RESUMEN
Hollow nerve guidance conduits are approved for clinical use for defect lengths of up to 3 cm. This is because also in pre-clinical evaluation they are less effective in the support of nerve regeneration over critical defect lengths. Hydrogel luminal fillers are thought to improve the regeneration outcome by providing an optimized matrix inside bioartificial nerve grafts. We evaluated here a modified hyaluronic acid-laminin-hydrogel (M-HAL) as luminal filler for two clinically approved hollow nerve guides. Collagen-based and chitosan-based nerve guides were filled with M-HAL in two different concentrations and the regeneration outcome comprehensively studied in the acute repair rat sciatic nerve 15 mm critical defect size model. Autologous nerve graft (ANG) repair served as gold-standard control. At 120 days post-surgery, all ANG rats demonstrated electrodiagnostically detectable motor recovery. Both concentrations of the hydrogel luminal filler induced improved regeneration outcome over empty nerve guides. However, neither combination with collagen- nor chitosan-based nerve guides resulted in functional recovery comparable to the ANG repair. In contrast to our previous studies, we demonstrate here that M-HAL slightly improved the overall performance of either empty nerve guide type in the critical defect size model.
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Regeneración Tisular Dirigida/métodos , Ácido Hialurónico/química , Hidrogeles/química , Laminina/química , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos/cirugía , Animales , Células Cultivadas , Femenino , Ratas , Ratas Endogámicas LewRESUMEN
The familial disease of hereditary multiple exostoses is characterized by abnormal skeletal deformities requiring extensive surgical procedures. In hereditary multiple exostoses patients there is a shortage in the pericellular glycosaminoglycan (GAG) of heparan sulfate (HS), related to defective activity of HS glycosyltransferases, mainly in the pericellular regions of chondrocytes. This study searched for a novel approach employing xylosides with different aglycone groups priming a variety of GAG chains, in attempting to alter the GAG compositional profile. Cell cultures of patients with osteochondroma responded to p-nitrophenyl ß-D-xyloside by a significant increase in total GAG synthesis, expressed mainly in the extracellular domains, limited to chondroitin sulfate). The different ß-D-xylosides, in addition to increasing the synthesis of extracellular GAGs, led to a significant depletion of the intracellular GAG domains. In mouse chondrocyte cultures, ß-D-xylosides with different aglycones created a unique distribution of the GAG pools. Of special interest was the finding that the naphthalene methanol ß-D-xyloside showed the highest absolute levels of HS-GAGs in both extracellular and intra-pericellular moieties compared with other ß-D-xylosides and with controls without xyloside. In summary, ß-D-xylosides can be utilized in chondrocyte cultures to modify the distribution of GAGs between the extracellular and intracellular compartments. In addition, xylosides may alter the profile of specific GAG chains in each moiety.
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Condrocitos/efectos de los fármacos , Sulfatos de Condroitina/biosíntesis , Glicósidos/farmacología , Animales , Animales Recién Nacidos , Línea Celular , Condrocitos/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Naftalenos/farmacología , Osteocondroma , Células Tumorales CultivadasRESUMEN
This study aimed to investigate the innovative antigliotic guiding regenerative gel (AGRG) as reviving matrix for reconnection of spinal cord defect in rat models of complete acute and chronic spinal cord injury (SCI). In acute SCI, a 2 mm segment of the spinal cord (SC) was removed at Th7-Th8. Then AGRG was injected to the gap or left untreated. In chronic SCI, a 1 mm segment of the spinal cord (SC) was removed at Th7-Th8. One month later, the injured area was cleaned from connective and scar tissue, creating a gap of 2-3 mm. Then, AGRG was injected to the gap or left untreated. Functional, electrophysiological, histological and immunohistochemical assessments were performed. In acute SCI, at week 24, 75% of AGRG group showed a somatosensory evoked potential (SEP) signal. Appearance of myelin basic protein (MBP) was observed in the injured area in the AGRG group (p < 0.1), compared to the untreated group. In chronic SCI, 24 weeks after 2nd surgery, appearance of MBP, indicating presence of myelinated axons, was observed in AGRG group, compared to the untreated group (p < 0.01). These preliminary results suggest that AGRG can serve as a vital bridging station inducing regeneration of injured SC in acute and chronic cases of paraplegia.
