeiro JM, de Assis GF, Garlet GP, Taga R. Bone repair and augmentation using block of sintered bovine-derived anorganic bone graft in cranial bone defect model.Clin. Oral Impl. Res. 20, 2009; 340-350.doi: 10.1111/j.1600-0501.2008.01659.x.

Comparative analysis of the replication of bovine herpesvirus 1 (BHV1) and BHV5 in bovine derived-neuron-like cells

Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50 % of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37 % of cells being in the early stages of apoptosis; 63.69 % of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.

Lateral ridge augmentation using equine- and bovine-derived cancellous bone blocks: a feasibility study in dogs

OBJECTIVES: The aim of the present study was to histologically evaluate and compare a new prototype collagen type I/III-containing equine- (EB) and a bovine- (BB) derived cancellous bone block in a dog model. MATERIALS AND METHODS: Four standardized box-shaped defects were bilaterally created at the buccal aspect of the alveolar ridge in the lower jaws of five beagle dogs and randomly allocated to either EB or BB. Each experimental site was covered by a native (non-crosslinked) collagen membrane and left to heal in a submerged position for 12 weeks. Dissected blocks were processed for semi-/and quantitative analyses. RESULTS: Both groups had no adverse clinical or histopathological events (i.e. inflammatory/foreign body reactions). BB specimens revealed no signs of biodegradation and were commonly embedded in a fibrous connective tissue. New bone formation and bony graft integration were minimal. In contrast, EB specimens were characterized by a significantly increased cell (i.e. osteoclasts and multinucleated giant cells)-mediated degradation of the graft material (P<0.001). The amount and extent of bone ingrowth was consistently higher in all EB specimens, but failed to reach statistical significance in comparison with the BB group (P>0.05). CONCLUSIONS: It was concluded that the application of EB may not be associated with an improved bone formation than BB.

Adsorption of Enamel Matrix Proteins to a Bovine Derived Bone Grafting Material and its Regulation of Cell Adhesion, Proliferation and Differentiation

The use of various combinations of enamel matrix derivative (EMD) and grafting materials has been shown to promote periodontal wound healing/regeneration. However, the downstream cellular behavior of periodontal ligament (PDL) cells and osteoblasts has not yet been studied. Furthermore, it is unknown to what extent the bleeding during regenerative surgery may influence the adsorption of exogenous proteins to the surface of bone grafting materials and the subsequent cellular behavior. In the present study, the aim is to test EMD adsorption to the surface of natural bone mineral (NBM) particles in the presence of blood and determine the effect of EMD coating to NBM particles on downstream cellular pathways, such as adhesion, proliferation, and differentiation of primary human osteoblasts and PDL cells.

Effects of enamel matrix proteins in combination with a bovine-derived natural bone mineral for the repair of bone defects.

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

A bayesian network meta-analysis on comparisons of enamel matrix derivatives, guided tissue regeneration and their combination therapies

Aims: Guided tissue regeneration (GTR) and enamel matrix derivatives (EMD) are two popular regenerative treatments for periodontal infrabony lesions. Both have been used in conjunction with other regenerative materials. We conducted a Bayesian network meta-analysis of randomized controlled trials on treatment effects of GTR, EMD and their combination therapies. Material and Methods: A systematic literature search was conducted using the Medline, EMBASE, LILACS and CENTRAL databases up to and including June 2011. Treatment outcomes were changes in probing pocket depth (PPD), clinical attachment level (CAL) and infrabony defect depth. Different types of bone grafts were treated as one group and so were barrier membranes. Results: A total of 53 studies were included in this review, and we found small differences between regenerative therapies which were non-significant statistically and clinically. GTR and GTR-related combination therapies achieved greater PPD reduction than EMD and EMD-related combination therapies. Combination therapies achieved slightly greater CAL gain than the use of EMD or GTR alone. GTR with BG achieved greatest defect fill. Conclusion: Combination therapies performed better than single therapies, but the additional benefits were small. Bayesian network meta-analysis is a promising technique to compare multiple treatments. Further analysis of methodological characteristics will be required prior to clinical recommendations.

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

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

Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer.

