RESUMO
There is increasing evidence that the physicochemical nature of any given material is a dominant factor for the release of cytokines by innate immune cells, specifically of macrophages, and thus majorly influences their interaction with other cell types. Recently, we could show that the 3D structure of star shaped polytheylene oxide-polypropylene oxide co-polymers (sP(EO-stat-PO))-hydrogel coated substrates has a stronger influence on the release pattern of cytokines after 7 days of culture than surface chemistry. Here, we focused on the analysis of cytokine release over time and a more detailed analysis of cell morphology by scanning electron microscopy (SEM). Therefore, we compared different strategies for SEM sample preparation and found that using osmium tetroxide combined with aqua bidest led to best preparation results. For cytokine release we show significant changes from day 3 to day 7 of cell culture. After 3 days, the sP(EO-stat-PO)-coated substrates led to an induction of pro-angiogenic CCL3 and CCL4, and of low amounts of the anti-inflammatory IL10, which declined at day 7. In contrast, pleiotropic IL6 and the pro-inflammatory TNFα and IL1ß were expressed stronger at day 7 than at day 3.
Assuntos
Citocinas/metabolismo , Leucócitos Mononucleares/metabolismo , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/toxicidade , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Microscopia Eletrônica de Varredura , Nanofibras/química , Nanofibras/toxicidade , Polímeros/química , Fatores de TempoRESUMO
Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer(®) LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level.
Assuntos
Materiais Biocompatíveis/química , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Células Germinativas , Imuno-Histoquímica , Ácido Láctico/química , Camundongos , Microscopia Eletrônica de Varredura , Células-Tronco Pluripotentes/metabolismo , Poliésteres , Polímeros/química , Politetrafluoretileno/química , Polivinil/química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Calcific uraemic arteriolopathy (CUA) or calciphylaxis is a rare, life-threatening disease predominantly occurring in patients with end-stage renal disease. Its pathogenesis has been suggested to include ectopic osteogenesis in soft tissue and the vasculature associated with extracellular matrix (ECM) remodelling. METHODS: To gain further insights into the pathogenesis of CUA, we performed systematic analyses of skin specimens obtained from seven CUA patients including histology, immunohistochemistry, electron microscopy, electron dispersive X-ray analysis (EDX) and quantitative real-time RT-PCR. Skin specimens of (i) seven patients without chronic kidney disease and without CUA and (ii) seven dialysis patients without CUA served as controls. RESULTS: In the CUA skin lesions, we observed a significant upregulation of bone morphogenic protein 2 (BMP-2), its target gene Runx2 and its indirect antagonist sclerostin. Furthermore, we detected an increased expression of inactive uncarboxylated matrix Gla protein (Glu-MGP). The upregulation of osteogenesis-associated markers was accompanied by an increased expression of osteopontin, fibronectin, laminin and collagen I indicating an extensive remodelling of the subcutaneous ECM. EDX analysis revealed calcium/phosphate accumulations in the subcutis of all CUA patients with a molar ratio of 1.68 ± 0.06 matching that of hydroxyapatite mineral. Widespread media calcification in cutaneous arterioles was associated with destruction of the endothelial layer and partial exfoliation of the endothelial cells (ECs). CD31 immunostaining revealed aggregates of ECs contributing to intraluminal obstruction and consecutive malperfusion resulting in the clinical picture of ulcerative necrosis in all seven patients. CONCLUSIONS: Our data indicate that CUA is an active osteogenic process including the upregulation of BMP-2 signalling, hydroxyapatite deposition and extensive matrix remodelling of the subcutis.