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Traumatismos de la Médula Espinal , Ratas , Animales , Traumatismos de la Médula Espinal/cirugíaRESUMEN
The microstructure and chemical composition of the calcite shell of the sea barnacle Tetraclita rufotincta (Pilsbry, 1916) were investigated using microscopic and analytical methods. The barnacle shell was separated mechanically into its three substructural units: outer, interior, and inner layers. The organic matrices of these structural parts were further separated into soluble and insoluble constituents and their characteristic functional groups were studied by FTIR. Investigation of the mechanical properties of the interior mass of the shell reveals remarkable viscoelastic behavior. In general, the mechanical behavior of the shell is a function of its geometry as well as of the material, of which it is constructed. In the case of T. rufotincta, as calcite is a brittle material, the elastic behavior of the shell is apparently related to its micro- and macroarchitecture. The latter enables the shell to fulfill its primary function which is to protect the organism from a hostile environment and enables its survival. Our detailed identification of the similarities and differences between the various structural components of the shell in regard to the composition and properties of the organic component will hopefully throw light on the role of organic matrices in biomineralization processes.
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Exoesqueleto/química , Carbonato de Calcio/química , Thoracica/química , Exoesqueleto/ultraestructura , Animales , Microscopía de Fuerza Atómica , Espectroscopía Infrarroja por Transformada de Fourier , Thoracica/ultraestructuraRESUMEN
Among the objectives in writing the current chapter were the curiosity and the interest in allocating the sites and routes of migration of the reservoirs of the mesenchymal precartilaginous stem cells of the developing limbs in health and in disease. We chose to emphasize the events believed to initiate in these regions of stem cells, which may lead to growth retardation disorders. Thus, this narrow niche touches an enlarged scope of developmental biology angles and fields. The enclosed coverage sheds light on part of the musculoskeletal system, skeletogenesis, organogenesis of mobile structures and organs, the limbs, joints and digits (arthrology). It appears that the key role of the cartilage-bone regions is their responsibility to replenish the physis with committed chondrocytes, during the developmental, maturation and puberty periods. We shall start by outlining the framework of normal limb formation, the modalities, signals and the agents participating in this biological creation and regulation, illustrating potential sites that might deviate from normal development during the growth periods.
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Desarrollo Óseo/fisiología , Epífisis/embriología , Trastornos del Crecimiento/etiología , Células Madre Mesenquimatosas/fisiología , Animales , Enfermedades Óseas/fisiopatología , Proteínas Morfogenéticas Óseas/fisiología , Neoplasias Óseas/fisiopatología , Cartílago/embriología , Extremidades/embriología , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiologíaRESUMEN
Background and Aims: The aim of this study was to investigate the innovative guiding regenerative gel (GRG) and antigliotic GRG (AGRG) fillings for nerve conduits, prepared with Food and Drug Administration (FDA)-approved agents and expected to provide an alternative to autologous nerve graft and to enable reconnection of massive nerve gaps in a rabbit model of chronic peripheral nerve injury with massive loss defect that simulates the human condition of chronic injury with a large gap. Methods: The components and dosimetry for GRG and AGRG formulations were investigated in vitro on nerve cell culture and in vivo on 10-mm reconstructed sciatic nerves of 72 rats using different concentrations of agents and completed on a rabbit model of delayed (chronic) complete peripheral nerve injury with a 25-mm gap. Forty rabbits underwent delayed (9 weeks after complete injury of the tibial portion of the sciatic nerve) nerve tube reconstruction of a gap that is 25 mm long. GRG and AGRG groups were compared with autologous and empty tube reconstructed groups. Rats and rabbits underwent electrophysiological and histochemical assessments (19 weeks for rats and 40 weeks for rabbits). Results: Application of AGRG showed a significant increase of about 78% in neurite length per cell and was shown to have the most promising effect on neuronal outgrowth, with total number of neurites increasing by 4-fold. The electrophysiological follow-up showed that AGRG treatment is most promising for the reconstruction of the tibial portion of the sciatic nerve with a critical gap of 25 mm. The beneficial effect of AGRG was found when compared with the autologous nerve graft reconstruction. Thirty-one weeks post the second surgery (delayed reconstruction), histochemical observation showed significant regeneration after using AGRG neurogel, compared with the empty tube, and succeeded in significantly regenerating the nerve, as well as the autologous nerve graft, which was almost similar to a healthy nerve. Conclusion: We demonstrate that in the model of delayed peripheral nerve repair with massive loss defect, the application of AGRG led to a stronger nerve recovery and can be an alternative to autologous nerve graft.
