RESUMEN
Fibrin-based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid-phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non-invasive molecular imaging systems enable real-time tracking of processes in the living animal. In this study, a non-invasive fluorescence based imaging system was applied to follow and quantify site-specific degradation of fibrin sealant. To enable non-invasive tracking of fibrin in vivo, fibrin-matrix was labelled by incorporation of a fluorophore-conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore-labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16 days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site.
Asunto(s)
Materiales Biocompatibles/química , Fibrina/química , Microscopía Fluorescente/métodos , Animales , Ácido Edético/química , Femenino , Fibrinólisis , Cinética , Ratones , Ratones Endogámicos BALB C , Modelos Teóricos , Óptica y Fotónica , Ingeniería de Tejidos/métodosRESUMEN
Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co-culture with adipose-derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co-cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase-14. Moreover, we showed OEC/ASC-induced vessel-like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co-culture-induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system.
Asunto(s)
Adipocitos/citología , Tejido Adiposo/citología , Células Endoteliales/citología , Fibrina/química , Células Madre/citología , Técnicas de Cultivo de Célula , Técnicas de Cocultivo/métodos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucocitos Mononucleares/citología , Metaloproteinasa 14 de la Matriz/metabolismo , Microscopía Fluorescente , Neovascularización Patológica , Ingeniería de Tejidos/métodosRESUMEN
Fibrin biomatrices have been used for many years for hemostasis and sealing and are a well-established surgical tool. The objective of the present study was to compare two commercially available fibrin biomatrices regarding the effect of their thrombin concentration on keratinocytes and wound healing in vitro and in vivo. Keratinocytes showed significant differences in adhesion, viability, and morphology in the presence of the fibrin matrices in vitro. A high thrombin concentration (800-1,200 IU/mL) caused deteriorated cell compatibility. By using a thrombin inhibitor, those differences could be reversed. In a rat excisional wound healing model, we observed more rapid wound closure and less wound severity in wounds treated with a fibrin matrix containing a lower concentration of thrombin (4 IU/mL). Furthermore, fewer new functional vessels and a lower level of vascular endothelial growth factor were measured in wounds after 7 days treated with the matrix with higher thrombin concentration. These in vivo results may be partially explained by the in vitro biocompatibility data. Additionally, results show that low thrombin biomatrices were degraded faster than the high thrombin material. Hence, we conclude that the composition of fibrin biomatrices influences keratinocytes and therefore has an impact on wound healing.
Asunto(s)
Materiales Biocompatibles/farmacología , Adhesivo de Tejido de Fibrina/farmacología , Piel/efectos de los fármacos , Trombina/farmacología , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológico , Animales , Adhesión Celular , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas In Vitro , Queratinocitos , Masculino , Ratas , Ratas Sprague-Dawley , Piel/lesiones , Piel/patologíaRESUMEN
Human amniotic membrane (hAM) represents a tissue that is well established as biomaterial in the clinics with potential for new applications in regenerative medicine. For tissue engineering (TE) strategies, cells are usually combined with inductive factors and a carrier substrate. We have previously recognized that hAM represents a natural, preformed sheet including highly potent stem cells. In the present approach for cartilage regeneration we have induced chondrogenesis in hAM in vitro. For this, hAM biopsies were cultured for up to 56 days under chondrogenic conditions. The induced hAM was characterized for remaining viability, glycosaminoglycan (GAG) accumulation using histochemical analysis, and a quantitative assay. Collagen I, II and X was immunohistochemically determined and cartilage-specific mRNA expression of (sex determining region Y-) box 9, cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), versican (CSPG2), COL1A1, COL9A2, melanoma inhibitory activity (MIA), and cartilage-linking protein 1 (CRTL1) analyzed by quantitative real-time polymerase chain reaction. Human AM was successfully induced to accumulate GAG, as demonstrated by Alcianblue staining and a significant (p < 0.001) increase of GAG/viability under chondrogenic conditions peaking in a 29.9 ± 0.9-fold induction on day 56. Further, upon chondrogenic induction collagen II positive areas were identified within histological sections and cartilage-specific markers including COMP, AGC1, CSPG2, COL1A1, COL9A2, MIA, and CRTL1 were found upregulated at mRNA level. This is the first study, demonstrating that upon in vitro induction viable human amnion expresses cartilage-specific markers and accumulates GAGs within the biomatrix. This is a promising first step towards a potential use of living hAM for cartilage TE.
