Effects of Silica Based Biomaterials on Bone Marrow Derived Cells - Material Aspects of Bone Regeneration

Silica based biomaterials, such as melt-derived bioactive glasses and sol-gel glasses, have been used for a long time in bone healing applications because of their ability to form hydroxyapatite and to stimulate stem cell proliferation and differentiation. In this study, bone marrow derived cells were cultured with bioactive glass and sol-gel silica, and seeded into porous polymer composite scaffolds that were then implanted femorally and subcutaneously in rats to monitor their migration inside host tissue. Bone marrow derived cells were also injected intraperitoneally. Transplanted cells migrated to various tissues inside the host, including the lung, liver spleen, thymus and bone marrow. The method of transplantation affected the time frame of cell migration, with intraperitoneal injection being the fastest and femoral implantation the slowest, but not the target tissues of migration. Transplanted donor cells had a limited lifetime in the host and were later eliminated from all tested tissues. Bioactive glass, however, affected the implanted cells negatively. When it was present in the scaffold no donor cells were found in any of the tested host tissues. Bioactive glass S53P4 was found to support both osteoblastic and osteoclastic phenotype of bone marrow derived cells, but it was resistant to the resorbing effect of osteoclastic bone marrow derived cells, showing that bioactive glass is rather dissolved through physicochemical reactions than resorbed by cells. Fast-dissolving silica sol gel in microparticulate form was found to increase collagen formation by bone marrow derived cells, while slow dissolving silica microparticles enhanced their proliferation, suggesting that the dissolution rate of silica controls the response of bone marrow derived cells.

Molecular markers for progression of squamous cell carcinoma of the skin

Incidence of nonmelanoma skin cancer (NMSC) is increasing. Ultraviolet (UV) –light is a major risk factor for the development of cutaneous SCC. Cutaneous SCCs that develop to chronic ulcers are known to progress and metastasize more easily than UV-induced SCCs. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes which are suggested to have a role in cancer growth and invasion. The molecular background for progression of cutaneous SCC was examined by immunohistochemistry (IHC) using tissue samples of recessive dystrophic epidermolysis bullosa (RDEB) –associated SCC, sporadic UV-induced SCC, and SCC precursors. IHC studies using tissue microarray (TMA) technique revealed overexpression of MMP-7 and MMP-13 in SCC tumor cells. MMP-7 expression was enhanced especially in the SCC tumor cells of the RDEB –associated SCCs. Studies with SCC cell lines showed that tumor cell derived MMP-7 activated heparin binding epidermal growth factor –like growth factor (HB-EGF) which enhanced the growth of SCC tumor cells. Further, it was shown that type VII collagen (COL7) is expressed in sporadic SCC tumor cells. Interestingly, it was shown that SCC –associated MMP-13 is capable of cleaving COL7 in vitro. COL7 cleavage may have a role in the progression of cutaneous SCC. Studies on serine proteinase inhibitor gene family using SCC tumor cell gene array, quantitative real-time PCR, SCC cell lines, normal human epidermal keratinocytes and IHC of TMA samples showed that serine proteinase inhibitor clade A, member 1 (serpinA1, alpha-1-antitrypsin) is expressed and produced by human SCC tumor cells but not by normal keratinocytes. Moreover, serpinA1 expression was shown to correlate with the progression of cutaneous SCC using transformed HaCaT-cell lines and mouse chemically induced skin SCC model. SerpinA1 may serve as a novel biomarker for the progression of cutaneous SCC. This study elucidated putative mechanisms of the progression of cutaneous SCC and revealed novel biomarker candidates for the progression of SCC of the skin.

Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops

Abstract: Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.

Evidence for an inhibitory role of bone morphogenetic protein(s) in the follicular-luteal transition in cattle