RESUMEN
In the current study we investigated the suitability of a novel hyaluronic acid-laminin hydrogel (HAL) as luminal filler and carrier system for co-transplanted cells within a composite chitosan-based nerve graft (CNG) in a rat critical nerve defect model. The HAL was meant to improve the performance of our artificial nerve guides by giving additional structural and molecular support to regrowing axons. We filled hollow CNGs or two-chambered nerve guides with an inserted longitudinal chitosan film (CNG[F]s), with cell-free HAL or cell-free HA or additionally suspended either naïve Schwann cells (SCs) or fibroblast growth factor 2-overexpressing Schwann cells (FGF2-SCs) within the gels. We subjected female Lewis rats to immediate 15 mm sciatic nerve gap reconstruction and comprehensively compared axonal and functional regeneration parameters with the gold standard autologous nerve graft (ANG) repair. Motor recovery was surveyed by means of electrodiagnostic measurements at 60, 90, and 120 days post-reconstruction. Upon explantation after 120 days, lower limb target muscles were harvested for calculation of muscle-weight ratios. Semi-thin cross-sections of nerve segments distal to the grafts were evaluated histomorphometrically. After 120 days of recovery, only ANG treatment led to full motor recovery. Surprisingly, regeneration outcomes revealed no regeneration-supportive effect of HAL alone and even an impairment of peripheral nerve regeneration when combined with SCs and FGF2-SCs. Furthermore, complementary in vitro studies, conducted to elucidate the reason for this unexpected negative result, revealed that SCs and FGF2-SCs suspended within the hydrogel relatively downregulated gene expression of regeneration-supporting neurotrophic factors. In conclusion, cell-free HAL in its current formulation did not qualify for optimizing regeneration outcome through CNG[F]s. In addition, we demonstrate that our HAL, when used as a carrier system for co-transplanted SCs, changed their gene expression profile and deteriorated the pro-regenerative milieu within the nerve guides.
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Ácido Hialurónico/farmacología , Laminina/metabolismo , Nervios Periféricos/trasplante , Células de Schwann/efectos de los fármacos , Nervio Ciático/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Quitosano/farmacología , Hidrogeles/farmacología , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Ratas , Células de Schwann/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: The present study focuses on the effect of 780 nm laser irradiation on the growth of embryonic rat brain cultures embedded in NVR-Gel (cross-linked hyaluronic acid with adhesive molecule laminin and several growth factors). Dissociated neuronal cells were first grown in suspension attached to cylindrical microcarriers (MCs). The formed floating cell-MC aggregates were subsequently transferred into stationary cultures in gel and then laser treated. The response of neuronal growth following laser irradiation was investigated. MATERIALS AND METHODS: Whole brains were dissected from 16 days Sprague-Dawley rat embryos. Cells were mechanically dissociated, using narrow pipettes, and seeded on positively charged cylindrical MCs. After 4-14 days in suspension, the formed floating cell-MC aggregates were seeded as stationary cultures in NVR-Gel. Single cell-MC aggregates were either irradiated with near-infrared 780 nm laser beam for 1, 4, or 7 minutes, or cultured without irradiation. Laser powers were 10, 30, 50, 110, 160, 200, and 250 mW. RESULTS: 780 nm laser irradiation accelerated fiber sprouting and neuronal cell migration from the aggregates. Furthermore, unlike control cultures, the irradiated cultures (mainly after 1 minute irradiation of 50 mW) were already established after a short time of cultivation. They contained a much higher number of large size neurons (P<0.01), which formed dense branched interconnected networks of thick neuronal fibers. CONCLUSIONS: 780 nm laser phototherapy of embryonic rat brain cultures embedded in hyaluronic acid-laminin gel and attached to positively charged cylindrical MCs, stimulated migration and fiber sprouting of neuronal cells aggregates, developed large size neurons with dense branched interconnected network of neuronal fibers and, therefore, can be considered as potential procedure for cell therapy of neuronal injury or disease.
