Dispersão da lidocaína administrada por via epidural em cães posicionados em decúbito lateral ou esternal / [Dispersion of lidocaine epidurally administered to dogs positioned on lateral or external decubitus]

O objetivo do estudo foi verificar clinicamente a dispersão da lidocaína no espaço epidural de cães posicionados em diferentes decúbitos. Foram utilizados 16 cães, com peso médio de 17,5 quilogramas. Esses foram tranquilizados com acepromazina, anestesiados com propofol e alocados em dois grupos, conforme o decúbito de posicionamento: decúbito esternal (GE) e decúbito lateral direito (GLD). Ambos os grupos receberam lidocaína a 2%, no volume de 0,25mL/kg, e permaneceram no mesmo decúbito por 20 minutos. Em seguida, avaliou-se o bloqueio dos membros pélvicos e a extensão do bloqueio, a partir da sétima vértebra lombar, por meio de pinçamento interdigital e do panículo paravertebral. Foi, então, realizada cirurgia de orquiectomia. Após tal procedimento, avaliou-se o tempo total de bloqueio dos membros pélvicos. Todos os cães apresentaram bloqueio bilateral, sem diferenças quanto à extensão cranial entre os grupos, sendo a mediana de 7,5 (1-14) vértebras para GE e de 4 (1-14) para GLD. O tempo de bloqueio dos membros direito e esquerdo foi de 123 ± 26 e 130 ± 20 minutos, para GE, e de 120 ± 21 e 121 ± 20 minutos, para GLD, sem diferenças entre os grupos ou entre os membros. Conclui-se que o decúbito não interfere na distribuição da lidocaína administrada por via epidural.(AU)

The aim of this study was to clinically verify the dispersion of lidocaine in the epidural space of dogs placed in different positions. Sixteen dogs with an average weight of 17.5 kilograms were used. These were tranquilized with acepromazine, anesthetized with propofol and allocated to two groups: sternal decubitus (GE) and right lateral decubitus (GLD). Both groups received 2% of lidocaine in the volume of 0.25mL/kg and remained in the same position for 20 minutes. The blocking of the pelvic limbs and the extension of it from the seventh lumbar vertebra were evaluated by means of interdigital and paravertebral panniculus clamping. Orchiectomy surgery was then performed. Afterwards, the total blocking time of the pelvic limbs was evaluated. All dogs presented bilateral blocking, with no differences in cranial extension between groups, with a median of 7.5 (1-14) vertebrae for GE and 4 (1-14) for GLD. The blocking time of the right and left limbs were 123 ± 26 and 130 ± 20 minutes for GE, and 120 ± 21 and 121 ± 20 minutes for GLD with no difference between groups or between limbs. It is concluded that the decubitus does not interfere with the epidural lidocaine distribution.(AU)

Inpatient dermatology consultations in a tertiary care hospital in Spain: A retrospective study of 750 patients. / Interconsultas dermatológicas en pacientes hospitalizados en un hospital terciario en España: un estudio retrospectivo de 750 pacientes.

OBJECTIVE: Medical consultations are a healthcare task that is generally undervalued and understudied. The main objective of this study was to describe and analyse the consultations requested of dermatology departments and the aspects that are involve in resolving the consultations. PATIENTS AND METHODS: We conducted an observational retrospective study that collected the consultations for patients hospitalised over a period of 36 months. We analysed variables related to the management of patients and the quality of the care. RESULTS: The study included 750 patients with a mean age of 64.85 (±20.23) years. The medical departments requested the most consultations, mainly internal medicine departments (37.9%). The most common topic for the consultations was inflammatory dermatoses (36.2%). The most common individual diagnosis was drug rash (8.1%). CONCLUSIONS: Consultation activities have a significant impact on the management of hospitalised patients, resulting in improved quality of care and reduced healthcare costs. These activities are also of training value for resident physicians.

A combined CoMFA and CoMSIA 3D-QSAR study of benzamide type antibacterial inhibitors of the FtsZ protein in drug-resistant Staphylococcus aureus.

