RESUMO
OBJECTIVE: To investigate the effects of different doses of topical dexamethasone (Dex) on sciatic nerves with simulated compressive neuropathy. METHODS: Thirty-two Wistar rats were divided into four groups of 8: Sham group: no compression of the sciatic nerve + no treatment; Saline: chronic compression of the left sciatic nerve for 4 weeks + saline; 0.8% Dex: chronic compression + 0.8 mg of Dex; 3.2% Dex: chronic compression + 3.2 mg of Dex. Two sponge strips soaked with saline or Dex were placed under and over the nerve for 30 min in both Dex groups. Mixed-nerve-elicited somatosensory evoked potentials (M-SSEPs) and compound muscle action potentials (CMAPs) were measured to verify the compressive neuropathy in post-treatment follow-up. Behavioral observations of thermal hyperalgesia tests were quantified before electrophysiological examinations. Treated and contralateral nerves were harvested for histomorphological analysis. RESULTS: M-SSEP and CMAP amplitudes significantly decreased and latencies were significantly prolonged on postcompression thermal hyperalgesia tests. Rats in both Dex groups showed significant improvement in both sensory and motor conductive values and in neurological function, as well as increased mean myelin diameter on the final histomorphological examination. For rats in the saline group, these parameters showed incomplete recovery compared with the Sham group and the precompression baseline. Moreover, the changes after Dex treatment were not dose-dependent. CONCLUSIONS: Topical Dex reversed electrophysiological, behavioral, and structural changes in chronically compressed sciatic nerves. Differences between the beneficial effects of high-dose and low-dose Dex were nonsignificant.
Assuntos
Anti-Inflamatórios/administração & dosagem , Dexametasona/administração & dosagem , Síndromes de Compressão Nervosa/patologia , Neuropatia Ciática/tratamento farmacológico , Potenciais de Ação/efeitos dos fármacos , Administração Tópica , Animais , Modelos Animais de Doenças , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Nervo IsquiáticoRESUMO
Dopaminergic PC12 cells can synthesize and release dopamine, providing a good cellular model for investigating dopamine regulation. Optogenetic stimulation of channelrhodopsin-2 provides high spatial and temporal precision for selective stimulation as a powerful neuromodulation tool for neuroscience studies. The aim of this study is to measure dopamine release from dopaminergic PC12 cells under optogenetic stimulation using electrochemical recording of self-assembled monolayers modified microelectrode with amperometric measurement in real time. The activation of PC12 cells under various optogenetic stimulation schemes are characterized by measuring single-cell Ca(2+) imaging. After 10 seconds of optogenetic stimulation, the evoked intracellular Ca(2+) level and dopamine current of channelrhodopsin-2-transfected PC12 cells were 1.6- and 3.5-fold higher than those of the control cells. The optogenetic stimulation effects on Ca(2+) influx and dopamine release were 81% and 63% inhibition by using a Ca(2+) channel antagonist Nifedipine. The results indicate that optogenetic stimulation can evoke voltage-gated Ca(2+) channel-dependent dopamine exocytosis from PC12 cells in a cell specific, temporally precise and dose-dependent manner. This proposed dopamine recording system can be developed to be a good cell model for dopamine regulation and drug screening in vitro, or dopaminergic cell implantation therapy in vivo using optogenetic stimulation in a precise and convenient way.
Assuntos
Dopamina/metabolismo , Técnicas Eletroquímicas , Exocitose/fisiologia , Optogenética , Animais , Técnicas Biossensoriais , Cálcio/metabolismo , Channelrhodopsins , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microeletrodos , Células PC12 , RatosRESUMO
Chitosan-gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan-gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1WSC constructs (0.187±0.095 µg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329±0.660 µg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.
Assuntos
Cartilagem , Quitosana/química , Gelatina/química , Engenharia Tecidual , Alicerces Teciduais , Animais , Microscopia Eletrônica de Varredura , CoelhosRESUMO
Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-ß3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.