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Movimiento Celular/efectos de la radiación , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia por Luz de Baja Intensidad , Neuronas/fisiología , Neuronas/efectos de la radiación , Fototerapia , Animales , Encéfalo/citología , Encéfalo/embriología , Células Cultivadas , Neuronas/citología , Ratas , Ratas Sprague-DawleyRESUMEN
The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non-familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction.
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Condrocitos/metabolismo , Exostosis Múltiple Hereditaria/genética , Exostosis/genética , N-Acetilglucosaminiltransferasas/genética , Especificidad de Anticuerpos , Secuencia de Bases , Estudios de Casos y Controles , Células Cultivadas , Condrocitos/patología , Análisis Mutacional de ADN , Exostosis/patología , Expresión Génica , Glucuronidasa/análisis , Glucuronidasa/genética , Glicosaminoglicanos/análisis , Glicosaminoglicanos/genética , Humanos , Immunoblotting/métodos , Inmunohistoquímica , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/análisis , N-Acetilglucosaminiltransferasas/inmunología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Hyaluronic acid (HA), a major component of the extracellular matrix, is an attractive material for various medical applications. Yet, its low mechanical rigidity and fast in vivo degradation hinder its utilization. Here, we demonstrate the reinforcement of HA by its integration with a low-molecular-weight peptide hydrogelator to produce a composite hydrogel. The formulation of HA with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide, one of the most studied self-assembling hydrogel-forming building blocks, showing notable mechanical properties, resulted in the formation of stable, homogeneous hydrogels. Electron microscopy analysis demonstrated a uniform distribution of the two matrices in the composite forms. The composite hydrogels showed improved mechanical properties and stability to enzymatic degradation while maintaining their biocompatibility. Moreover, the storage modulus of the FmocFF/HA composite hydrogels reached up to 25 kPa. The composite hydrogels allowed sustained release of curcumin, a hydrophobic polyphenol showing antioxidant, anti-inflammatory, and antitumor activities. Importantly, the rate of curcumin release was modulated as a function of the concentration of the FmocFF peptide within the hydrogel matrix. This work provides a new approach for conferring mechanical rigidity and stability to HA without the need of cross-linking, thus potentially facilitating its utilization in different clinical applications, such as sustained drug release.
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Materiales Biocompatibles/química , Matriz Extracelular/química , Ácido Hialurónico/química , Hidrogeles/química , Péptidos/química , Materiales Biocompatibles/síntesis química , Péptidos/síntesis químicaRESUMEN
BACKGROUND: The increasing number of newborns requiring intubation and artificial ventilation in the sophisticated premature and intensive care units of recent years has been followed by a concomitant increase in the number of children who develop tracheal stenosis as a sequela of prolonged intubation, with a consequent increasing need for tracheal surgical repair. This study was designed to evaluate the ability of a new tissue-engineered biodegradable membrane to tightly seal significant tracheal defects. MATERIALS AND METHODS: A surgically induced tracheal defect of 10 x 5 mm was repaired in rabbits using the NVR-7 membrane--a cross-linked copolymer derived from a dextran sulphate gelatin construct. The unique features of this new membrane are biocompatibility, biodegradability, elasticity, and suturability, as well as a smooth sterilization process. The animals were sacrificed and the tracheas examined at 2, 3, 4, and 8 weeks postsurgery. RESULTS: Seven (7) of 8 rabbits undergoing tracheal surgery survived, with a tight air seal and an almost normal airway. Macroscopic and microscopic studies of the removed specimens showed variable degrees of immunogenic reaction toward the membrane. In the long term (2-3 months), a complete regeneration of all the tracheal layers occurred, simulating the original structure and orderly arrangement of a normal trachea. CONCLUSIONS: The surgical correction using the above membrane enabled the operated animals to overcome any respiratory distress, adequately correcting the induced tracheal defect. From this experimental study, we conclude that the new NVR-7 membrane appears to be a promising therapeutic adjunct in the treatment of patients with tracheal defects.