A major problem today is bacterial resistance to antibiotics and the small number of new therapeutic agents approved in recent years. The development of new antibiotics capable of acting on new targets is urgently required. The filamenting temperature-sensitive Z (FtsZ) bacterial protein is a key biomolecule for bacterial division and survival. This makes FtsZ an attractive new pharmacological target for the development of antibacterial agents. There have been several attempts to develop ligands able to inhibit FtsZ. Despite the large number of synthesized compounds that inhibit the FtsZ protein, there are no quantitative structure-activity relationships (QSAR) that allow for the rational design and synthesis of promising new molecules. We present the first 3D-QSAR study of a large and diverse set of molecules that are able to inhibit the FtsZ bacterial protein. We summarize a set of chemical changes that can be made in the steric, electrostatic, hydrophobic and donor/acceptor hydrogen-bonding properties of the pharmacophore, to generate new bioactive molecules against FtsZ. These results provide a rational guide for the design and synthesis of promising new antibacterial agents, supported by the strong statistical parameters obtained from CoMFA (r(2)(pred) = 0.974) and CoMSIA (r(2)(pred) = 0.980) analyses.

Artrodesis del raquis. Ciencia básica / Spinal arthrodesis. Basic science

Una artrodesis vertebral consiste en la combinación de un sistema de estabilización mecánica de 2 o más segmentos vertebrales con una sustancia biológica que promueva la osteogénesis, con el objetivo de conseguir la fusión permanente entre zonas más o menos extensas de dichos segmentos. En una artrodesis vertebral, el aporte biológico por excelencia es el autoinjerto; sin embargo, su obtención genera una alta incidencia de morbilidad y, en casos de artrodesis de más de un espacio intervertebral, la cantidad disponible suele ser insuficiente. El tiempo de extracción e implantación prolonga la intervención quirúrgica, aumentando la exposición y riesgo a sangrado e infección. Por ello, actualmente hay una búsqueda de sustancias que posean las propiedades del autoinjerto evitando la morbilidad y tiempo de cirugía añadido que genera extraer el autoinjerto. En este trabajo se estudia la interacción biomecánica-biología en la artrodesis vertebral (AU)

Spinal arthrodesis consists of a combination of a system of mechanical stabilisation of one or more vertebral segments with a biological substance that promotes osteoneogenesis, with aim of achieving the permanent fusion between areas more or less the same size of these segments. In spinal arthrodesis, the biological support par excellence is the autograft. However, obtaining this involves a high incidence of morbidity and, in cases of arthrodesis of more than one intervertebral space, the quantity available is usually insufficient. The extraction and implantation time prolongs the surgery, increasing the exposure to and risk of bleeding and infection. For these reasons, there is a search for substances that possess the properties of the autograft, avoiding the morbidity and added surgical time required to extract the autograft. The biomechanical-biological interaction in vertebral arthrodesis has been studied in this article (AU)

[Spinal arthrodesis. basic science]. / Artrodesis del raquis. Ciencia básica.

Spinal arthrodesis consists of a combination of a system of mechanical stabilisation of one or more vertebral segments with a biological substance that promotes osteoneogenesis, with aim of achieving the permanent fusion between areas more or less the same size of these segments. In spinal arthrodesis, the biological support par excellence is the autograft. However, obtaining this involves a high incidence of morbidity and, in cases of arthrodesis of more than one intervertebral space, the quantity available is usually insufficient. The extraction and implantation time prolongs the surgery, increasing the exposure to and risk of bleeding and infection. For these reasons, there is a search for substances that possess the properties of the autograft, avoiding the morbidity and added surgical time required to extract the autograft. The biomechanical-biological interaction in vertebral arthrodesis has been studied in this article.

Uso de biomateriales con fines neurorregenerativos. Aplicación en modelo experimental de traumatismo craneoencefálico / Use of biomaterials in neural regeneration. Application in an experimental model of head injury

Objetivos: Evaluar el rendimiento de los biomateriales poliméricos basados en ácido hialurónico y su utilidad en el Sistema Nervioso Central, sirviendo como soporte, para la supervivencia y diferenciación celular. Material y Metodos: Con el fin de evaluar la viabilidad de los soportes poliméricos y acanalados, se realizaron experimetos in vitro e in vivo mediante el implante en corteza cerebral de ratas Wistar. Mediante técnicas inmunocitoquímicas e histológicas se procedió al análisis de la viabilidad de los soportes. Resultados: Tras el cultivo pudimos constatar la viabilidad celular sobre los biomateriales, asi como su potencial utilidad para la regeneración in vivo de estructuras vasculares y neurales. Conclusiones: La posibilidad de regenerar estructuras vasculares y neurales a través del implante de biomateriales basados en ácido hialurónico, constituye un avance en la utilización de biomateriales en el Sistema Nervioso Central (AU)