Assuntos
Calciofilaxia/patologia , Cálcio/metabolismo , Matriz Extracelular/metabolismo , Falência Renal Crônica/patologia , Osteogênese/fisiologia , Dermatopatias/patologia , Uremia/patologia , Adulto , Idoso , Apoptose , Western Blotting , Calciofilaxia/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Técnicas Imunoenzimáticas , Falência Renal Crônica/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dermatopatias/metabolismo , Uremia/metabolismo , Adulto JovemRESUMO
Currently, ex vivo expansion of hematopoietic stem cells (HSC) is still insufficient. Traditional approaches for HSC expansion include the use of stromal cultures, growth factors, and/or bioreactors. Biomaterial-based strategies provide new perspectives. We focus on identifying promising two-dimensional (2D) polymer candidates for HSC expansion. After a 7-day culture period with cytokine supplementation, 2D fibrin, poly(D,L-lactic-co-glycolic acid; Resomer® RG503), and Poly(É-caprolactone; PCL) substrates supported expansion of cord blood (CB)-derived CD34⺠cells ex vivo. Fibrin cultures achieved the highest proliferation rates (>8700-fold increase of total nuclear cells, p<0.001), high total colony-forming units (3.6-fold increase, p<0.001), and highest engraftment in NSG mice (7.69-fold more donor cells compared with tissue culture polysterene, p<0.001). In addition, the presence of multiple human hematopoietic lineages such as myeloid (CD13âº), erythroid (GypCâº), and lymphoid (CD20âº/CD56âº) in murine transplant recipients confirmed the multilineage engraftment potential of fibrin-based cultures. Filopodia development in fibrin-expanded cells was a further indicator for superior cell adhesion capacities. We propose application of fibrin, Resomer® RG503, and PCL for future strategies of CB-CD34⺠cell expansion. Suitable polymers for HSC expansion might also be appropriate for future drug discovery applications or for studies aimed to develop hematological therapies.
Assuntos
Técnicas de Cultura de Células/métodos , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Polímeros/farmacologia , Animais , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular , Sobrevivência Celular , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Imuno-Histoquímica , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Especificidade de Órgãos/efeitos dos fármacos , Ensaio Tumoral de Célula-TroncoRESUMO
Expansion of multipotent, undifferentiated and proliferating cord blood (CB)-hematopoietic stem cells (HSC) in vitro is limited and insufficient. Bone marrow (BM) engineering in vitro allows mimicking the main components of the hematopoietic niche compared to conventional expansion strategies. In this study, four different 3D biomaterial scaffolds (PCL, PLGA, fibrin and collagen) were tested for freshly isolated cord blood (CB)-CD34(+) cell expansion in presence of (i) efficient exogenous cytokine supplementation and (ii) umbilical cord (UC)-mesenchymal stem cells (MSC). Cell morphology, growth and proliferation were analyzed in vitro as well as multi-organ engraftment and multilineage differentiation in a murine transplantation model. All scaffolds, except 3D PLGA meshes, supported CB-CD34(+) cell expansion, which was additionally stimulated by UC-MSC support. CB-CD34(+) cells cultured on human-derived 3D fibrin scaffolds with UC-MSC support i) reached the highest overall growth (5 × 10(8)-fold expansion of total nuclear cells after fourteen days and 3 × 10(7)-fold expansion of CD34(+) cells after seven days, p < 0.001), ii) maintained a more primitive immunophenotype for more cell divisions, iii) exhibited superior morphological, migratory and adhesive properties, and iv) showed the significantly highest numbers of engraftment and multilineage differentiation (CD45, CD34, CD13, CD3 and CD19) in BM, spleen and peripheral blood in long-term transplanted NSG mice compared to the other 3D biomaterial scaffolds. Thus, the 3D fibrin scaffold based BM-mimicry strategy reveals optimal requirements for translation into clinical protocols for CB expansion and transplantation.
Assuntos
Sangue Fetal/citologia , Fibrina/química , Células-Tronco Hematopoéticas/citologia , Alicerces Teciduais/química , Animais , Antígenos CD34/biossíntese , Materiais Biocompatíveis , Células da Medula Óssea/citologia , Proliferação de Células , Transplante de Células/métodos , Células Cultivadas/citologia , Citocinas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Eletrônica de Varredura/métodos , Fenótipo , Engenharia Tecidual/métodos , Cordão Umbilical/citologiaRESUMO
Immune cells are present in the blood and in resident tissues, and the nature of their reaction towards biomaterials is decisive for materials success or failure. Macrophages may for example be classically activated to trigger inflammation (M1), or alternatively activated which supports healing and vascularisation (M2). Here, we have generated 3D nanofibrous meshes in different porosities and precisely controlled surface chemistries comprising PLGA, hydrogel-coated protein repellant and protein repellant endowed with the bioactive peptide sequences GRGDS or GLF. We also prepared 2D substrates with corresponding surface chemistry for a systematic evaluation of primary human macrophage adhesion, migration, transcriptome expression, cytokine release and surface marker expression. Our data show that material morphology is a powerful means in biomaterial design to influence immune cell response. Flat substrates lead to an increased number of M2 classified CD163(+) macrophages. However, these M2 cells released large amounts of pro-inflammatory cytokines. In contrast, 3D nanofibres with corresponding surface chemistry yielded M1 classified 27E10(+) macrophages with a significantly increased release of pro-angiogenic chemokines and angiogenesis related molecules and a strong decrease of pro-inflammatory cytokines. We thus suggest that, for macrophages in contact with biomaterials, cytokine release is taken as main criterion instead of surface-markers for macrophage classifications.