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Implantes Absorbibles , Materiales Biocompatibles , Membranas Artificiales , Enfermedades de la Tráquea/cirugía , Animales , Materiales Biocompatibles/química , Condrocitos/patología , Sulfato de Dextran/química , Modelos Animales de Enfermedad , Elasticidad , Epitelio/patología , Fibroblastos/patología , Gelatina/química , Polímeros/química , Conejos , Procedimientos de Cirugía Plástica , Regeneración/fisiología , Esterilización , Propiedades de Superficie , Suturas , Tráquea/patología , Tráquea/cirugía , Cicatrización de Heridas/fisiologíaRESUMEN
OBJECTIVE: Dental pulp is soft connective tissue maintaining the vitality of the tooth, while odontoblasts form the dentin. Our earlier DNA microarray analysis revealed expression of putative tumour suppressor exostosin 1 (EXT-1) in odontoblasts. EXT-1 is essential for heparan sulphate synthesis, which may play a role in the dentin mineralization. Since the absence of the functional EXT-1 causes bone tumours, expression in odontoblasts is interesting. Our aim was to analyse further the EXT-1 expression in human tooth. DESIGNS: DNA microarray and PCR techniques were used to study the EXT-1 expression in mature native human odontoblasts and pulp tissue as well as in newly-differentiated cultured odontoblast-like cells. Immunohistochemistry was performed to study EXT-1 protein in mature human teeth, teeth with incomplete root and developing teeth. RESULTS: Markedly higher EXT-1 was observed in mature odontoblasts than in pulp at mRNA level with DNA microarray and PCR techniques. Immunohistochemistry of mature tooth revealed EXT-1 both in odontoblasts and the predentin but not in the dentin. EXT-1 was also observed in the odontoblasts of incomplete root, but the localization of the staining was different. In developing foetal tooth, staining was detected in ameloblasts and the basal lamina. CONCLUSIONS: The detection of EXT-1 in both mature and newly-differentiated cells indicates a role in the odontoblast function, and EXT-1 staining in the predentin indicates a function in the dentin formation. Detection of EXT-1 in developing teeth indicates a role in tooth development.
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N-Acetilglucosaminiltransferasas/metabolismo , Odontoblastos/metabolismo , Ameloblastos/metabolismo , Células Cultivadas , Pulpa Dental/metabolismo , Dentina/metabolismo , Dentinogénesis/fisiología , Humanos , Técnicas para Inmunoenzimas , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Aberrant glomerular polyanionic charge of glycosaminoglycans (GAGs) and sialic acid expression has been observed in proteinuric human and experimental glomerular diseases. Angiotensin-converting enzyme inhibitors (ACEI) lower proteinuria and amend renal function deterioration via hemodynamic mechanisms. We tested the hypothesis that ACEI modulate proteinuria additionally by modifying glomerular GAGs. METHODS: In this study, we explored the effects of the ACEI enalapril on proteinuria and GAG synthesis in puromycin aminonucleoside (PAN)-treated rats. We employed cationic colloidal gold (CCG) localization in glomerular basement membranes (GBM) to identify GAGs by electron microscopy and determined sialic acid residues by immunohistochemical staining with lectins. To clarify ACEI effects on GAG production in vitro, we studied de novo GAG synthesis into newly synthesized proteoglycans in podocytes and mesangial cells using 35S incorporation. Cells were incubated with or without PAN, and with increasing doses of the ACEI enalaprilat. RESULTS: PAN rats developed severe proteinuria that was significantly improved by enalapril treatment. In non-treated PAN rats GBM GAGs were reduced, whereas in the enalapril-treated group GBM GAGs were significantly increased to control levels. Enalapril did not affect glomerular sialic acid. Furthermore, in cultured podocytes and mesangial cells PAN decreased de novo GAG synthesis, an effect which was significantly ameliorated by enalaprilat treatment. CONCLUSION: Treatment with ACEI improves permselectivity properties of the glomerular capillary wall by maintaining its GAG content. This finding provides an additional new mechanism, whereby ACEI exert anti-proteinuric effects.