Objetives: To evaluate the performance of polymeric biomaterials based on hyaluronic acid and their usefulness in the central nervous system as support for cell differentiation and survival. Material and methods: With the purpose of assessing the viability of polymeric cannulated scaffolds, in vitro and in vivo experiments were made involving implantation in the Wistar rate brain cortex. Immunocytochemical and histological techniques were used to analyze scaffold viability. Results: Following culture, cell viability on the biomaterials was confirmed, together with the potential usefulness of the latter for the in vivo regeneration of vascular and neural structures. Conclusions: The possibility of regenerating vascular and neural structures through the implantation of biomaterials based on hyaluronic acid constitutes an advance in the use of biomaterials in the central nervous system (AU)

Uso de biomateriales en medicina regenerativa, aspectos básicos y aplicaciones en el Sistema Nervioso / Use of biomaterials in regenerative medicine, basic aspects and applications in the Nervous System

Objetivo: Analizar algunos de los aspectos físico-químicos y estructurales más importantes en el diseño de biomateriales destinados a la reparación tisular con un enfoque hacia su posible utilidad y potencialidad en el sistema nervioso. Diseño: Se analizan diversos estudios enfocados a la síntesis y diseño de biomateriales destinados a la reconstrucción tisular a partir de matrices porosas, sistemas nanoestructurados y combinación de biomateriales y células madre con fines regenerativos. Conclusiones: La práctica en el empleo de biomateriales con fines regenerativos constituye hoy día un hecho evidente y un gran desafío para la medicina neuroregenerativa (AU)

Objective: To analyze some of the physical-chemical and structural most important aspect in the design of biomaterials for tissue repair with an approach to its possible usefulness in the nervous system. Design: We analyzed several studies regarding the synthesis and design of biomaterials for tissue reconstruction by using porous scaffolds, nanostructured systems, and combination of biomaterials and stem cells. Conclusions: Nowadays the use of biomaterials for regenerative processes is an evident fact, and a great challenge for neuroregenerative medicine (AU)

Selection and induction of rat skeletal muscle-derived cells to the chondro-osteogenic lineage.

Bone marrow (BM) has been long established as the main source of pluripotential mesenchymal stem cells (MSCs), and has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The existence of an osteoprogenitor cell in other connective tissues such as skeletal muscle has been reported. In light of its availability and because of the relative ease of muscle cell isolation, skeletal muscle is an attractive source of cells for use in tissue engineering applications. The aim of this study was to explore the potential to differentiate into the chondro--osteoblastic lineage of a plastic adhering cell population, referred t as skeletal muscle-derived cells (SMDCs), obtained from biopsies of rat skeletal muscle. SMDCs displayed a fibroblast-like morphology. Our study revealed that the isolated cell population had a mesenchymal origin as indicated by abundant expression of STRO-1 and CD166. Osteogenic markers like osteocalcin (OC), bone sialoprotein (BSP) and osteopontin (OP) gene expressions were detected by RT-PCR. When these cells were cultured in the presence of an osteo-inductive culture medium, positive staining for alkaline phosphatase (ALP) and formation of mineralized matrix were increased. Furthermore SMDCs formed bone and cartilage tissues in vivo when placed inside of diffusion chambers and in demineralized bone matrix (DBM) cylinders, implanted subdermically into the backs of rat for 28 days. In conclusion, this experimental procedure is capable of selecting a cell population obtained from the skeletal muscle that is able to complete the differentiation pathway leading to the formation of cartilage and bone. In this respect SMDCs resemble BM stromal cells (BMSCs) and have demonstrated a potential application for cartilage and bone tissue engineering.

Adult stem cells applied to tissue engineering and regenerative medicine.

Regeneration takes place in the body at a moment or another throughout life. Bone, cartilage, and tendons (the key components of the structure and articulation in the body) have a limited capacity for self-repair and, after traumatic injury or disease, the regenerative power of adult tissue is often insufficient. When organs or tissues are irreparably damaged, they may be replaced by an artificial device or by a donor organ. However, the number of available donor organs is considerably limited. Generation of tissue-engineered replacement organs by extracting stem cells from the patient, growing them and modifying them in clinical conditions after re-introduction in the body represents an ideal source for corrective treatment. Mesenchymal stem cells (MSCs) are the multipotential progenitors that give rise to skeletal cells, vascular smooth muscle cells, muscle (skeletal and cardiac muscle), adipocytes (fat tissue) and hematopoietic (blood)-supportive stromal cells. MSCs are found in multiple connective tissues, in adult bone marrow, skeletal muscles and fat pads. The wide representation in adult tissues may be related to the existence of a circulating blood pool or that MSCs are associated to the vascular system.