Assuntos
Cartilagem/crescimento & desenvolvimento , Condrogênese/genética , Colágeno Tipo II/metabolismo , Células-Tronco Mesenquimais/citologia , Adulto , Animais , Cartilagem/química , Cartilagem/metabolismo , Diferenciação Celular/genética , Colágeno Tipo II/química , Feminino , Humanos , Ácido Hialurônico/química , Células-Tronco Mesenquimais/metabolismo , Camundongos , Gravidez , Suínos , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Different intraoperative neuromonitoring modalities (mixed-nerve somatosensory-evoked potential [M-SSEP], dermatomal somatosensory-evoked potential [D-SSEP], compound motor-evoked potential [CMEP], electromyography [EMG], and the Hoffmann reflex [H-reflex]) have been developed for early detection of nerve root injury, for timely revision, and for damage reduction. In this study, we discuss the advantages and disadvantages of M-SSEP and D-SSEP by reviewing experimental evidence from animal models and clinical practice.
Assuntos
Potencial Evocado Motor/fisiologia , Potenciais Somatossensoriais Evocados/fisiologia , Reflexo H/fisiologia , Monitorização Intraoperatória/métodos , Raízes Nervosas Espinhais/fisiologia , Animais , Eletromiografia , Humanos , Procedimentos Neurocirúrgicos/efeitos adversos , Traumatismos dos Nervos Periféricos/prevenção & controle , Raízes Nervosas Espinhais/cirurgiaRESUMO
Fibroblast-like synoviocytes (FLS) play an important role in the pathologic processes of destructive arthritis by producing a number of catabolic cytokines and metalloproteinases (MMPs). The expression of these mediators is controlled at the transcriptional level. The purposes of this study were to evaluate the anti-arthritic effects of magnolol (5,5'-Diallyl-biphenyl-2,2'-diol), the major bioactive component of the bark of Magnolia officinalis, by examining its inhibitory effects on inflammatory mediator secretion and the NF-κB and AP-1 activation pathways and to investigate its therapeutic effects on the development of arthritis in a rat model. The in vitro anti-arthritic activity of magnolol was tested on interleukin (IL)-1ß-stimulated FLS by measuring levels of IL-6, cyclooxygenase-2, prostaglandin E(2), and matrix metalloproteinases (MMPs) by ELISA and RT-PCR. Further studies on how magnolol inhibits IL-1ß-stimulated cytokine expression were performed using Western blots, reporter gene assay, electrophoretic mobility shift assay, and confocal microscope analysis. The in vivo anti-arthritic effects of magnolol were evaluated in a Mycobacterium butyricum-induced arthritis model in rats. Magnolol markedly inhibited IL-1ß (10 ng/mL)-induced cytokine expression in a concentration-dependent manner (2.5-25 µg/mL). In clarifying the mechanisms involved, magnolol was found to inhibit the IL-1ß-induced activation of the IKK/IκB/NF-κB and MAPKs pathways by suppressing the nuclear translocation and DNA binding activity of both transcription factors. In the animal model, magnolol (100 mg/kg) significantly inhibited paw swelling and reduced serum cytokine levels. Our results demonstrate that magnolol inhibits the development of arthritis, suggesting that it might provide a new therapeutic approach to inflammatory arthritis diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Artrite/tratamento farmacológico , Compostos de Bifenilo/farmacologia , Lignanas/farmacologia , Membrana Sinovial/patologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite/patologia , Compostos de Bifenilo/uso terapêutico , Citocinas/antagonistas & inibidores , Citocinas/sangue , Modelos Animais de Doenças , Fibroblastos , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Interleucina-1beta/farmacologia , Lignanas/uso terapêutico , Metaloproteinases da Matriz/efeitos dos fármacos , Ratos , Resultado do TratamentoRESUMO
BACKGROUND: Tendon-bone tunnel healing is crucial for long term success in anterior cruciate ligament (ACL) reconstruction. The periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondroblasts during tendon-bone healing. We developed a scaffold-free method using polymerized fibrin-coated dishes to make functional periosteal progenitor cell (PPC) sheets. Bioengineered PPC sheets for enhancing tendon-bone healing were evaluated in an extra-articular bone tunnel model in rabbit. METHODS: PPC derived from rabbit tibia periosteum, cultivated on polymerized fibrin-coated dishes and harvested as PPC sheet. A confocal microscopy assay was used to evaluate the morphology of PPC sheets. PPC sheets as a periosteum to wrap around hamstring tendon grafts were pulled into a 3-mm diameter bone tunnel of tibia, and compared with a tendon graft without PPC sheets treatment. Rabbits were sacrificed at 4 and 8 weeks postoperatively for biochemical as-say and histological assay to demonstrate the enhancement of PPC sheets in tendon-bone healing. RESULTS: PPC spread deposit on fibrin on the dish surface with continuous monolayer PPC was ob-served. Histological staining revealed that PPC sheets enhance collagen and glycosaminoglycans deposition with fibrocartilage formation in the tendon-bone junction at 4 weeks. Collagen fiber with fibrocartilage formation at tendon-bone junction was also found at 8 weeks. Matured fibrocartilage and dense collagen fiber were formed at the tendon-bone interface at 8 weeks by Masson trichrome and Safranin-O staining. CONCLUSIONS: Periosteal progenitor cell monolayer maintains the differentiated capacity and osteochondral potential in order to promote fibrocartilage formation in tendon-bone junction. Bioengineered PPC sheets can offer a new feasible therapeutic strategy of a novel approach to enhance tendon-bone junction healing.