Bone morphogenetic proteins (BMPs) and their receptors are expressed in ovarian theca cells (TC) and granulosa cells (GC) and BMPs have been implicated in the regulation of several aspects of follicle development including thecal androgen production and granulosal oestrogen production. Their potential involvement in luteal function has received less attention. in this study, we first compared relative abundance of mRNA transcripts for BMPs, activin-beta A and BMP/activin receptors in bovine corpus luteum (CL) and follicular theca and granulosa layers before undertaking functional in vitro experiments to test the effect of selected ligands (BMP6 and activin A) on luteinizing bovine TC and GC. Relative to P-actin transcript abundance, CL tissue contained more BMP4 and -6 mRNA than granulosa, more BMP2 mRNA than theca but much less activin-beta A mRNA than both granulosa and theca. Transcripts for all seven BMP/activin receptors were readily detected in each tissue and two transcripts (BMPRII, ActRIIA) were more abundant in CL than either theca or granulosa, consistent with tissue responsiveness. In vitro luteinization of TC and GC from antral follicles (4-6 mm) was achieved by culturing with 5% serum for 6 days. Treatment with BMP6 (0, 2, 10, and 50 ng/ml) and activin A (0, 2, 10 and 50 ng/ml) under these conditions dose-dependently suppressed forskolin-induced progesterone (P-4) secretion from both cell types without affecting cell number. BMP6 reduced forskolin-stimulated upregulation of STAR mRNA and raised 'basal' CYP17A1 mRNA level in theca-lutein cells without affecting expression of CYP11A1 or hydroxy-Delta-5-steroid dehydrogenase, 3 beta- and steroid Delta-isomerase 1 (HSD3B1). In granulosa-lutein cells, STAR transcript abundance was not affected by BMP6, whereas forskolin-induced expression of CYP11A1, HSD3B1, CYP19A1 and oxytocin transcripts was reduced. In both cell types, follistatin attenuated the suppressive effect of activin A and BMP6 on forskolin-induced P4 secretion but had no effect alone. Granulosa-lutein cells secreted low levels of endogenous activin A (similar to 1 ng/ml); BMP6 reduced this, while raising follistatin secretion thus decreasing activin A:follistatin ratio. Collectively, these findings support inhibitory roles for BMP/activin signalling in luteinization and steroidogenesis in both TC and GC.

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

Evidence supports local roles for TGFβ superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signaling receptors is likely modulated by extracellular binding proteins (BP). In this study we compared expression of four BPs (chordin, gremlin, noggin, follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1-18mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P<0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P<0.01) gremlin and follistatin mRNA abundance, with a significant cell-type x follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P<0.05) in GC of large 'E-active' than 'E-inactive' follicles while follistatin mRNA level was higher (P<0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP-BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin or FSHR mRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin, and gremlin mRNA levels up to 20-fold (P<0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signaling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signaling.

Culture of mature trophoblastic giant cells from bovine placentomes

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentim and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined. (c) 2006 Elsevier B.V. All rights reserved.

Actinic cheilitis and squamous cell carcinoma of the lip: clinical, histopathological and immunogenetic aspects

Queilite actínica é a principal lesão pré-neoplásica do lábio. O carcinoma espinocelular do lábio é incluído nas estatísticas brasileiras junto com os cânceres de boca e, em conjunto, somam 40% dos cânceres de cabeça e pescoço. Há certo desconhecimento médico e odontológico em geral quanto aos fatores relacionados à carcinogênese e à progressão de tumores de boca. Genes de supressão tumoral e proteínas regulatórias de proliferação celular exercem papel na evolução da queilite actínica para carcinoma espinocelular e no comportamento biológico deste. O conhecimento de marcadores de diagnóstico e prognóstico e sua investigação têm utilidade no acompanhamento de tais pacientes.

Association between CD31 expression and histopathologic features in stage IB squamous cell carcinoma of the cervix

This study was undertaken to evaluate the association between the expression of CD31 in the tumor and the histopathologic findings in patients with carcinoma of the cervix. This study included prospectively 30 women, aged 46.6 +/- 10.7 years, with stage IB squamous cell carcinoma of the cervix submitted to radical hysterectomy from November 2001 to September 2002. Samples from the tumor were taken and immunohistochemically evaluated by a monoclonal antibody for CD31. Clinicopathologic characteristics such as stage, tumor size, grade of differentiation, lymphatic vascular space invasion (LVSI), parametrial involvement, and status of pelvic lymph nodes were also recorded. The clinical stage (FIGO) was IB1 in 22 patients (73.3%) and IB2 in 8 patients (26.7%). The expression of CD31 was significantly associated with tumor size and the presence of LVSI, but not with grade of differentiation and vaginal or parametrial involvement (P= 0.03, P= 0.032, P= 0.352, P= 0.208, and P= 0.242, respectively). on univariate analysis, the presence of pelvic lymph node metastasis was influenced by LVSI (P= 0.003) and CD31 expression (P= 0.032). However, on multivariate analysis, the presence of LVSI (P= 0.007) was the only independent predictor of pelvic lymph node metastasis. The CD31 expression in tumor is significantly associated with LVSI and tumor size in patients with early-stage squamous cell carcinoma of the cervix.

HLA-G polymorphism and transitional cell carcinoma of the bladder in a Brazilian population

The morphologic appearance and clinical behavior of the human urinary bladder papillary transitional cell carcinoma (TCC) probably result from a complex interaction between carcinogenic insults and host resistance during the patient's life. While the main recognized risk factors are of environmental origin (e.g. smoking), relatively little information exists about the susceptibility to TCC development. The human leukocyte antigen G (HLA-G) molecule plays an important role in immune response regulation and has been implicated in the inhibition of the cytolytic function of natural killer and cytotoxic T cells. Several lines of evidence indicate that HLA-G polymorphisms influence the expression level and production of different HLA-G isoforms. The aim of this study was to explore a possible influence of the HLA-G polymorphism on the susceptibility to urinary bladder TCC development and progression in smokers and nonsmokers Brazilian subjects. The HLA-G locus was found to be associated with susceptibility to TCC development and progression. The G*0104 allelic group (specially the G*010404 allele) and the G*0103 allele were associated with a tobacco-dependent influence on TCC development. The G*0104 group was associated with progression to high-grade tumors, irrespective of smoking habit, while the G*0103 allele was associated to high-grade tumor only in smoking patients. Our results are an evidence that the HLA-G locus itself, or as part of an extended haplotype encompassing this chromosome region (particularly the HLA-A given the high linkage disequilibrium observed between them in this data series), may be associated with TCC susceptibility and tumor progression, suggesting a tobacco-dependent influence of these polymorphisms.