The effect of type I collagen on osteochondrogenic differentiation in adipose-derived stromal cells in vivo.

BACKGROUND: Recent studies have demonstrated that adipose-derived adult stromal cells (ADASCs) offer great promise for cell-based therapies due to their ability to differentiate towards bone, cartilage and fat [corrected] The objective of this study was to investigate whether type I collagen would elicit in vivo bone formation of passaged rat adipose-derived adult stromal cells (ADASC) placed extraskeletally. METHODS: After expansion for 1-4 passages (P), cells were incubated in osteogenic medium containing dexamethasone, ascorbic acid and beta-glycerol phosphate for 2-4 weeks. Undifferentiated cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and von Kossa staining as well as by gene expression of ALP, osteopontin (OP), osteonectin (ON), osteocalcin (OC), collagen I (colI), collagen II (colII), bone sialoprotein (BSP), periostin (Postn), runx2, osterix (Osx), sox9, msx1 and msx2. Diffusion chambers were filled with 1x10(6) cells mixed with or without type I collagen gel and implanted subcutaneously into rats. Controls included chambers exposed to (1) undifferentiated cells (with or without collagen, (2) collagen without cells and (3) empty chambers (n=5 per group). RESULTS: Four weeks after implantation, in vivo bone and cartilage formation was demonstrated in implants containing 4-week osteo-induced P1 and P4 cells wrapped in the collagen gel, as confirmed by Goldner's trichrome and Alcian blue staining, respectively. Newly formed bone stained positive for type I collagen. Control implants had no bone or cartilage and were primarily filled with fibrous tissue at that time interval. DISCUSSION: Recent studies have demonstrated that ADASC offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage and fat. However, the influence of different matrices on the in vivo osteogenic capability of ADASC is not fully understood. These findings suggest that type I collagen may support the survival and expression of osteogenic and chondrogenic phenotypes in passaged rat ADASC in vivo.

A modified rhTGF-beta1 and rhBMP-2 are effective in initiating a chondro-osseous differentiation pathway in bone marrow cells cultured in vitro.

Rat bone marrow cells were cultured in vitro in a collagen-gel medium at 0.5% fetal bovine serum concentration for 10 days in the presence of recombinant human transforming growth factor-beta-1, genetically engineered to contain a collagen binding domain (rhTGF-beta1-F2), or a commercial rhTGF-beta1. To compare the effects of TGF-betas with other growth factors in which the osteogenic capacity has been widely documented, a recombinant human bone morphogenetic protein (rhBMP-2) was evaluated. Once serum conditions compatible with growth were re-established, the selected cells were cultured for 6 more days in the presence of the growth factor. In the last 2 days, dexamethasone (dex) and beta-glycerophosphate (beta-GP) were added to promote osteogenesis. After this 16-day period, cells were placed into diffusion chambers or demineralized bone matrix (DBM) implants, and implanted subdermally on the backs of rats for 28 days. Biochemical, histological, and immunohistochemistry analysis provided evidence of cartilage (commercial rhTGF-beta1-treated cells), osteoid (rhTGF-beta1-F2-treated cells), and bone tissues (rhBMP-2 treated cells), inside the diffusion chambers, whereas bone, cartilage, and osteoid were observed inside the DBM implants under any of the three growth factors effect. Our study advances the technology capable of selecting a cell population from bone marrow that, in the presence of rhTGF-beta1 or rhBMP-2 in vitro, achieves chondro-osteogenic potential in vitro and in vivo.

Células madre e ingeniería tisular ósea. Bases celulares y perspectivas terapéuticas / Stem cells and bone tissue engineering