Assuntos
Ligamento Cruzado Anterior/transplante , Osso e Ossos/cirurgia , Periósteo/cirurgia , Células-Tronco/citologia , Tendões/cirurgia , Tíbia/transplante , Cicatrização , Animais , Ligamento Cruzado Anterior/cirurgia , Osteogênese/fisiologia , Periósteo/patologia , Coelhos , Tendões/patologia , Tíbia/cirurgia , Transplante Autólogo/métodos , Cicatrização/fisiologiaRESUMO
STUDY DESIGN: We used a severe contusive spinal cord injury (SCI) model and electrophysiologic, motor functional, immunohistochemical, and electron microscopic examinations to analyze the neuroprotective effects of delayed granulocyte colony-stimulating factor (G-CSF) treatment. OBJECTIVE: To determine the neuroprotective effects of delayed G-CSF treatment using multimodality evaluations after severe contusive SCI in rats. SUMMARY OF BACKGROUND DATA: Despite some reports that G-CSF treatment in the acute stage of different central nervous system injury models was neuroprotective, it has not been determined whether delayed G-CSF treatment can promote neural recovery in severe contusive SCI. METHODS: Rats with severe contusive SCI were divided into 2 groups: G-CSF group rats were given serial subcutaneous injections of G-CSF, and control group rats (controls) were given only saline injections on postcontusion days 9 to 13. Using the Basso-Beattie-Bresnahan scale and cortical somatosensory evoked potentials, we recorded functional evaluations weekly. The spinal cords were harvested for protein and immunohistochemical analysis, and for electron microscopy examination. RESULTS: The preserved spinal cord area was larger in G-CSF group rats than in control group rats. Both sensory and motor functions improved after G-CSF treatment. Detachment and disruption of the myelin sheets in the myelinated axons were significantly decreased, and axons sprouted and regenerated. There were fewer microglia and macrophages in the G-CSF group than in the control group. The levels of brain-derived neurotrophic factor were comparable between the 2 groups. CONCLUSION: Delayed G-CSF treatment at the subacute stage of severe contusive SCI promoted spinal cord preservation and improved functional outcomes. The mechanism of G-CSF's protection may be related in part to attenuating the infiltration of microglia and macrophages.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Fármacos Neuroprotetores/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Animais , Contusões , Modelos Animais de Doenças , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Feminino , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Microglia/efeitos dos fármacos , Microglia/patologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/reabilitação , Fatores de TempoRESUMO
Due to the potential for increasing ocean temperatures to detrimentally impact reef-building corals, there is an urgent need to better understand not only the coral thermal stress response, but also natural variation in their sub-cellular composition. To address this issue, while simultaneously developing a molecular platform for studying one of the most common Taiwanese reef corals, Seriatopora hystrix, 1,092 cDNA clones were sequenced and characterized. Subsequently, RNA, DNA and protein were extracted sequentially from colonies exposed to elevated (30°C) temperature for 48 hours. From the RNA phase, a heat shock protein-70 (hsp70)-like gene, deemed hsp/c, was identified in the coral host, and expression of this gene was measured with real-time quantitative PCR (qPCR) in both the host anthozoan and endosymbiotic dinoflagellates (genus Symbiodinium). While mRNA levels were not affected by temperature in either member, hsp/c expression was temporally variable in both and co-varied within biopsies. From the DNA phase, host and Symbiodinium hsp/c genome copy proportions (GCPs) were calculated to track changes in the biological composition of the holobiont during the experiment. While there was no temperature effect on either host or Symbiodinium GCP, both demonstrated significant temporal variation. Finally, total soluble protein was responsive to neither temperature nor exposure time, though the protein/DNA ratio varied significantly over time. Collectively, it appears that time, and not temperature, is a more important driver of the variation in these parameters, highlighting the need to consider natural variation in both gene expression and the molecular make-up of coral holobionts when conducting manipulative studies. This represents the first study to survey multiple macromolecules from both compartments of an endosymbiotic organism with methodologies that reflect their dual-compartmental nature, ideally generating a framework for assessing molecular-level changes within corals and other endosymbioses exposed to changes in their environment.