Asunto(s)
Amnios/citología , Diferenciación Celular , Linaje de la Célula/fisiología , Condrogénesis/fisiología , Placenta/citología , Cartílago/citología , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Embarazo , Células Madre/citologíaRESUMEN
Thermoreversible hydrogels for tissue engineering (TE) purposes have gained increased attention in recent years as they can be combined with cells and drugs and directly injected into the body. Following the fate of transplanted cells in situ is essential in characterizing their distribution and survival, as well as the expression of specific markers or cell-matrix interactions. Existing histological embedding methods, such as paraffin wax embedding, can mechanically damage some biomaterials during processing. In this study, we describe a broadly applicable preparation protocol that allows the handling of delicate, thermoreversible scaffolds for histological sectioning. The gelatin solution permits the embedding of samples at 37 °C, which suits the solid phase of most TE scaffolds. A thermoreversible scaffold of polycaprolactone microparticles, combined with poly(polyethylene glycol methacrylate ethyl ether) and containing human adipose-derived stem cells, was prepared for histology by an initial gelatin embedding step in addition to the standard cryosectioning and paraffin processing protocols. Sections were evaluated by hematoxylin eosin staining and immunostaining for human vimentin. The gelatin embedding retained the scaffold particles and permitted the complete transfer of the construct. After rapid cooling, the solid gelatin blocks could be cryosectioned and paraffin infiltrated. In contrast to direct cryosectioning or paraffin infiltration, the extended protocol preserved the scaffold structure as well as the relevant cell epitopes, which subsequently allowed for immunostaining of human cells within the material. The gelatin embedding method proposed is a generalizable alternative to standard preparations for histological examination of a variety of delicate samples.
Asunto(s)
Gelatina/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Tejido Adiposo/metabolismo , Linaje de la Célula , Criopreservación/métodos , Eosina Amarillenta-(YS)/química , Epítopos/química , Hematoxilina/química , Humanos , Hidrogeles/química , Metacrilatos/química , Parafina/química , Poliésteres/química , Polietilenglicoles/química , Polímeros/química , Células Madre/citología , Temperatura , Vimentina/químicaRESUMEN
OBJECTIVE: To assess the time-dependent treatment effects of extracorporeal shock wave therapy (ESWT) in a standard rodent ischemic epigastric flap model. BACKGROUND: ESWT has been shown to accelerate tissue repair in acute and chronic wounds and improve graft survival, but the mechanism remains incompletely understood. METHODS: Shock waves at 0.1 mJ/mm and 5 impulses/s (total 300 impulses) were applied to the epigastric flap ischemic region at various times pre-, immediately and 24 hours postischemic insult. Flap survival; vascular perfusion; vessel number; von Willebrand factor and smooth muscle actin protein expression as well as in vivo vascular endothelial growth factor receptor 2 expression were evaluated at 1, 3, and 7 days postoperatively in ESWT-treated and untreated controls. RESULTS: Flap perfusion, microvessel number, and survival (through reduced flap contraction and necrosis) were significantly enhanced in the treated groups compared with controls, irrespective of timing of shock wave treatment (preischemia vs. postischemia). Vascular endothelial growth factor receptor 2 expression was dynamically upregulated in response to ESWT. CONCLUSION: Shock wave preconditioning and treatment postischemic insult improves skin flap survival through neovascularization and early upregulation of angiogenesis-related growth factors.
