Telomere length variation in normal epithelial cells adjacent to tumor: potential biomarker for breast cancer local recurrence.

A better understanding of the risk of local recurrence (LR) will facilitate therapeutic decision making in the management of early breast cancers. In the present study, we investigated whether telomere length in the normal breast epithelial cells surrounding the tumor is predictive of breast cancer LR; 152 women who were diagnosed with breast cancer at the Lombardi Comprehensive Cancer Center were included in this nested case-control study. Cases (patients had LR) and controls (patients had no LR) were matched on year of surgery, age at diagnosis and type of surgery. Telomere fluorescent in situ hybridization was used to determine the telomere length using formalin fixed paraffin-embedded breast tissues. Small telomere length variation (TLV), defined as the coefficient variation of telomere lengths among examined cells, in normal epithelial cells adjacent to the tumor was significantly associated with a 5-fold (95% confidence interval = 1.2-22.2) increased risk of breast cancer LR. When the subjects were categorized into quartiles, a significant inverse dose-response relationship was observed with lowest versus highest quartile odds ratio of 15.3 (P(trend) = 0.012). Patients who had large TLV had significantly better 10 year recurrence free survival rate compared with patients who had small TLV (80 versus 33%). The present study revealed that TLV in normal epithelial cells adjacent to tumor is a strong predictor of breast cancer LR. If confirmed by future studies, TLV in normal epithelial cells adjacent to tumor has the potential to become a promising biomarker for predicting breast cancer LR after breast conserving surgery.

Vitalized guided bone regeneration membrane from marrow stromal cells.

PURPOSE: Various materials have been used to make guided bone regeneration membranes. The purpose of this study was to create a novel osteogenic membrane without synthetic material. The osteogenic potential of the membrane was evaluated by both in vitro and in vivo testing. MATERIALS AND METHODS: The membrane was obtained by continuous culture of marrow stromal cells. The structure of the membrane was characterized by staining with hematoxylin-eosin, von Kossa, and carboxyfluorescein diacetate; immunohistochemical staining against collagen type I; electron microscopy; and energy-dispersive spectrometry. The osteogenic potential and bone augmentation effect of the membrane were investigated by implantation of the membrane and a membrane/natural coral composite into nude mice, respectively. RESULTS: The membrane was composed of living cells and a dense matrix of collagen type I. Mineral deposition was apparent through electronic microscopic observation and von Kossa staining. Energy-dispersive spectrometry indicated that the calcium:phosphorus ratio of mineral was 1.71 in the membrane. The membrane had formed a thin layer of bone 2 months after implantation subcutaneously. In the bone augmentation specimens, new bone was observed histologically on the surface and in the pores of natural coral in all specimens of membrane-coral composite. CONCLUSIONS: This study developed a novel strategy to produce a vital guided bone regeneration membrane without synthetic material. Membrane derived from marrow stromal cells was osteogenic and had an optimizing bone augmentation effect.

A minimal common osteochondrocytic differentiation medium for the osteogenic and chondrogenic differentiation of bone marrow stromal cells in the construction of osteochondral graft.

To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approach, controllable differentiation of bone marrow stromal cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. This study proposes to test a minimum common osteochondrocytic differentiation medium (MCDM) formulated by including common soluble supplements (dexamethasone and ascorbic acid) used to induce chondrogenic and osteogenic differentiation. The MCDM coupled with supplemented growth factors was tested for its ability to differentiate BMSCs into osteogenic and chondrogenic lineages in both two-dimensional and three-dimensional culture systems. When transforming growth factor beta3 was added to MCDM, BMSCs differentiated to chondrocyte-like cells, evidenced by the expression of glycosaminoglycans and type II collagen, whereas osteogenic differentiation was induced by supplementing osteogenic protein-1, resulting in detectable expression of osteopontin and osteocalcin. These chondrogenic and osteogenic differentiation markers were significantly enhanced in the three-dimensional cultures compared to the two-dimensional monolayer cultures. The results achieved in this study lay a foundation for future development of osteochondral graft, which could be engineered from bilayered scaffold with spatially loaded growth factors to control BMSC differentiation.

The role of lipase and alpha-amylase in the degradation of starch/poly(epsilon-caprolactone) fiber meshes and the osteogenic differentiation of cultured marrow stromal cells.

The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.

Dose-dependent osteogenic effect of octacalcium phosphate on mouse bone marrow stromal cells.

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.

Production of a new tissue-engineered adipose substitute from human adipose-derived stromal cells.