Assuntos
Antozoários/genética , Regulação da Expressão Gênica/fisiologia , Temperatura Alta , Chaperonas Moleculares/genética , Animais , Antozoários/fisiologia , DNA/isolamento & purificação , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/genética , Chaperonas Moleculares/análise , Proteínas/análise , RNA/isolamento & purificação , TemperaturaRESUMO
BACKGROUND: The efficacy and tolerability of 500-730 kDa sodium hyaluronate (Hyalgan®) for treatment of osteoarthritis (OA) pain has been established in clinical trials, but few data are available in the Asian population. We conducted a randomized, double-blind, multicenter, placebo-controlled study to evaluate the efficacy and tolerability of this preparation in a Taiwanese population. METHODS: Two hundred patients with mild to moderate OA of the knee were randomized to receive five weekly intra-articular injections of sodium hyaluronate or placebo. The primary efficacy outcome was the change from baseline to Week 25 in patients' evaluation of pain using a 100-mm visual analog scale (VAS) during the 50-foot walking test. Additional outcomes included Western Ontario and McMaster Universities (WOMAC) scores, time on the 50-foot walking test, patient's and investigator's subjective assessment of effectiveness, acetaminophen consumption, and the amounts of synovial fluid. RESULTS: The Hyalgan® treatment group showed a significantly greater improvement from baseline to Week 25 in VAS pain on the 50-foot walking test than the placebo group (p = 0.0020). The Hyalgan® group revealed significant improvements from baseline to week 25 in WOMAC pain and function score than the placebo group (p = 0.005 and 0.0038, respectively) Other outcomes, such as time on the 50-foot walking test and subjective assessment of effectiveness, did not show any significant difference between groups. Both groups were safe and well tolerated. CONCLUSIONS: The present study suggests that five weekly intra-articular injections of sodium hyaluronate are well tolerated, can provide sustained relief of pain, and can improve function in Asian patients with osteoarthritis of the knee. TRIAL REGISTRATION: Therapeutic study, Level I-1a (randomized controlled trial with a significant difference).
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Ácido Hialurônico/uso terapêutico , Osteoartrite do Joelho/tratamento farmacológico , Adjuvantes Imunológicos/administração & dosagem , Idoso , Povo Asiático , Método Duplo-Cego , Teste de Esforço , Feminino , Nível de Saúde , Humanos , Ácido Hialurônico/administração & dosagem , Injeções Intra-Articulares , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/etnologia , Osteoartrite do Joelho/fisiopatologia , Medição da Dor , Recuperação de Função Fisiológica , Taiwan/epidemiologia , Resultado do Tratamento , CaminhadaRESUMO
Chitosan nanocomposites were prepared from chitosan and gold nanoparticles (AuNPs) or silver nanoparticles (AgNPs) of â¼5 nm size. Transmission electron microscopy (TEM) showed the NPs in chitosan did not aggregate until higher concentrations (120-240 ppm). Atomic force microscopy (AFM) demonstrated that the nanocrystalline domains on chitosan surface were more evident upon addition of AuNPs (60 ppm) or AgNPs (120 ppm). Both nanocomposites showed greater elastic modulus, higher glass transition temperature (T(g)) and better cell proliferation than the pristine chitosan. Additionally, chitosan-Ag nanocomposites had antibacterial ability against Staphylococcus aureus. The potential of chitosan-Au nanocomposites as hemostatic wound dressings was evaluated in animal (rat) studies. Chitosan-Au was found to promote the repair of skin wound and hemostasis of severed hepatic portal vein. This study indicated that a small amount of NPs could induce significant changes in the physicochemical properties of chitosan, which may increase its biocompatibility and potential in wound management.