RESUMO: A geleia de Wharton é uma fonte de células tronco mesenquimais (CTMs) que ainda não havia sido testada para a produção de embriões bovinos por transferência nuclear (TN). O objetivo deste estudo foi isolar, caracterizar e testar as CTMs derivadas da geleia de Wharton para produção de embriões e gestações por transferência nuclear em bovinos. O cordão umbilical foi coletado durante o nascimento e as células derivadas da geleia de Wharton (CGWs) foram isoladas por explante e cultivadas em Dulbecco?s Modified Eagle Medium. Fibroblastos (FB) da pele foram isolados após 6 meses de vida. As análises morfológicas foram realizadas pelas microscopias de campo claro e eletrônica de varredura durante o cultivo celular. Caracterização fenotípica e genotípica por citometria de fluxo, imunocitoquímica, RT-PCR e indução da diferenciação em linhagens celulares foi realizada com as CGWs. No procedimento de TN, ovócitos no estágio de metáfase II foram enucleados usando micromanipuladores, fusionados com CGWs ou FB e então ativados artificialmente. Micrografias de microscopia de varredura revelaram que CGWs tiveram forma variada sob cultivo. Os marcadores mesenquimais de CTMs (CD29+, CD73+, CD90+ and CD105+) foram expressos em cultura de CGWs bovina, como evidenciado por citometria de fluxo, imunocitoquímica e RT-PCR. Quando induzidas, estas células diferenciaram-se em osteócitos, condrócitos e adipócitos. Após classificação, as CGWs foram utilizadas na TN. A taxa de formação de blastocistos por TN com CGWs no sétimo dia de cultivo foi de 25,80±0,03%, similar a produção de blastócitos por TN com fibroblastos de pele (19,00±0,07). Gestações foram obtidas e mostraram que CGWs constituem um novo tipo celular para ser usado na clonagem animal. ABSTRACT: Wharton?s jelly is a source of mesenchymal stem cells (MSCs) that had not yet been tested for bovine embryo production by nuclear transfer (NT). Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton?s jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton?s jelly (WJCs) were isolated by explant and cultured in Dulbecco?s Modified Eagle Medium. Skin Fibroblasts (FB) were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM) during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+) were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%). Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

Transcriptional dissection of pancreatic tumors engrafted in mice.

BACKGROUND: Engraftment of primary pancreas ductal adenocarcinomas (PDAC) in mice to generate patient-derived xenograft (PDX) models is a promising platform for biological and therapeutic studies in this disease. However, these models are still incompletely characterized. Here, we measured the impact of the murine tumor environment on the gene expression of the engrafted human tumoral cells. METHODS: We have analyzed gene expression profiles from 35 new PDX models and compared them with previously published microarray data of 18 PDX models, 53 primary tumors and 41 cell lines from PDAC. The results obtained in the PDAC system were further compared with public available microarray data from 42 PDX models, 108 primary tumors and 32 cell lines from hepatocellular carcinoma (HCC). We developed a robust analysis protocol to explore the gene expression space. In addition, we completed the analysis with a functional characterization of PDX models, including if changes were caused by murine environment or by serial passing. RESULTS: Our results showed that PDX models derived from PDAC, or HCC, were clearly different to the cell lines derived from the same cancer tissues. Indeed, PDAC- and HCC-derived cell lines are indistinguishable from each other based on their gene expression profiles. In contrast, the transcriptomes of PDAC and HCC PDX models can be separated into two different groups that share some partial similarity with their corresponding original primary tumors. Our results point to the lack of human stromal involvement in PDXs as a major factor contributing to their differences from the original primary tumors. The main functional differences between pancreatic PDX models and human PDAC are the lower expression of genes involved in pathways related to extracellular matrix and hemostasis and the up- regulation of cell cycle genes. Importantly, most of these differences are detected in the first passages after the tumor engraftment. CONCLUSIONS: Our results suggest that PDX models of PDAC and HCC retain, to some extent, a gene expression memory of the original primary tumors, while this pattern is not detected in conventional cancer cell lines. Expression changes in PDXs are mainly related to pathways reflecting the lack of human infiltrating cells and the adaptation to a new environment. We also provide evidence of the stability of gene expression patterns over subsequent passages, indicating early phases of the adaptation process.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Resposta tecidual ao enxerto xenógeno inorgânico de osso bovino: avaliação histomorfológica e histomorfométrica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of Bio-Oss on osseointegration of dental implants surrounded by circumferential bone defects of different dimensions: an experimental study in the dog