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Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Enalapril/farmacología , Glicosaminoglicanos/biosíntesis , Glomérulos Renales/efectos de los fármacos , Nefrosis/metabolismo , Puromicina Aminonucleósido/toxicidad , Animales , Modelos Animales de Enfermedad , Inmunohistoquímica , Glomérulos Renales/metabolismo , Glomérulos Renales/ultraestructura , Masculino , Microscopía Electrónica de Transmisión , Nefrosis/patología , Podocitos/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/toxicidad , Ratas , Ratas WistarRESUMEN
A method for growth and maintenance of vital cartilaginous tissue is necessary for cartilage repair by in-vitro produced biologic implants. A previously tested perfusion system simulating joint activity was used. Whole epiphyses collected from thirty 11-day-old chick embryos were divided into two groups. One group was grown in a tissue culture dish for 10 days. The other group was placed in a perfusion system termed a joint-simulating device (JSD). After a period of 10 days, histology and immunohistochemistry were performed on five epiphyses from each group. Histologically, epiphyses grown in the device coalesced into a homogenous three-dimensional mass. The bridging tissue between individual epiphyses was highly cellular (PCNA staining positive) and was composed of mesenchymal stem cells as shown by expression of FGF receptor 3. No such tissue formed between epiphyses in the tissue culture dish and the epiphyseal cores were shown to be necrotic. The rest of the epiphyses were evaluated for radioactive sulfate incorporation into glycosaminoglycans (GAGs). A tenfold increase in sulfate incorporation occurred in epiphyses grown within the JSD as compared to the traditional culture method. In conclusion, embryonic epiphyses could be a suitable source for the ex-vivo growth of tissue-engineered cartilage constructs that might later be used as an in-vivo cartilage implant. The joint simulating device effectively maintains cartilage viability and bioactivity for as long as 10 days.
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Cartílago Articular/embriología , Epífisis/embriología , Técnicas de Cultivo de Órganos/métodos , Ingeniería de Tejidos/métodos , Animales , Células Cultivadas , Embrión de Pollo , Medios de Cultivo , Sulfatos/metabolismoRESUMEN
Microarray gene expression analysis was utilized to identify genes upregulated in primary rat calvaria cultures in response to mechanical force. One of the identified genes designated CMF608 appeared to be novel. The corresponding full-length cDNA was cloned and characterized in more details. It encodes a putative 2597 amino acid protein containing N-terminal signal peptide, six leucine-rich repeats (LRRs), and 12 immunoglobulin-like repeats, 10 of which are clustered within the C-terminus. Expression of CMF608 is bone-specific and the main type of CMF608-positive cells is mesenchymal osteochondroprogenitors with fibroblast-like morphology. These cells reside in the perichondral fibrous ring of La Croix, periosteum, endosteum of normal bone as well as in the activated periosteum and early fibrous callus generated postfracture. Expression of CMF608 is notably absent from the regions of endochondral ossification. Mature bone cell types do not produce CMF608 with the exception of chondrocytes of the tangential layer of the articular cartilage, which are thought to be under constant mechanical loading. Ectopic expression of CMF608 in HEK293T cells shows that the protein is subjected to post-translational processing and its N-terminal approximately 90 kDa polypeptide can be found in the conditioned medium. Ectopic expression of either the full-length cDNA of CMF608 or of its N-terminal region in CMF608-negative ROS17/2.8 rat osteosarcoma cells results in transfected clones displaying increased proliferation rate and the characteristics of less-differentiated osteoblasts compared to the control cells. Our data indicate that CMF608 is a unique marker of early osteochondroprogenitor cells. We propose that it could be functionally involved in maintenance of the osteochondroprogenitor cells pool and its down-regulation precedes terminal differentiation.
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Huesos/fisiología , Condrocitos/fisiología , Osteocitos/fisiología , Biosíntesis de Proteínas , Células Madre/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Fracturas Óseas/genética , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Cráneo/fisiología , Estrés Mecánico , Regulación hacia ArribaRESUMEN
OBJECTIVE: To evaluate whether satellite cells injected into infarct areas in rabbits remain viable during 6 weeks follow-up and can improve cardiac function as assessed by echocardiography. METHODS: Myocardial infarction was induced in 16 New Zealand white rabbits, by ligation of the marginalis sinistra artery. Tissue from gluteus muscle biopsies was dissected into small pieces and cultured. Within 2-3 weeks the cells were expanded by 2-3 orders of magnitude and were fluorescent labeled. Single cell pellets for resuspension at >10(6)/1 ml were directly injected into the infarct areas in 8 rabbits. In 8 additional rabbits, 1 ml saline was injected (control). Regional left ventricular function was assessed weekly by 2-D echocardiography until animals were sacrificed. Analysis was performed blind and independently by two experienced echocardiographers, based on the American Society of Echocardiography scheme. RESULTS AND DISCUSSION: Six treated and five control rabbits completed the study. One week after the artery occlusion, left ventricular function scoring did not differ between groups, mean 8.7+/-1.6 vs 8.3+/-1.9 (P=0.74). At 6 weeks post-injection, echocardiographic score was significantly better in the treated group, mean 2.6+/-0.9 vs 6.9+/-2.1 (P=0.002). The treated group showed significant gradual segmental improvement between the first week up to week 6. After sacrifice, macro and microscopic transmural areas showed typical changes of myocardial infarction. Histochemical staining identified viable grafted cells in high density 6 weeks post-transplantation in all grafted hearts. CONCLUSION: Autologous satellite cells (skeletal myofiber), can be successfully grafted into rabbit hearts following myocardial infarction and may induce improved regional left ventricular function.