Assuntos
Antibacterianos/química , Materiais Biocompatíveis/química , Quitosana/química , Nanocompostos/química , Animais , Antibacterianos/farmacologia , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quitosana/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Ouro/química , Ouro/farmacologia , Humanos , Masculino , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Nanocompostos/ultraestrutura , Ratos , Ratos Sprague-Dawley , Prata/química , Prata/farmacologia , Pele/efeitos dos fármacos , Pele/lesões , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Temperatura de Transição , Cicatrização/efeitos dos fármacosRESUMO
Human placenta-derived mesenchymal stem cells (hPMSCs) represent a promising source of stem cells. The application of hPMSCs in cartilage tissue engineering, however, was less reported. In this study, hPMSCs were grown in a three-dimensional (3D) environment for cartilage tissue formation in vitro. To select proper scaffolds for 3D culture of mesenchymal stem cells (MSCs), rat adipose-derived MSCs were initially employed to optimize the composition and condition of the 3D environment. The suitability of a poly(D,L-lactide-co-glycolide) (PLGA) precision scaffold previously developed for seeding and culture of primary chondrocytes was tested for MSCs. It was established that MSCs had to be embedded in alginate gel before seeded in the PLGA precision scaffold for cartilage-like tissue formation. The inclusion of nano-sized calcium-deficient hydroxyapatite (nCDHA) and/or a recombinant protein containing arginine-glycine-aspartate (RGD) into the alginate gel enhanced the chondrogenesis for both rat adipose-derived MSCs and hPMSCs. The amount of extracellular matrix such as glycosaminoglycan and type II collagen accumulated during a period of 21 days was found to be the greatest for hPMSCs embedded in the alginate/nCDHA/RGD gel and injected and cultivated in the precision scaffold. Also, histological analyses revealed the lacunae formation and extracellular matrix production from the seeded hPMSCs. Comparing human bone marrow-derived MSCs (hBMSCs) and hPMSCs grown in the previous composite scaffolds, the secretion of glycosaminoglycan was twice as higher for hPMSCs as that for hBMSCs. It was concluded that the alginate/nCDHA/RGD mixed gel in the aforementioned system could provide a 3D environment for the chondrogenesis of hPMSCs, and the PLGA precision scaffold could provide the dimensional stability of the whole construct. This study also suggested that hPMSCs, when grown in a suitable scaffold, may be a good source of stem cells for building up the tissue-engineered cartilage.
Assuntos
Cartilagem/fisiologia , Condrogênese , Células-Tronco Mesenquimais/citologia , Placenta/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Alginatos/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Cartilagem/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Feminino , Ácido Glucurônico/farmacologia , Glicosaminoglicanos/metabolismo , Ácidos Hexurônicos/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microesferas , Gravidez , Ratos , Coloração e RotulagemRESUMO
The immunological response of macrophages to physically produced pure Au and Ag nanoparticles (NPs) (in three different sizes) is investigated in vitro. The treatment of either type of NP at > or =10 ppm dramatically decreases the population and increases the size of the macrophages. Both NPs enter the cells but only AuNPs (especially those with smaller diamter) up-regulate the expressions of proinflammatory genes interlukin-1 (IL-1), interlukin-6 (IL-6), and tumor necrosis factor (TNF-alpha). Transmission electron microscopy images show that AuNPs and AgNPs are both trapped in vesicles in the cytoplasma, but only AuNPs are organized into a circular pattern. It is speculated that part of the negatively charged AuNPs might adsorb serum protein and enter cells via the more complicated endocytotic pathway, which results in higher cytotoxicity and immunological response of AuNPs as compared to AgNPS.
Assuntos
Apoptose/efeitos dos fármacos , Ouro/imunologia , Ouro/farmacologia , Macrófagos/imunologia , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , Prata/imunologia , Prata/farmacologia , Animais , Linhagem Celular , Macrófagos/patologia , Teste de Materiais , Camundongos , Tamanho da PartículaRESUMO
Highly porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds for cartilage tissue engineering were fabricated in this study using the fused deposition manufacturing (FDM) process and were further modified by type II collagen. The average molecular weight of PLGA decreased to about 60% of the original value after the melt-extrusion process. Type II collagen exhibited sponge-like structure and filled the macroporous FDM scaffolds. An increase of the fiber spacing resulted in an increase of the porosity. The storage modulus of FDM scaffolds with a large fiber spacing was comparable to that of the native porcine articular cartilage. Although the FDM hybrid scaffolds were swollen in various extents after 28 days of in vitro culture, the seeded chondrocytes were well distributed in the interior of the scaffolds with a large fiber spacing and neocartilage was formed around the scaffolds. The study also suggested that a low processing temperature may be required to produce PLGA precision scaffolds using FDM.