Assuntos
Hidrogéis , Macrófagos/imunologia , Nanofibras , Adesão Celular , Células Cultivadas , Citocinas/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Ácido Láctico , Macrófagos/citologia , Macrófagos/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/10(9) platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.
Assuntos
Anti-Infecciosos/metabolismo , Plaquetas/metabolismo , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/crescimento & desenvolvimento , beta-Defensinas/metabolismo , Plaquetas/citologia , Plaquetas/microbiologia , Feminino , Humanos , Imuno-Histoquímica , MasculinoRESUMO
Here, we propose a collagen-based three-dimensional (3D) environment for hematopoietic stem and progenitor cells (HPC) with mesenchymal stem cells (MSC) derived either from bone marrow (BM) or umbilical cord (UC), to recapitulate the main components of the BM niche. Mechanisms described for HPC homeostasis were systematically analyzed in comparison to the conventional liquid HPC culture. The 3D-cultivation allows dissecting two sub-populations of HPC: (I) HPC in suspension above the collagen gel and (II) migratory HPC in the collagen fibres of the collagen gel. The different sites represent distinct microenvironments with significant impact on HPC fate. HPC in niche I (suspension) are proliferative and a dynamic culture containing HPC (CD34(+)/CD38(-)), maturing myeloid cells (CD38(+), CD13(+), CAE(+)) and natural killer (NK) cells (CD56(+)). In contrast, HPC in niche II showed clonal growth with significant high levels of the primitive CD34(+)/CD38(-) phenotype with starting myeloid (CD13(+), CAE(+)) differentiation, resembling the endosteal part of the BM niche. In contrast, UC-MSC are not adequate for HSC expansion as they significantly enhance HPC proliferation and lineage commitment. In conclusion, the 3D-culture system using collagen and BM-MSC enables HPC expansion and provides a potential platform to dissect regulatory mechanisms in hematopoiesis.
Assuntos
Técnicas de Cocultura/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , ADP-Ribosil Ciclase 1/biossíntese , Antígenos CD34/biossíntese , Apoptose , Biotecnologia/métodos , Células da Medula Óssea/citologia , Antígenos CD13/biossíntese , Diferenciação Celular , Colágeno/química , Géis/química , Sistema Hematopoético/imunologia , Humanos , Fenótipo , Alicerces Teciduais/química , Cordão Umbilical/citologiaRESUMO
BACKGROUND: Mesenchymal stem cells (MSC) represent a particularly attractive cell type for bone tissue engineering because of their ex vivo expansion potential and multipotent differentiation capacity. MSC are readily differentiated towards mature osteoblasts with well-established protocols. However, tissue engineering frequently involves three-dimensional scaffolds which (i) allow for cell adhesion in a spatial environment and (ii) meet application-specific criteria, such as stiffness, degradability and biocompatibility. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we analysed two synthetic, long-term degradable polymers for their impact on MSC-based bone tissue engineering: PLLA-co-TMC (Resomer® LT706) and poly(ε-caprolactone) (PCL). Both polymers enhance the osteogenic differentiation compared to tissue culture polystyrene (TCPS) as determined by Alizarin red stainings, scanning electron microscopy, PCR and whole genome expression analysis. Resomer® LT706 and PCL differ in their influence on gene expression, with Resomer® LT706 being more potent in supporting osteogenic differentiation of MSC. The major trigger on the osteogenic fate, however, is from osteogenic induction medium. CONCLUSION: This study demonstrates an enhanced osteogenic differentiation of MSC on Resomer® LT706 and PCL compared to TCPS. MSC cultured on Resomer® LT706 showed higher numbers of genes involved in skeletal development and bone formation. This identifies Resomer® LT706 as particularly attractive scaffold material for bone tissue engineering.