Asunto(s)
Ondas de Choque de Alta Energía/uso terapéutico , Neovascularización Fisiológica/fisiología , Trasplante de Piel/métodos , Colgajos Quirúrgicos/irrigación sanguínea , Colgajos Quirúrgicos/patología , Animales , Biopsia con Aguja , Modelos Animales de Enfermedad , Arterias Epigástricas , Rechazo de Injerto , Supervivencia de Injerto , Inmunohistoquímica , Isquemia/prevención & control , Precondicionamiento Isquémico/métodos , Masculino , Necrosis/patología , Necrosis/prevención & control , Cuidados Posoperatorios/métodos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Trasplante de Piel/efectos adversos , Resultado del TratamientoRESUMEN
Tissue engineering strategies usually require cell isolation and combination with a suitable biomaterial. Human amniotic membrane (AM) represents a natural two-layered sheet comprising cells with proven stem cell characteristics. In our approach, we evaluated the differentiation potential of AM in toto with its sessile stem cells as alternative to conventional approaches requiring cell isolation and combination with biomaterials. For this, AM-biopsies were differentiated in vitro using two osteogenic media compared with control medium (CM) for 28 days. Mineralization and osteocalcin expression was demonstrated by (immuno)histochemistry. Alkaline phosphatase (AP) activity, calcium contents and mRNA expression of RUNX2, AP, osteopontin, osteocalcin, BMP-2 (bone morphogenetic protein), and BMP-4 were quantified and AM viability was evaluated. Under osteogenic conditions, AM-biopsies mineralized successfully and by day 28 the majority of cells expressed osteocalcin. This was confirmed by a significant rise in calcium contents (up to 27.4 ± 6.8 mg/dl d28), increased AP activity, and induction of RUNX2, AP, BMP-2 and BMP-4 mRNA expression. Relatively high levels of viability were retained, especially in osteogenic media (up to 78.3 ± 19.0% d14; 62.9 ± 22.3% d28) compared to CM (42.2 ± 15.2% d14; 35.1 ± 8.6% d28). By this strategy, stem cells within human AM can successfully be driven along the osteogenic pathways while residing within their natural environment.
Asunto(s)
Amnios/citología , Diferenciación Celular , Osteogénesis , Fosfatasa Alcalina/metabolismo , Amnios/efectos de los fármacos , Amnios/enzimología , Biomarcadores/metabolismo , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/enzimología , Osteogénesis/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The in vitro and in vivo efficiency of fibroin microparticles as a delivery carrier for bone morphogenetic protein-2 (BMP-2) was evaluated. BMP-2 was encapsulated in silk fibroin particles that were produced by a simple and very mild processing method. The dose-response of BMP-2-loaded fibroin particles was examined in C2C12 cells, after 5 days of culture. The BMP-2 retained most of its activity as observed by the increase in alkaline phosphatase activity, which was much higher when BMP-2 was encapsulated into the particles rather than just surface-adsorbed. After 2 weeks of culture, increased mineralization was observed with BMP-2-loaded particles in comparison to soluble added growth factor. No significant cytotoxicity was detected. When implanted in a rat ectopic model, bone formation was observed by in vivo micro-computed tomography after 2 and 4 weeks postimplantation, with particles loaded with 5 or 12.5 microg BMP-2. An increase in bone density was observed over time. Histology revealed further evidence of ectopic bone formation, observed by strong alizarin red staining and osteocalcin immunostaining. Our findings show that fibroin microparticles may present an interesting option for future clinical applications in the bone tissue engineering field, and therefore, further studies have been planned.
Asunto(s)
Proteína Morfogenética Ósea 2/administración & dosificación , Fibroínas/administración & dosificación , Seda/química , Animales , Desarrollo Óseo , Línea Celular , Portadores de Fármacos , Humanos , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Tomografía Computarizada por Rayos XRESUMEN
The literature provides linear regression formulas for dental age estimation that is based on radiological two-dimensional measurements of the pulp size. The aim of the present study was to explore whether the previously presented regression formulas could lead to statistically sound results and to appropriate repeatability when applied to young individuals. Orthopantomograms (OPGs) of 44 Austrian individuals, aged between 13 and 24 years, were selected at random. In accordance with the reported method, six teeth on each OPG were chosen to carry out the measurements. Statistical analysis was performed in order to assess the difference between the estimated and the true chronological age. The regression formulas reported by Kvaal et al. (1995) led to a consistent underestimation; the regression formulas reported by Paewinsky et al. (2005) resulted in a constant overestimation of age. The statistical analysis of intraobserver and interobserver variation revealed a variation width below 2%, respectively.