Assuntos
Condrócitos/citologia , Colágeno Tipo II , Alicerces Teciduais , Animais , Cartilagem Articular/crescimento & desenvolvimento , Proliferação de Células , Células Cultivadas , Imuno-Histoquímica , Ácido Láctico/química , Microscopia Eletrônica de Varredura , Peso Molecular , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Porosidade , Sus scrofa , Fatores de Tempo , Engenharia TecidualRESUMO
The fused deposition manufacturing (FDM) system has been used to fabricate tissue-engineered scaffolds with highly interconnecting and controllable pore structure, although the system is limited to a few materials. For this reason, the liquid-frozen deposition manufacturing (LFDM) system based on an improvement of the FDM process was developed. Poly(D,L-lactide-co-glycolide) (PLGA) precision scaffolds were fabricated using LFDM from PLGA solutions of different concentrations. A greater concentration of PLGA solution resulted in greater mechanical strength but also resulted in less water content and smaller pore size on the surface of the scaffolds. LFDM scaffolds in general had mechanical strength closer to that of native articular cartilage than did FDM scaffolds. Neocartilage formation was observed in LFDM scaffolds seeded with porcine articular chondrocytes after 28 days of culture. Chondrocytes in LFDM scaffolds made from low concentrations (15-20%) of PLGA solution maintained a round shape, proliferated well, and secreted abundant extracellular matrix. In contrast, the FDM PLGA scaffolds had low cell numbers and poor matrix production because of heavy swelling. The LFDM system offered a useful way to fabricate scaffolds for cartilage tissue-engineering applications.
Assuntos
Cartilagem Articular , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Materiais Biocompatíveis , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/lesões , Cartilagem Articular/fisiologia , Contagem de Células , Proliferação de Células , Forma Celular , Condrócitos/citologia , Condrócitos/fisiologia , Colágeno/metabolismo , Matriz Extracelular/fisiologia , Congelamento , Glicosaminoglicanos/metabolismo , Ácido Láctico , Microscopia Eletrônica de Varredura , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Suínos , Engenharia Tecidual/instrumentaçãoRESUMO
BACKGROUND: Tissue-engineered solutions for promoting the tendon graft incorporation within the bone tunnel appear to be promising. HYPOTHESIS: To determine the feasibility that conjugation of hyaluronic acid-tethered bone morphogenetic protein-2 can be used to stimulate periosteal progenitor cells direct fibrocartilagenous attachment and new bone formation in an extra-articular tendon-bone healing model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 42 mature New Zealand White rabbits were used. The long digitorum extensor tendon was transplanted into a bone tunnel of the proximal tibia. The tendon was pulled through a drill hole in the proximal tibia and attached to the medial aspect of the tibia. Photopolymerizable hydrogel based on poly (ethylene glycol) diacrylate with hyaluronic acid-tethered bone morphogenetic protein-2 was injected and photogelated in a bone tunnel. Histological and biomechanical examination of the tendon-bone interface was evaluated at postoperative weeks 3 and 6. RESULTS: Histological analysis showed an interface fibrocartilage and new bone formed by photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells at 6 weeks. Biomechanical testing revealed higher maximum pullout strength and stiffness in experimental groups with a statistically significant difference at 3 and 6 weeks after tendon transplantation. CONCLUSION: The healing tendon-bone interface undergoes a gradual remodeling process; it appears that photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells possesses a powerful inductive ability between the tendon and the bone to incorporate the healing in a rabbit model. CLINICAL RELEVANCE: Novel technologies, such as those described in this study, including photopolymerization and tissue engineering, may provide minimally invasive therapeutic procedures via arthroscopy to enhance biological healing after reconstruction of the anterior cruciate ligament.