OBJECTIVES: This study was designed to evaluate the effect of gap width and graft placement on bone healing around implants placed in simulated extraction sockets of various widths in four Labrador dogs. MATERIALS AND METHODS: Five Osseotite implants per dog were placed in the mandible of four dogs. Two implants were inserted into sites with a 2.37 mm and two with a 1 mm gap present between the implants and bone around the coronal 6 mm of the implants in each dog. For one of each gap sizes, the gap was filled with Bio-Oss, and the other two with blood alone. A fifth implant was inserted without a gap and used as a control. Ground sections were prepared from biopsies taken at 4 months and histometric measurements of osseointegration and bone between the threads made for the coronal 6 mm. RESULTS: The medians for osseointegration ranged from 5.2 mm for control to 1-2.6 mm for the test modalities. There were significant differences for linear measurements of osseointegration (chi(2) 18.27; df 4; P=0.0011) and bone area within threads (chi(2) 23.4; df 4; P=0.0001) between test modalities. CONCLUSIONS: The results suggest that the wider the gap around the implants, the less favourable the histological outcome at short time intervals following treatment. They also infer that bone grafting with an organic bovine bone xenograft seems to lead to a more favourable histological outcome for wider circumferential defects but not for narrower defects.

Isoellipticines: novel compounds for leukaemia treatment

Ellipticine, an anticancer agent, has had limited clinical success due to low solubility and toxic side effects. To overcome these limitations, a panel of novel ellipticine isomers were designed and synthesised with the aim of evaluating their anti-cancer effects on selected cancer cell lines. A preliminary NCI 60-cell screen demonstrated that these isoellipticines displayed promising anti-tumour activity across a number of different cell types, particularly leukaemia cell lines. We consequently examined the effect of these derivatives in detail on the Acute Myeloid Leukaemia (AML) cell line, MV4-11. Cell cycle analyses revealed that the compounds had a range of distinctive cell cycle effects on MV4-11 cells. 7-Hydroxyisoellipticine showed the most promise with respect to cytostatic activity. We demonstrated that this compound inhibited proliferation of leukaemia cells by preventing cells from progressing from G2 phase. Our research suggests that this is mediated by an induction of reactive oxygen species (ROS), which in turn activates the DNA damage response pathway. More extensive research on the source of ROS generated by the most potent derivative, 7-formyl-10-methylisoellipticine showed that this compounds cytotoxicity is partially mediated by an induction of mitochondrial derived reactive oxygen species (ROS). We showed that 7-formyl-10-methylisoellipticine has synergistic effects when used in combination with the clinically used AML drug, daunorubicin, as well as DPI, a Nox inhibitor. Additionally, combination experiments with other drugs served to give us a deeper insight into 7- formyl-10-methylisoellipticine mechanism of action. 7-Formyl-10-methylisoellipticine also displayed promising in vivo results. Treatment resulted in a lack of toxicity, as measured by body weight changes and liver enzyme analyses. Most importantly, 7-formyl-10-methylisoellipticine demonstrated potent anti-tumour activity in the in vivo xenograft mouse model, implying the potential of isoellipticines as novel chemotherapeutic agents in the treatment of leukaemia. In summary, this study provides for the first time detailed cellular information on the potential use of isoellipticines as chemotherapeutic agents. Our study documents for the first time, the therapeutic potential of an isoellipticine compound in a subcutaneous AML cell-derived xenograft (CDX) model. By probing the mechanism of action of this novel compound class we have uncovered a potential clinical application in the field of adjuvant therapy. We anticipate that the recent research on ellipticine derivatives, such as this study, will lead the development of an ellipticine analogue that may be employed clinically.

Repair of critical-size defects with autogenous periosteum-derived cells combined with bovine anorganic apatite/collagen: an experimental study in rat calvaria

The aim of this study was to evaluate the bone repair using autogenous periosteum-derived cells (PDC) and bovine anorganic apatite and collagen (HA-COL). PDC from Wistar rats (n=10) were seeded on HA-COL discs and subjected to osteoinduction during 6 days. Critical-size defects in rat calvarias were treated with blood clot (G1), autogenous bone (G2), HA-COL (G3) and HA-COL combined with PDC (G4) (n=40), and then analyzed 1 and 3 months after surgeries. Radiographic analysis exhibited no significant temporal change. G1 and G2 had discrete new marginal bone, but the radiopacity of graft materials in G2, G3 and G4 impaired the detection of osteogenesis. At 3 months, histopathological analysis showed the presence of ossification islets in G1, which was more evident in G2, homogeneous new bone around HA-COL in G3 and heterogeneous new bone around HA-COL in G4 in addition to moderate presence of foreign body cells in G3 and G4. Histomorphometric analysis showed no change in the volume density of xenograft (p>0.05) and bone volume density in G2 was twice greater than in G1 and G4 after 3 months (p<0.05), but similar to G3. The PDC did not increase bone formation in vivo, although the biomaterial alone showed biocompatibility and osteoconduction capacity.