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Infarto del Miocardio/terapia , Células Satélite del Músculo Esquelético/trasplante , Función Ventricular Izquierda/fisiología , Animales , Técnicas de Cultivo de Célula , Ecocardiografía , Electrocardiografía , Estudios de Seguimiento , Conejos , Resultado del TratamientoRESUMEN
Combined modality treatment of musculoskeletal tumors led to improved patient survival. As survival improves, more consideration is given to the functional outcome of treatment, and interest is focused on the development of less mutilating and extensive surgery. One modality that can reduce patient disability significantly is cryosurgery, as it allows minimally invasive surgery based on marginal resection and tumor interface sterilization instead of wide resection of certain neoplasms. Classical cryosurgery as developed by Marcove involves pouring of liquid nitrogen into the tumor bed. This approach revolutionized the treatment of some tumors such as giant cell tumor of bone, allowing intra-lesional resection to substitute the wide-resection method used up to that time. However, complications of this method of treatment are common, including nitrogen emboli, fractures of the bone due to extensive necrosis and damage to neurovascular elements. A recent development in the field of cryosurgery has been the argon-based system allowing controlled formation of an ice-ball surrounding a metallic probe. The system is computer controlled and allows precise evaluation of the tumor bed interface as well as surrounding structures that need to be protected. Prior to application of this method in humans it is important to ensure that interface sterilization is indeed achieved using cryosurgery. To evaluate this question, a Swarm rat chondrosarcoma was used. Cell viability was assessed following ice-ball formation. Histological evaluation indicated that cell death occurs up to 5 millimeters from the ice-ball if temperatures of -40 degrees Celsius at the metallic probe are achieved. A further evaluation was performed on samples obtained from patients during surgery. A minimum of two freezing cycles was shown to be necessary to achieve tissue viability similar to that of boiled tissue. Twenty-seven patients were operated to date using an argon-based cryosurgery system. The patients included 7 cases of grade I chondrosarcoma, 5 cases of giant cell tumor of bone, 14 cases of a metastatic lytic bone lesion and a single case of osseous-fibrous dysplasia. None of the patients suffered nerve injury during the operation. After a minimal follow-up period of 2 years only two of the surviving patients had a recurrence (a giant cell tumor of the proximal fibula, and the patient with the osseous-fibrous dysplasia whose tumor recurred as a frankly malignant adamantimoma). There were no pathological fractures. This method appears practical and allows close monitoring of the surrounding tissue to reduce the chances of recurrence.
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Neoplasias Óseas/terapia , Crioterapia/métodos , Neoplasias de los Músculos/terapia , Animales , Argón/química , Argón/metabolismo , Neoplasias Óseas/secundario , Línea Celular Tumoral , Supervivencia Celular , Condrosarcoma/terapia , Tumor Óseo de Células Gigantes/terapia , Humanos , Metástasis de la Neoplasia , Nitrógeno/metabolismo , Ratas , Factores de TiempoRESUMEN
This pilot study examined the effects of composite implants of cultured embryonal nerve cells and laser irradiation on the regeneration and repair of the completely transected spinal cord. Embryonal spinal cord nerve cells dissociated from rat fetuses and cultured on biodegradable microcarriers and embedded in hyaluronic acid were implanted in the completely transected spinal cords of 24 adult rats. For 14 consecutive post-operative days, 15 rats underwent low power laser irradiation (780 nm, 250 mW), 30 min daily. Eleven of the 15 (73%) showed different degrees of active leg movements and gait performance, compared to 4 (44%) of the 9 rats with implantation alone. In a controlgroup of seven rats with spinal cord transection and no transplantation or laser, six (86%) remained completely paralyzed. Three months after transection, implantation and laser irradiation, SSEPs were elicited in 69% of rats (p = 0.0237) compared to 37.5% in the nonirradiated group. The control group had no SSEPs response. Intensive axonal sprouting occurred in the group with implantation and laser. In the control group, the transected area contained proliferating fibroblasts and blood capillaries only. This suggests: 1. These in vitro composite implants are a regenerative and reparative source for reconstructing the transected spinal cord. 2. Post-operative low power laser irradiation enhances axonal sprouting and spinal cord repair.