Adipose tissue is an accessible and abundant source of mesenchymal stem cells for soft-tissue reconstruction. In an attempt to create a novel, entirely autologous tissue-engineered adipose substitute, we extracted human stromal cells from either lipoaspirated or resected fat, and assessed their capacity to produce a three-dimensional adipose tissue using an adapted "self-assembly" culture methodology. This strategy involved a concomitant induction of adipogenic differentiation whilst ascorbic acid supplementation stimulated the stromal cells to produce and organize their own "biomaterial" in the form of extracellular matrix, forming manipulatable sheets that are then assembled into thicker reconstructed adipose tissues. When compared to resected fat, lipoaspiration-derived cells featured an increased adipogenic potential and the enhanced ability to recreate an adipose substitute in vitro. When viewed by scanning electron microscopy, the appearance of these reconstructed adipose tissues was strikingly similar to subcutaneous fat. Furthermore, these substitutes secreted adipokines and mediated beta-adrenergic receptor-stimulated lipolysis, hence reproducing known major biological functions of white adipose tissue. Therefore, our cell-based tissue engineering strategy led to the production of a functional and entirely natural reconstructed adipose tissue, which offers the potential to be used for specific in vitro applications as well as for autologous soft-tissue reconstruction.

Osteogenic differentiation of marrow stromal cells cultured on nanoporous alumina surfaces.

A major goal in orthopedic biomaterials research is to design implant surfaces, which will enhance osseointegration in vivo. Several microscale as well as nanoscale architectures have been shown to significantly affect the functionality of bone cells i.e., osteoblasts. In this work, nanoporous alumina surfaces fabricated by a two-step anodization process were used. The nanostructure of these surfaces can be controlled by varying the voltage used for anodization process. Marrow stromal cells were isolated from mice and seeded on nanoporous and amorphous (control) alumina surfaces. Cell adhesion, proliferation, and viability were investigated for up to 7 days of culture. Furthermore, the cell functionality was investigated by calcein staining. The cells were provided with differentiation media after 7 days of culture. The alkaline phosphatase (ALP) activity and matrix production were quantified using a colorimetric assay and X-ray photoelectron spectroscopy (XPS) for up to 3 weeks of culture (2 weeks after providing differentiation media). Further, scanning electron microscopy (SEM) was used to investigate osteoblast morphology on these nanoporous surfaces. Over the 3-week study, the nanoporous alumina surfaces demonstrated approximately 45% increase in cell adhesion, proliferation, and viability, 35% increase in ALP activity, and 50% increase in matrix production when compared with the control surfaces.

Application of energy-dispersive X-ray microanalysis on the diagnosis of atypical calcific band keratopathy.

We describe pathological, ultrastructural, and spectrographic analyses of surgically resected cornea from a man with atypical corneal opacification, and discuss the corneal pathogenesis, and the utility of energy-dispersive X-ray microanalysis (EDXA). The histopathologic features of a case presenting with longstanding bilateral corneal clouding are reported, which was difficult to be diagnosed clinically as calcific band keratopathy. The patient underwent keratoplasty on his right eye. Paraffin sections of the host corneal button were subjected to hematoxylin and eosin (H&E). An adjacent section was studied by means of EDXA. Plastic sections were observed under a transmission electron microscope (TEM). H&E staining revealed flattened corneal epithelial cells and disappearing Bowman's membrane accompanied by numerous basophilic granular deposits within the underlying corneal stroma. EDXA demonstrated the elevated peaks of calcium and phosphorus within the area of granular deposits. TEM revealed electron dense material consistent with extracellular calcospherites. Given the patient's past medical and family history, and the serologic test results, the clinical presentation and histological findings were most consistent with calcific band keratopathy associated with preexisting minimal syphilitic keratitis. Extensive histologic studies including EDXA on resected corneal tissue can be helpful for the differential diagnosis and may elucidate the pathogenesis of corneal diseases.

Utilidad de la expresión de Ki67 en las biopsias preoperatorias para predecir la recidiva bioquímica del cáncer de próstata después de la prostatectomía radical / The usefulness of Ki67 expression in the biopsy specimens, to predict the biochemical progresion of the prostate cancer after radical prostatectomy

OBJETIVO: Evaluar la utilidad de la expresión de Ki67 de las biopsias diagnósticas preoperatorias, para predecir la recidiva bioquímica del cáncer de próstata después de la prostatectomía radical. MATERIAL Y MÉTODOS: Analizamos la expresión de Ki67 en las biopsias ecodirigidas de 103 pacientes a los que se les practicó prostatectomía radical. El tiempo medio de seguimiento es de 3,4 años (1,3-8,8 años). Correlacionamos la recidiva bioquímica con los factores pronósticos clásicos como el PSA (>10/=7/3%/3%/3%/10/=7/<7) y clasificación pT (pT3/pT0-2), para predecir la progresión bioquímica del cáncer de próstata después de la prostatectomía radical