Los avances en el conocimiento de las proteínas morfogenéticas de hueso (BMP) han propiciado su utilización directa en las reparaciones óseas, planteando la falta de control sobre el tiempo que estas proteínas permanecen en la lesión, la actividad real de las mismas y la imprescindible necesidad de células osteogénicas en el lugar de aplicación, efectores últimos de la acción inductora. Por lo tanto, cuando la falta de células sea el problema fundamental, la aplicación directa de factores osteoinductores, aun cuando en otras circunstancias puedan ser más útiles, no producirá los resultados esperados, siendo el aporte de células osteoprogenitoras la línea de actuación más apropiada, bien directamente o a través de un material transportador osteoconductor, sin descartar la posibilidad de inyección sistémica. Entre las aplicaciones directas a la cirugía ortopédica de esta ingeniería tisular, está la consecución de artrodesis del raquis como tratamiento de inestabilidades de origen diverso. Los problemas actuales se centran en el fracaso de la fusión y en la morbilidad de la zona donante de autoinjerto. En las artroplastias la ingeniería tisular muestra también un campo de aplicación inmediato, si bien antes es necesario solucionar los problemas relativos a la estabilidad primaria. En cualquier caso, la validez de las conclusiones de la ingeniería tisular pasará por su verificación en modelos clínicos humanos con diseños epidemiológicos prospectivos metodológicamente correctos. Los problemas éticos y legales serán, en fin, los condicionantes fundamentales para la generalización de la ingeniería tisular como propuesta terapéutica. En este trabajo se realiza una revisión conceptual de estos problemas (AU)

Production of a recombinant human basic fibroblast growth factor with a collagen binding domain.

Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by [3H]thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. The high-affinity binding was demonstrated by the binding of [3H]collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column. The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications.

Immunocytochemical detection of Reissner's fiber-like glycoproteins in the subcommissural organ and the floor plate of wildtype and cyclops mutant zebrafish larvae.

The subcommissural organ (SCO) and the floor plate (FP) secrete high molecular weight glycoproteins that polymerize in the form of the Reissner's fiber (RF). To study to what extent the absence of the FP affects the expression of these glycoproteins, we have investigated the brain and spinal cord of 48-h and 72-h wildtype and cyclops (cyc) mutant zebrafish larvae by using a polyclonal antiserum against bovine RF. Wildtype larvae showed immunoreactivity in the SCO at the dorsal forebrain-midbrain boundary. In the ventricle, over the SCO surface, thin immunoreactive fibers aggregated into an RF that ran along the third and fourth ventricles and the central canal of the spinal cord until, at its caudal end, the fiber disintegrated and formed a strongly immunoreactive massa caudalis that left the neural tube and invaded the surrounding tissues of the tail fin. The rostral end of the FP, lining the pontine flexure, was also strongly immunoreactive, as was the caudal third of the FP. Cyc mutants showed an immunoreactive SCO and fibrous material in the ventricle, but an RF was missing. There was no label in the ventral midline of the neural tube except in some specimens in which the caudal FP persisted and was immunoreactive. It is concluded that the product of the cyc gene is not required for the expression of SCO glycoproteins but for their polymerization into an RF in the brain ventricles.

Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein.

Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen-binding domain, and (iii) cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains a collagen-binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured using a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factors was demonstrated by their ability to stimulate proliferation of human vein endothelial cells (HVEC), monitored by [3H]-thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. Binding of the renatured hbFGF-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of [3H]-collagen to the rhbFGF-F2 protein immobilized on a Ni-NTA column. The rhbFGF-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen-binding domain effectively targets the recombinant growth factor to type I collagen. The clinical effect of rhbFGF-F2 on wound healing is also studied in streptozotocin-induced diabetic rats and evaluated by histological examination comparing with rhbFGF-F1 and commercial bFGF effects. The highly beneficial effects of rhbFGF-F2 on wound healing is suggested to be due to its extremely potent angiogenesis and granulation tissue formation activities, leading to a rapid reepithelialization of the wound. Topical application of rhbFGF-F2 mixed with type I collagen is a more effective method in accelerating closure of full-thickness excisional skin-wound in diabetic rats when compared with the fusion protein alone or commercial hbFGF at the same doses. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins as well as to develop new strategies for specific biomedical applications.

Selection and amplification of a bone marrow cell population and its induction to the chondro-osteogenic lineage by rhOP-1: an in vitro and in vivo study.