Assuntos
Proteínas Morfogenéticas Ósseas/administração & dosagem , Remodelação Óssea/efeitos dos fármacos , Transplante de Células-Tronco/métodos , Traumatismos dos Tendões/terapia , Tendões/transplante , Fator de Crescimento Transformador beta/administração & dosagem , Animais , Fenômenos Biomecânicos , Proteína Morfogenética Óssea 2 , Sobrevivência Celular , Ácido Hialurônico/administração & dosagem , Hidrogéis/administração & dosagem , Injeções Intralesionais , Microscopia de Fluorescência , Periósteo/citologia , Polietilenoglicóis/administração & dosagem , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/fisiologia , Células-Tronco/efeitos da radiação , Traumatismos dos Tendões/patologia , Tendões/patologia , Raios UltravioletaRESUMO
The nanocomposites (denoted "CII-Au") of porcine type II collagen (CII) with 0.05, 0.1, 0.5, 1, or 2.5% (wt/wt) Au nanoparticles ( approximately 5 nm) were fabricated for potential use in cartilage tissue engineering. Au formed clusters on the surface of all nanocomposites and appeared to distribute along the collagen fibrils inside the matrix. The addition of Au at low concentrations (< or =0.5%) increased the modulus and viscosity, as well as the free radical-scavenging ability. These effects decreased at higher concentrations of Au. The chondrocytes on CII-Au became spindle-like with lamellipodia formation. Cell proliferation on CII-Au 0.1% was promoted. Nitric oxide (NO) in the culture medium was reduced by CII-Au 0.05% and CII-Au 0.1%. Type I collagen, aggrecan, and Sox 9 gene expressions increased with an increased Au content, but slightly decreased at 2.5% Au. There was no significant difference in the CII gene expression. The cellular uptake of Au was observed but less than that which occurred when 10 ppm of Au was added in culture medium. Chondrocytes cultured with < or =10 ppm of Au nanoparticles showed neither cytotoxicity nor change in gene expression. Au at an appropriate amount could be well dispersed in CII, and enhanced the material modulus, antioxidant effect, as well as the chondrocyte growth and matrix production.
Assuntos
Cartilagem Articular/citologia , Condrócitos/efeitos dos fármacos , Colágeno Tipo II/farmacologia , Ouro/química , Nanopartículas , Animais , Materiais Biocompatíveis/farmacologia , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/química , Citoesqueleto/efeitos dos fármacos , Sequestradores de Radicais Livres/análise , Expressão Gênica , Óxido Nítrico/análise , Reologia , Suínos , Engenharia TecidualRESUMO
STUDY DESIGN: We evaluated electrophysiologic changes of the cauda equina after lumbar laminotomy in rats. OBJECTIVE: To clarify immediate and long-term electrophysiologic and neurologic responses in an experimental postlaminotomy animal fibrosis model. SUMMARY OF BACKGROUND DATA: Postspinal surgery-induced epidural fibrosis is assessed using either Gadolinium- enhanced magnetic resonance imaging (MRI) or intraoperative observations. In experimental animal models mimicking this complication, many approaches are used: advanced imaging (computed tomography, CT; and MRI), functional observations, biomechanical techniques, and histologic examinations. However, no study has reported the substantial neurophysiologic changes of the cauda equina in such a model. METHODS: Rats were given a sham operation (laminar exposure only), a left L5 hemilaminotomy alone, or a left L5 hemilaminotomy with extradural topical collagen. Mixed-nerve-elicited somatosensory-evoked potentials (M-SSEPs) and dermatomal (D)-SSEPs were recorded at the thoracolumbar junction after percutaneous stimulation of the posterior tibial nerve at the bilateral medial ankles and the L5 dermatomal field, respectively. Potentials recorded on the operated and nonoperated sides before surgery and then 30 minutes, 2 weeks, and 1, 2, and 3 months after surgery were compared. Walking track and thermal hyperalgesia test results and a final histologic analysis of perineural fibrosis were correlated. RESULTS: Electrical stimulation yielded reproducible responses in all rats on all tests. Preoperative and postoperative measurements showed no statistically significant differences in M-SSEP or D-SSEP. Postoperative D-SSEPs in both experimental groups showed significant reductions in relative amplitude, but the M-SSEPs of all groups and D-SSEPs of the control groups remained constant. CONCLUSION: SSEP is valuable for detecting electrophysiologic changes after laminotomy fibrosis, but acceptable accuracy requires proper stimulation and recording settings. D-SSEP monitoring provided reliable, useful information about the functional integrity of the cauda equina in this animal model. We recommend D-SSEP monitoring as a supplemental tool for quantifying the effect of postlaminotomy fibrosis on neuropathy.