OBJETIVE: To evaluate the usefulness of Ki67 expression in the biopsy specimens, to predict the biochemical progression of the prostate cancer after radical prostatectomy. MATERIAL AND METHODS: We analyse the Ki67 expression in the biopsy specimens of 103 patients treated with radical prostatectomy. The mean follow up is 3.4 years (1.3-8.8 years). We correlate the biochemical progression with traditional prognostic factors as the PSA (>10/=7/3%/3%/3%/10/=7/<7) and pT ification (pT3/pT0-2), to predict the biochemical progression of the prostate cancer after radical prostatectomy

Comparison of human bone marrow stromal cells seeded on calcium-deficient hydroxyapatite, beta-tricalcium phosphate and demineralized bone matrix.

The aim of this study was to compare three resorbable biomaterials regarding seeding efficacy with human bone marrow stromal cells (BMSCs), cell penetration into the matrix, cell proliferation and osteogenic differentiation. Calcium-deficient hydroxyapatite (CDHA), beta-tricalcium phosphate (beta-TCP), and demineralized bone matrix (DBM) were seeded with human BMSCs and kept in human serum and osteogenic supplements for 3 weeks. Morphologic and biochemical evaluations were performed on day 1, 7, 14 and 21. The allograft DBM and CDHA exhibited both an excellent seeding efficacy while the performance of beta-TCP was lower when compared. The total protein content and the values for specific alkaline phosphatase (ALP) increased on all matrices and no significant difference was found for these two markers. BMSCs in monolayer had a significant increase of protein, but not of ALP. Osteocalcin (OC) values increased significantly higher for BMSC in cultures on DBM when compared to CDHA and beta-TCP. The OC levels decreased significantly in the BMSC monolayer culture. BMSCs were found inconsistently within the synthetic materials, whereas in DBM they were found more homogeneously distributed throughout the matrix. All three matrices promoted BMSC proliferation and differentiation to osteogenic cells. DBM allografts seem to be more favorable with respect to cell ingrowth tested by histology, and osteogenic differentiation ascertained by an increase of OC. CDHA with its high specific surface area showed more favorable properties than beta-TCP regarding reproducibility of the seeding efficacy.

Ultrastructural and biophysical studies on protein conformations of epithelium and stroma in benign prostatic hyperplasia before and after transurethral resection of the prostate.

The alterations in protein conformation of epithelium and stroma in human benign prostatic hyperplasia (BPH) undergoing prostatic needle biopsy (PNB) and transurethral resection of the prostate (TURP) were isolated in vitro and investigated using attenuated total reflectance (ATR)/Fourier transform infrared (FT-IR) microspectroscopy. Second-derivative spectral analysis and a curve-fitting program were used to verify and obtain the protein secondary conformation and composition of the samples. TURP uses an electric cutting heat but PNB is a nonheating process. The results indicate a predominantly higher proportion of beta-sheet conformation of the epithelium or stroma in BPH tissue at 1633 or 1630 cm(-1) in the amide I band, but after treatment with TURP, both maximum peaks shifted to 1644 or 1646 cm(-1) due to random coil structure, respectively. This conversion suggests that the heat generated by the cutting loop could increase the temperature in the tissue surrounding the prostate, thereby modifying the protein conformational structure of epithelium and stroma in BPH tissue.

Molecular biological ontogenesis of the thymic reticulo-epithelial cell network during the organization of the cellular microenvironment.