The differentiation and maturation of osteoprogenitor cells into osteoblasts are processes which are thought to be modulated by transforming growth factors-beta (TGF-beta) as well as by bone morphogenetic proteins (BMPs). Osteogenic protein-1 (OP-1, also known as BMP-7) is a member of the BMP family, and it is considered to have important regulatory roles in skeletal embryogenesis and bone healing. Rat bone marrow cells were cultured in vitro in a collagen-gel medium containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of 40 ng/ml recombinant human OP-1 (rhOP-1). Under these conditions, survival of the bone marrow cell population was dependent on the presence of rhOP-1. Subsequently, the selected cells were cultured-for 6 days in medium containing 40 ng rhOP-1 and 10% FBS. During the last 2 days, dexamethasone (10(-8) M) and beta-glycerophosphate (2 mM) were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels, colony number and size were determined. Chondro-osteogenic differentiation in vitro was evaluated in terms of the expression of alkaline phosphatase, the production of osteocalcin and the formation of mineralized matrix. After culturing in vitro, cells were placed inside diffusion chambers or inactivated demineralized bone matrix (DBM) cylinders and implanted subdermically into the backs of old rats for 28 days. Biochemical, histological and immunocytochemical analyses provided evidence of cartilage and osteoid tissue inside the diffusion chambers, whereas bone was also observed inside the DBM implants. In conclusion, this experimental procedure is capable of selecting a cell population from bone marrow which, in the presence of rhOP-1, achieves skeletogenic potential under in vitro as well as in vivo environments.

A recombinant human TGF-beta1 fusion protein with collagen-binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells.

A continuous source of osteoblasts for normal bone maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the bone marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-beta (TGF-beta1 and TGF-beta2) and TGF-beta-related bone morphogenetic proteins (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-beta1-F2, a fusion protein engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-beta1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the 10-day selection period, the surviving cells in the rhTGF-beta1-F2 test groups proliferated rapidly in response to serum factors (10% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative bone cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and beta-glycerophosphate (beta-GP). Although rhTGF-beta1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-beta1-F2 fusion protein, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized bone matrix (iDBM) and/or diffusion chambers into older rats they are able to produce bone and cartilage. The population of progenitor cells captured by rhTGF-beta1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.

Floor plate and the subcommissural organ are the source of secretory compounds of related nature: comparative immunocytochemical study.

The subcommissural organ of vertebrates secretes glycoproteins into the third ventricle that condense to form Reissner's fiber (RF). Antibodies raised against the bovine RF-glycoproteins reacted with the floor plate (FP) cells of two teleost (Oncorhynchus kisutch, Sparus aurata) and two amphibian (Xenopus laevis, Batrachyla taeniata) species. At the ultrastructural level, the immunoreactivity was confined to secretory granules, mainly concentrated at the apical cell pole. In the rostro-caudal axis, a clear zonation of the FP was distinguished, with the hindbrain FP being the most, or the only (Batrachyla taeniata), immunoreactive region of the FP. In all the species studied, the caudal FP lacked immunoreactivity. Both the chemical nature of the immunoreactive material and the rostro-caudal zonation of the FP appear to be conservative features. Evidence was obtained that the FP secretes into the cerebrospinal fluid a material chemically related to the RF-glycoproteins secreted by the subcommissural organ. Thus, in addition to being the source of contact-mediated and diffusible signals, the FP might also secrete compounds into the cerebrospinal fluid that may act on distant targets.

Nitric oxide mediates hyperglycemia-induced defective migration in cultured endothelial cells.

PURPOSE: To examine the effects of elevated glucose on the migration and proliferation of vascular endothelial cells in an in vitro wound model and to investigate whether nitric oxide (NO) mediates the effects of elevated glucose. METHODS: Migration was investigated in monolayers of bovine aortic endothelial cells wounded by scraping and measuring the distance, the number of cells migrating, and the area covered by the migrating cells in the presence of various concentrations of glucose. The effects of NO were evaluated by adding to the cultures NG-monomethyl arginine (NMMA), an inhibitor of NO synthase, or S-nitrosylated penicillamine, which is a slow-release agent of NO. Proliferation was investigated in the presence of various concentrations of serum, glucose, or both. RESULTS: Elevated glucose levels (16.5 and 27.7 mmol/L) inhibited endothelial cell migration in a dose-dependent manner compared with cells cultured in the presence of 5.5 mmol/L glucose. Inhibition of migration was also observed when wounded mono-layers cultured in 5.5 mmol/L glucose were treated with S-nitrosylated penicillamine, which generates NO. Inhibition of NO synthase by NMMA prevented the inhibition of migration observed in media containing 27.7 mmol/L glucose. Elevated glucose levels did not affect cell proliferation except in the presence of 20% fetal bovine serum. CONCLUSIONS: An elevated glucose level inhibits endothelial cell migration in an in vitro wound model, and the inhibition appears to be mediated by increased levels of NO.