Assuntos
Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Poliésteres/farmacologia , Polímeros/farmacologia , Transcrição Gênica , Antraquinonas/farmacologia , Materiais Biocompatíveis/química , Adesão Celular , Diferenciação Celular , Corantes/farmacologia , Fibronectinas/química , Humanos , Microscopia Eletrônica de Varredura/métodos , Poliésteres/química , Poliestirenos/química , Engenharia Tecidual/métodos , Vitronectina/químicaRESUMO
OBJECTIVE: Medial artery calcification in patients with chronic kidney disease proceeds through intramembranous ossification resulting from osteoblast-induced calcification of the collagen extracellular matrix. The current study is based on the hypothesis that mesenchymal stem cells (MSC) constitute critical cells for procalcific extracellular matrix remodeling in patients with chronic kidney disease. METHODS AND RESULTS: Human MSC were cultured in media supplemented with pooled sera from either healthy or uremic patients (20%). Exposure to uremic serum enhanced the proliferation of MSC (cell counting, BrdU incorporation) whereas apoptosis and necrosis were not affected (annexin V and 7-amino-actinomycin staining). Uremic serum-exposed MSC recapitulated osteogenesis by matrix calcification and expression of bone-related genes (bone morphogenetic protein [BMP]-2 receptor, alkaline phosphatase, osteopontin, and Runx2) in 35 days. The uremic serum-induced osteogenesis was completely blocked by a BMP-2/4 neutralizing antibody or the natural antagonist NOGGIN. Calcification and matrix remodeling were further analyzed in a collagen-embedded osteogenesis model recapitulating the vascular collagen I/III environment. The uremic serum-induced calcification was shown to occur along collagen fibers as shown by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and von Kossa staining and was accompanied by extensive matrix remodeling. CONCLUSIONS: Uremic serum induced in a BMP-2/4-dependent manner an osteoblast-like phenotype in MSC accompanied by matrix remodeling and calcification.
Assuntos
Calcinose/etiologia , Células-Tronco Mesenquimais/patologia , Uremia/sangue , Adulto , Idoso , Apoptose , Proteína Morfogenética Óssea 2/fisiologia , Proteína Morfogenética Óssea 4/fisiologia , Proliferação de Células , Matriz Extracelular/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miofibroblastos/fisiologia , Osteogênese , FenótipoRESUMO
Recently, a new generation of dermal equivalents (DE) was presented which are solely generated on a human fibroblast-derived dermal matrix. Since human mesenchymal stem cells from bone marrow (BM-MSC) and Wharton's Jelly of the umbilical cord (UC-MSC) are characterised by a distinct biosynthetic and paracrine activity, they are an appealing alternative approach for generating cell-based DE. This study compares the epithelial-mesenchymal interaction and extracellular matrix (ECM) remodelling of cell-based and collagen-based DE using fibroblasts, BM-MSC or UC-MSC, respectively, in co-culture with the keratinocyte cell line HaCaT. While fibroblast-based DE exhibit normal matrix synthesis, proliferation and differentiation of keratinocytes, mesenchymal stem cell-based DE resulted in excessive production of inhomogenous matrix aggregates, loss of polarisation of the epidermal cell layer and an inconstant paracrine activity. In contrast, collagen-embedded MSC revealed a homogenous growth pattern as well as a constant expression of growth factors and ECM proteins without a negative influence on the epidermal layer as shown by histology, electron microscopy, immunohistochemistry and realtime-RT-PCR. These results indicate the necessity of an instructive biomaterial-based scaffold to direct stem cell differentiation, proliferation, paracrine activity as well as regulation of ECM deposition.
Assuntos
Materiais Biocompatíveis/farmacologia , Derme/fisiologia , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Engenharia Tecidual/métodos , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Colágeno/farmacologia , Derme/citologia , Derme/efeitos dos fármacos , Derme/ultraestrutura , Células Epidérmicas , Epiderme/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Pele Artificial , Cordão Umbilical/citologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vimentina/metabolismoRESUMO
Human mesenchymal stem cells (hMSC) are able to differentiate into mature cells of various mesenchymal tissues. Recent studies have reported that hMSC may even give rise to cells of ectodermal origin. This indication of plasticity makes hMSC a promising donor source for cell-based therapies. This study explores the differentiation potential of hMSC in a tissue-specific microenvironment simulated in vitro. HMSC were cultured air-exposed on dermal equivalents (DEs) consisting of collagen types I and III with dermal fibroblasts and subjected to conditions similar to those used for tissue engineering of skin with keratinocytes. Culture conditions were additionally modified by pre-treating the cells with 5-azacytidine or supplementing the medium with all trans retinoic acid (RA). HMSC were capable of adaptation to epidermis-specific conditions without losing their mesenchymal multipotency. However, despite the viability and evident three-dimensional epidermis-like growth pattern, hMSC showed a persistent expression of mesenchymal but not of epithelial markers, thus indicating a lack of epidermal (trans) differentiation. Further, electron microscopy and immunohistochemical analyses demonstrated that hMSC cultured under epidermis-specific conditions adopted a myofibroblastic phenotype and function, promoted in particular by air exposure. In conclusion, multipotent hMSC failed to differentiate into E-cadherin- or cytokeratin-expressing cells under optimized organotypic culture conditions for keratinocytes but differentiated into myofibroblast-like cells contracting the extracellular matrix, a phenomenon that was enhanced by RA and 5-azacytidine. These results indicate that hMSC might contribute to wound-healing processes by extracellular matrix reorganization and wound contraction but not by differentiation into keratinocytes.
Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Engenharia Tecidual/métodos , Actinas/metabolismo , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Pele/citologiaRESUMO
OBJECTIVE: Treatment strategies of cerebral aneurysms include surgical clipping and endovascular therapies. To determine the long-term results of these therapeutic strategies, the vessel wall reaction close to the former aneurysm was studied according to the assumption that an intact endothelial layer over the former aneurysm neck constitutes complete vessel wall reconstruction and stable aneurysm obliteration. METHODS: Aneurysms were created in 40 rabbits by intraluminal elastase incubation of the common carotid artery. Five animals each were assigned to the following groups: untreated, porous stents, polyurethane covered stentgrafts, porous stents with subsequent coiling. Ten animals were treated with coils alone, 10 with clips. After 6 months, angiography, histology, and scanning electron microscopy was performed. RESULTS: Porous stents did not obliterate the aneurysm, whereas stentgrafts did; in-stent stenosis of up to 60% was present because of neointimal multilayer proliferation. After coiling, the aneurysm dome was occluded with fibrinous and collagenous material, whereas the aneurysm neck was not covered by an endothelial lining. Coil loops lay bare within the vessel, with fresh thrombus material on their surface. After clipping, a thin layer of endothelial lining bridging the two attached vessel walls was present, thereby completely obliterating the aneurysm and reconstructing the vessel wall. CONCLUSION: This study demonstrates complete and stable aneurysm obliteration with vessel wall reconstruction after clipping, a sufficient obliteration of the aneurysm dome using endovascular techniques, but a failed healing response of the aneurysm neck that might correlate to its associated higher risk of rebleed. Whether or not this is counterbalanced by the better immediate outcome after endovascular treatment remains a matter of debate.
Assuntos
Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/terapia , Artéria Carótida Primitiva/patologia , Aneurisma Intracraniano/patologia , Aneurisma Intracraniano/terapia , Animais , Prótese Vascular , Doenças das Artérias Carótidas/induzido quimicamente , Doenças das Artérias Carótidas/fisiopatologia , Artéria Carótida Primitiva/diagnóstico por imagem , Angiografia Cerebral , Constrição Patológica/etiologia , Embolização Terapêutica/efeitos adversos , Embolização Terapêutica/instrumentação , Endotélio Vascular/patologia , Aneurisma Intracraniano/induzido quimicamente , Aneurisma Intracraniano/fisiopatologia , Microscopia Eletrônica de Varredura , Procedimentos Neurocirúrgicos , Elastase Pancreática , Período Pós-Operatório , Coelhos , Stents/efeitos adversos , Trombose/etiologia , Fatores de Tempo , CicatrizaçãoRESUMO
We evaluate the feasibility of nanosecond-pulsed and femtosecond-pulsed lasers for otologic surgery. The outcome parameters are cutting precision (in micrometers), ablation rate (in micrometers per second), scanning speed (in millimeters per second), and morphological effects on human middle ear ossicles. We examine single-spot ablations by a nanosecond-pulsed, frequency-tripled Nd:YAG laser (355 nm, beam diameter 10 microm, pulse rate 2 kHz, power 250 mW) on isolated human mallei. A similar system (355 nm, beam diameter 20 microm, pulse rate 10 kHz, power 160-1500 mW) and a femtosecond-pulsed CrLi:SAF-Laser (850 nm, pulse duration 100 fs, pulse energy 40 microJ, beam diameter 36 microm, pulse rate 1 kHz) are coupled to a scanner to perform bone surface ablation over a defined area. In our setups 1 and 2, marginal carbonization is visible in all single-spot ablations of 1-s exposures and longer: With an exposure time of 0.5 s, precise cutting margins without carbonization are observed. Cooling with saline solution result is in no carbonization at 1500 mW and a scan speed of 500 mms. Our third setup shows no carbonization but greater cutting precision, although the ablation volume is lower. Nanosecond- and femtosecond-pulsed laser systems bear the potential to increase cutting precision in otologic surgery.