Assuntos
Cauda Equina/fisiopatologia , Eletrodiagnóstico , Espaço Epidural/patologia , Potenciais Somatossensoriais Evocados , Laminectomia/efeitos adversos , Vértebras Lombares/cirurgia , Doenças do Sistema Nervoso Periférico/diagnóstico , Animais , Dor nas Costas/etiologia , Dor nas Costas/patologia , Dor nas Costas/fisiopatologia , Comportamento Animal , Modelos Animais de Doenças , Estimulação Elétrica , Fibrose/diagnóstico , Fibrose/etiologia , Testes Neuropsicológicos , Doenças do Sistema Nervoso Periférico/complicações , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Valor Preditivo dos Testes , Radiculopatia/etiologia , Radiculopatia/patologia , Radiculopatia/fisiopatologia , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Fatores de TempoRESUMO
STUDY DESIGN: We investigated changes in spinal somatosensory-evoked potential (SSEP) and nerve action potential (NAP), correlated behavior, and associated pathologic observation in experimental radiculopathy. OBJECTIVES: To create a rat model of sacrococcygeal radiculopathy for determining the validity of SSEP and NAP. SUMMARY OF BACKGROUND DATA: We examined the diagnostic sensitivity and value of electrophysiologic tests for evaluating lumbosacral root disease conflict. An appropriate animal model can help verify the value of these tests. METHODS: Preganglionic lesion group rats were given 2 loose ligatures around the cauda equina at the sacrum, and postganglionic lesion group rats were given 2 loose ligatures on the conjunction of the sacrococcygeal nerve roots and the caudalis nerve after they had received a laminectomy. Control group rats received a sham operation. SSEPs and NAPs were recorded preligature and postligature, and 3 times after surgery. These electrophysiologic observations were compared and correlated with tail-flick reflex and histology. RESULTS: All experimental group rats developed thermal hyperalgesia on day 14, as indicated by a significant reduction in TFL (tail-flick latency), which continued for 3 months. Amplitude decreased significantly and latency increased significantly in all SSEP recordings immediately after the operation; these changes persisted for 3 months. There were no significant differences between the experimental groups, but there were significant differences between the control and experimental groups. NAP amplitude and latency from the caudalis nerves did not change in any group in the first 2 postoperative weeks. From the second postoperative week until the 3-month follow-up, amplitude was significantly decreased and latency prolonged in the postganglionic group but unchanged in the others. CONCLUSIONS: Both SSEP and NAP are useful for evaluating electrophysiologic changes after various radiculopathies. The data also suggest that the conductivity of the peripheral nerve (NAP) was affected by the postganglionic compression of the corresponding nerve root, but not by the preganglionic lesion.
Assuntos
Fibras Autônomas Pós-Ganglionares/fisiologia , Fibras Autônomas Pré-Ganglionares/fisiologia , Radiculopatia/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Modelos Animais de Doenças , Eletrofisiologia , Potenciais Somatossensoriais Evocados/fisiologia , Hiperalgesia/fisiopatologia , Ratos , Ratos Sprague-Dawley , Região Sacrococcígea/inervação , Cauda/inervação , Cauda/fisiologiaRESUMO
We describe a biomimetic mode of insoluble signaling stimulation to provide target delivery of bone morphogenetic protein-2 (BMP-2), with the aim of prolonging the retention of BMP-2 use in bone tissue engineering and to enable its localized release in response to cellular activity. In our novel localization process, we used heterobifunctional acrylate-N-hydroxysuccinimide poly(ethylene glycol) (PEG) as a spacer to tether BMP-2 onto a poly(lactide-co-glycolide) scaffold. Use of PEG-tethered BMP-2 was feasible because BMP-2 retained its activity after covalent conjugation. The PEG-tethered BMP-2 conjugate sustained stimulation and retained its mitogenic activity, notably affecting pluripotent stem cell proliferation and differentiation. We seeded the scaffolds with bone marrow-derived mesenchymal stromal cells as progenitor cells to evaluate their morphology and phenotypic expression. We also created bilateral, full-thickness cranial defects in rabbits to investigate the osteogenic effect of cultured mesenchymal stromal cells on bone regeneration in vivo. Histomorphometry and histology demonstrated that the PEG-tethered BMP-2 conjugate enhanced de novo bone formation after surgery. Our work revealed the potential for biomimetic surface engineering by entrapping signaling growth factor to stimulate osteogenesis. Our technique may provide a new platform for bone-engineered stem cell therapies.