Asunto(s)
Implantes Absorbibles , Terapia por Luz de Baja Intensidad , Paraplejía/cirugía , Médula Espinal/trasplante , Animales , Células Cultivadas , Potenciales Evocados Somatosensoriales/fisiología , Trasplante de Tejido Fetal , Ácido Hialurónico , Regeneración Nerviosa , Neuronas/trasplante , Paraplejía/patología , Paraplejía/fisiopatología , Proyectos Piloto , Ratas , Ratas Wistar , Recuperación de la Función , Médula Espinal/patología , Médula Espinal/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
UNLABELLED: In this work we evaluated the efficacy of biodegradable composite co-polymer guiding neurotube, based on tissue-engineering technology, for the treatment of complete peripheral nerve injury where the nerve defect is significant. The right sciatic nerve of 12 three-month-old rats was completely transected and peripheral nerve segment was removed. A 2.2-cm biodegradable co-polymer neurotube containing viscous gel (NVR-N-Gel) with survival factors, neuroprotective agents and Schwann cells was placed between the proximal and the distal parts of the transected nerve for reconnection a 2-cm nerve defect. The proximal and distal parts of the nerve were fixed into the neurotube using 10-0 sutures. Ultrasound observation showed growth of the axons into the composite neurotube 2 months after the surgery. Electrophysiological study indicated compound muscle action potentials in nine out of 12 rats, 2-4 months after peripheral nerve reconstructive surgery. The postoperative follow-up (up to 4 months) on the operated rats that underwent peripheral nerve reconstruction using composite co-polymer neurotube, showed beginning of re-establishment of active foot movements. The tube was dissolved and nerve showed complete reconnection. Histological observation of the nerve showed growth of myelinated axons into the site where a 2-cm nerve defect replaced by composite co-polymer neurotube and into the distal part of the nerve. IN CONCLUSION: (1) an innovative composite neurotube for reconstruction of significant loss of peripheral nerve segment is described; (2) a viscous gel, containing survival factors, neuroprotective agents and Schwann cells served as a regenerative environment for repair. Further investigations of this reconstructive procedure are being conducted.
Asunto(s)
Materiales Biocompatibles , Implantes Experimentales/tendencias , Traumatismos de los Nervios Periféricos , Nervios Periféricos/fisiología , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/farmacología , Masculino , Nervios Periféricos/efectos de los fármacos , Ratas , Ratas Wistar , Procedimientos de Cirugía Plástica/métodos , Procedimientos de Cirugía Plástica/tendencias , Ingeniería de Tejidos/tendenciasRESUMEN
This study compared the articular cartilage repair potential of cultured chondrocytes transplantation with bone-cartilage paste-graft in the resurfacing of full-thickness defects without breaching of the subchondral bone plate in rabbit knees. A 5 x 5-mm articular cartilage defect was created in the patellar groove of the femur. Three months following creation, the defect was filled with cultured autologous chondrocytes (group 1) or bone-cartilage paste (group 2). A control group of untreated defects was followed for 1 year. The reparative tissue was analyzed macroscopically, histologically, and by immunohistochemistry 3-12 months post-transplantation. The surfaces of the reparative tissue in group 1 were smooth, and the defects were filled with reparative tissue that resembled hyaline cartilage. The composition of the repair tissue more closely resembled cartilage, as demonstrated by cartilage-specific stains. In contrast, the reparative tissue in group 2 was fibrous and exhibited markers of mesenchymal stem cells and bone formation. Transplantation of cultured chondrocytes into a full-thickness defect in the rabbit generates a biologic substitute tissue that resembles native articular cartilage with living cells capable of synthesizing the surrounding cartilage matrix. In contrast, analysis of the healing response to the paste-graft technique failed to show cartilage-like characteristics. This information may be clinically applicable to direct the use of these treatments in chondral injuries.