Autologous implant of bone marrow mononuclear cells as treatment of induced equine tendinitis

Superficial digital flexor tendonitis is an important cause of lameness in horses and its incidence ranges from 13% to 30%, depending on the horse's activity. This injury can occur in yearlings and compromise its carriers by reinjury or even impossibility to return to athletic life. In spite of the long period required for tendon repair, the scar tissue presents lack of elasticity and stiffness. As current treatment strategies produce only marginal results, there has been great interest in research of therapies that influence the quality or the speed of tendon repair. Stem cell therapy has shown promising results in degenerative diseases and cases of deficient healing processes. This study aims to evaluate the influence of autologous mesenchymal bone marrow stem cells in tendon healing, comparing treated and non-treated tendons. Superficial digital flexor tendonitis lesions were induced by collagenase infiltration in both forelimbs of 6 horses, followed by autologous implant in one of the forelimbs of each animal. The horses were evaluated using clinical, ultrasonographic, histopathologic, and immunohistochemical parameters. Tendon biopsies were performed at Day 48. Results found in the treatment group, such as high inflammatory cells infiltration, extracellular matrix synthesis, reduced amount of necrosis areas, small increase in cellular proliferation (KI-67/MIB-1), and low immunoreactivity to transforming growth factor P I, suggested the acceleration of tendon repair in this group. Further studies should be conducted in order to verify the influence of this treatment on later phases of tendon repair. Overall, after analysis of the results, we can conclude that cellular therapy with the mononuclear fraction of bone marrow has accelerated tendon repair at 48 days after treatment.

Bilateral Leydig cell tumor in stallion

The authors report a rare case of bilateral Leydig cell tumor in a noncryptorchid stallion, describing the gross. and microscopic findings. An 8-year-old Appaloosa stallion was examined for a unilateral enlargement of the testis associated with discomfort during palpation and signs of colic. General health conditions were good. Fine-needle aspiration cytology of the testis provided the diagnosis of bilateral Leydig cell tumor. Bilateral orchiectomy was performed and the testes were submitted to histopathologic examination that confirmed the diagnosis of Leydig cell tumor.

Hemangioendothelioma of bone in a patient with a constitutional supernumerary marker

A 13-year old girl was diagnosed as having a bone hemangioendothelioma. Cytogenetic studies identified the presence of a small supernumerary marker chromosome in this patient. Classical cytogenetic methods using G-, C-, Ag-NOR-banding were supplemented by spectral karyotyping (SKY) and fluorescence in situ hybridization to reveal a karyotype 47,XX,+mar.ish der(22)(D22S543+) karyotype in cells derived from the tumor and lymphocytes. These findings suggest that the supernumerary marker chromosome originated from the proximal centromeric region of chromosome 22, and that trisomy of the region 22q11: was not associated with adverse phenotypic effects, but that the presence of trisomy 22q11 may be related to the development of this tumor. (C) Elsevier B.V., 1999. All rights reserved.

Fresh-frozen human bone allograft in vertical ridge augmentation: clinical and tomographic evaluation of bone formation and resorption

The aim of the current study is to evaluate fresh-frozen human bone allografts (FHBAs) used in vertical ridge augmentation clinically and by computed tomography, and to analyze the resulting bone formation and graft resorption. Sixteen FHBAs were grafted in the maxillae and mandibles of 9 patients. The FHBAs, which were provided by the Musculoskeletal Tissue Bank of Marilia Hospital (Unioss), were frozen at -80A degrees C. After 7 months, dental implants were placed and bone parameters were evaluated. Vertical bone formation was measured by computerized tomography before (T0) and at 7 months (T1) after the surgical procedure. Bone graft resorption was measured clinically from a landmark screw head using a periodontal probe. The results were analyzed by Student's t-test. Significant differences existed in the bone formation values at T0 and T1, with an average change of 4.03 +/- A 1.69 mm. Bone graft resorption values were 1.0 +/- A 0.82 mm (20%). Implants were placed with varying insertion torque values (35-45 Ncm), and achieved primary stability. This study demonstrates that FHBAs promote satisfactory vertical bone formation with a low resorption rates, good density, and primary implant stability.