The thymus provides an optimal humoral microenvironment for the development of immunocompetent T cells. Although yolk sac derived pre-T, committed hematopoietic stem cells enter the thymus using a homing receptor, the immigration process also requires secretion of a peptide called thymotaxin by the cells of the reticulo-epithelial (RE) network of the thymic cellular microenvironment. The majority of RE cells have a round or irregular pale nucleus, which contains few, scattered, chromatin granules with a defined, spherical nucleolus, rich in basic histones. Their cytoplasm occasionally displays RNP granules, and is rich in non-histone proteins, fine phospholipid, lipid or cholesterin granules, and vacuoles filled with secreted substances. The cells of the subcapsular, endocrine RE cell layer (giant or nurse cells), characterized by PAS positive granules, express A2B5/TE4 cell surface antigens and MHC Class I (HLA A, B, C) molecules. In contrast to medullar RE cells, these subcapsular nurse cells also produce thymosins beta 3 beta 4. Thymic nurse cells (TNCs) display a neuroendocrine cell specific immunophenotype (IP): Thy-1+, A2B5+, TT+, TE4+, UJ13/A+, UJ127.11+, UJ167.11+, UJ181.4+, and presence of common leukocyte antigen (CLA+). Medullar RE cells display MHC Class II (HLA-DP, HLA-DQ, HLA-DR) molecule restriction. These cells also contain transforming growth factor-beta (TGF-beta) type II receptors and participate in the positive selection of T cells. Transmission electron-microscopic (TEM) observations have defined four functional subtypes of medullar RE cells: undifferentiated, squamous, villous, and cystic. All subtypes are connected by desmosomes. Immunocytochemical observations have shown that the secreted thymic hormones, thymosin alpha 1 and thymopoietin (and its short form, thymopentin or TP5), are produced by the same RE cells. Thymic RE cells also produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF that likely are important in various stages of thymocyte activation and differentiation. The co-existence of pituitary hormone and neuropeptide secretion, such as growth hormone, prolactin, adrenocorticotropic hormone, thyroid stimulating hormone, triiodothyronine, somatostatin, oxytocin, follicle stimulating hormone, luteinizing hormone, arginine vasopressin, growth hormone releasing hormone, corticotropin releasing hormone, nerve growth factor, vasoactive intestinal peptide, (pro) enkephalin, and beta-endorphin, production of a number of interleukins and growth factors, as well as the expression of receptors for all, by the same RE cell is an unique molecular biological phenomenon. These data illustrate the immensely important and diverse immuno-neuroendocrine functions of the thymic RE cellular network. Based on our systematic observations of the thymus in humans and other mammalian species, we suggest that the thymic RE cell network represents an extremely important cellular and humoral microenvironment in homeopathic regulatory mechanisms of the multicellular organism. Intrathymic T lymphocyte selection is a complex, multistep process, influenced by several functionally specialized RE cell subtypes and under constant immuno-neuroendocrine regulation, reflecting the dynamic changes of the organism.

Ultrastructural study on the in vitro interaction between haemopoietic cells and stromal cells. A new method using gel technique.

The ultrastructural study of the interaction between stroma and haemopoiesis is not an easy task because the loose attachment may be damaged during manipulation. This paper describes a technique by which the loose connection between preestablished stromal layer and attached haemopoietic cells (derived from blast colony forming cells) can be preserved and studied ultrastructurally. Stromal cultures were obtained from human bone marrow cells. Blast colony forming cells were studied by co-incubating the stroma with fetal calf serum supplemented McCoy's medium containing bovine plasma, thrombin and calcium to form a gel ('plasma clot'). Colony formers attached to the stroma formed myeloid colonies within 6 days. The semisolid plasma clot which solidifiers rapidly on the addition of glutaraldehyde or formaldehyde entraps the blastic colonies and haemopoietic cells in their position. Even the non-attached or mobile cells can be entrapped by this technique. The immature cells were found to be attached to the stromal surface and/or to the extracellular matrix, while the more mature cells migrated either to the surface of the colony or attached to the non-covered areas of the plastic surface. This method may offer a special technique to study dynamic interactions in other situations (e.g. chemotaxis etc.), too.

Realization of estradiol effects in the uterus of ovariectomized rats under acute stress.

The aim of this study was to examine the influence of acute stress on the realization of the effects of estrogens in the uterus of ovariectomized rats. The rats were immobilized for 6 h and given a single injection of estradiol dipropionate (E2; 10 micrograms/rat, i.m.). E2-only treated rats, olive oil injected and stressed rats, olive oil-only treated rats and untreated ovariectomized animals were used as controls. The effect of E2 was assessed by the volumes of cells, nuclei and nucleouli (morphometry), DNA content (Feulgen technique) and proliferative activity (mitotic index) in luminal epithelium, in glandular epithelium and in stromal cells of endometrium 24, 36 and 48 h after the injection of E2 or olive oil. All the effects of E2 were reduced in E2-treated rats subjected to stress in all the uterine structures. In olive oil treated rats, stress induced certain increase in the volume of cells and nuclei and slight increase in the DNA content in all the structures. It is suggested that the influence of stress on the uterus is mediated by impairment of some septs in the mechanism of action of estrogen on the uterus, which leads to a decrease in the sensitivity of the uterine structures to estrogens. Without estrogen stimulation the effect of stress on the uterus is realized via estrogen-independent pathways.