RESUMO
Successful periodontal repair and regeneration requires the coordinated responses from soft and hard tissues as well as the soft tissue-to-bone interfaces. Inspired by the hierarchical structure of native periodontal tissues, tissue engineering technology provides unique opportunities to coordinate multiple cell types into scaffolds that mimic the natural periodontal structure in vitro. In this study, we designed and fabricated highly ordered multicompartmental scaffolds by melt electrowriting, an advanced 3-dimensional (3D) printing technique. This strategy attempted to mimic the characteristic periodontal microenvironment through multicompartmental constructs comprising 3 tissue-specific regions: 1) a bone compartment with dense mesh structure, 2) a ligament compartment mimicking the highly aligned periodontal ligaments (PDLs), and 3) a transition region that bridges the bone and ligament, a critical feature that differentiates this system from mono- or bicompartmental alternatives. The multicompartmental constructs successfully achieved coordinated proliferation and differentiation of multiple cell types in vitro within short time, including both ligamentous- and bone-derived cells. Long-term 3D coculture of primary human osteoblasts and PDL fibroblasts led to a mineral gradient from calcified to uncalcified regions with PDL-like insertions within the transition region, an effect that is challenging to achieve with mono- or bicompartmental platforms. This process effectively recapitulates the key feature of interfacial tissues in periodontium. Collectively, this tissue-engineered approach offers a fundament for engineering periodontal tissue constructs with characteristic 3D microenvironments similar to native tissues. This multicompartmental 3D printing approach is also highly compatible with the design of next-generation scaffolds, with both highly adjustable compartmentalization properties and patient-specific shapes, for multitissue engineering in complex periodontal defects.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Humanos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Periodonto/cirurgia , Periodonto/fisiologia , Impressão Tridimensional , Ligamento PeriodontalRESUMO
Whispering-gallery mode (WGM) microdisk lasers show great potential for highly sensitive label-free detection in large-scale sensor arrays. However, when used in practical applications under normal ambient conditions, these devices suffer from temperature fluctuations and photobleaching. Here we demonstrate that these challenges can be overcome by a novel referencing scheme that allows for simultaneous compensation of temperature drift and photobleaching. The technique relies on reference structures protected by locally dispensed passivation materials, and can be scaled to extended arrays of hundreds of devices. We prove the viability of the concept in a series of experiments, demonstrating robust and sensitive label-free detection over a wide range of constant or continuously varying temperatures. To the best of our knowledge, these measurements represent the first demonstration of biosensing in active WGM devices with simultaneous compensation of both photobleaching and temperature drift.
Assuntos
Técnicas Biossensoriais/instrumentação , Lasers , Fotodegradação , Temperatura , Técnicas Biossensoriais/métodosRESUMO
The cellular microenvironment is extremely complex, and a plethora of materials and methods have been employed to mimic its properties in vitro. In particular, scientists and engineers have taken an interdisciplinary approach in their creation of synthetic biointerfaces that replicate chemical and physical aspects of the cellular microenvironment. Here the focus is on the use of synthetic materials or a combination of synthetic and biological ligands to recapitulate the defined surface chemistries, microstructure, and function of the cellular microenvironment for a myriad of biomedical applications. Specifically, strategies for altering the surface of these environments using self-assembled monolayers, polymer coatings, and their combination with patterned biological ligands are explored. Furthermore, methods for augmenting an important physical property of the cellular microenvironment, topography, are highlighted, and the advantages and disadvantages of these approaches are discussed. Finally, the progress of materials for prolonged stem cell culture, a key component in the translation of stem cell therapeutics for clinical use, is featured.
Assuntos
Materiais Biocompatíveis/química , Bioengenharia/métodos , Animais , Bioengenharia/instrumentação , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Desenho de Equipamento , Humanos , Células-Tronco Pluripotentes/citologia , Polímeros/química , Propriedades de SuperfícieRESUMO
Thiol-yne click chemistry is used to covalently link a ferrocenyl derivative to the pore walls of a fully organic porous polymer coating (SURGEL). By cyclic voltammetry, it is demonstrated that the ferrocene bound to the SURGEL via a flexible alkyl linker can be reversibly reduced and oxidised. Surprisingly, when adding ferrocene as an electrolyte, a Nernstian diffusion limited process is observed. We explain this observation in terms of a high permeability of the SURGELs for ferrocene after the post synthetic modification.
Assuntos
Técnicas Eletroquímicas , Compostos Ferrosos/química , Polímeros/química , Difusão , Eletrólitos/química , Metalocenos , Estrutura Molecular , Porosidade , Propriedades de SuperfícieRESUMO
Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self-renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self-renewal and differentiation. When combined with three-dimensional cultures in bioreactors, these systems will also enable large-scale expansion for future clinical applications.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Reatores Biológicos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno , Combinação de Medicamentos , Matriz Extracelular , Células Alimentadoras , Humanos , Laminina , Camundongos , ProteoglicanasRESUMO
Human-induced pluripotent stem cells (hiPSCs) may represent an ideal cell source for research and applications in regenerative medicine. However, standard culture conditions that depend on the use of undefined substrates and xenogeneic medium components represent a significant obstacle to clinical translation. Recently, we reported a defined culture system for human embryonic stem cells using a synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), in conjunction with xenogeneic-free culture medium. Here, we tested the hypothesis that iPSCs could be maintained in an undifferentiated state in this xeno-free culture system and subsequently be differentiated into mesenchymal stem cells (iPS-MSCs). hiPSCs were cultured on PMEDSAH and differentiated into functional MSCs, as confirmed by expression of characteristic MSC markers (CD166+, CD105+, CD90+,CD73+, CD31-, CD34-, and CD45-) and their ability to differentiate in vitro into adipogenic, chondrogenic, and osteoblastic lineages. To demonstrate the potential of iPS-MSCs to regenerate bone in vivo, the newly derived cells were induced to osteoblast differentiation for 4 days and transplanted into calvaria defects in immunocompromised mice for 8 weeks. MicroCT and histologic analyses demonstrated de novo bone formation in the calvaria defects for animals treated with iPS-MSCs but not for the control group. Moreover, positive staining for human nuclear antigen and human mitochondria monoclonal antibodies confirmed the participation of the transplanted hiPS-MSCs in the regenerated bone. These results demonstrate that hiPSCs cultured in a xeno-free system have the capability to differentiate into functional MSCs with the ability to form bone in vivo.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Animais , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Técnicas Citológicas/métodos , Feminino , Humanos , Hospedeiro Imunocomprometido , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Especificidade por SubstratoRESUMO
PURPOSE: Does hirudin coating improve the patency of iliac artery endoprostheses in comparison to non-hirudin-coated endoprostheses? MATERIALS AND METHODS: Nitinol stents and stentgrafts covered with polytetrafluoroethylene (PTFE) were coated with the polymer polyamino-p-xylylene-co-poly-p-xylylene using chemical vapor deposition (CVD) technique. Hirudin was covalently bound to the surface of the endoprostheses via the amino-group. External factors (mounting of the prosthesis, sterilization, storage time and temperature, release) affecting the hirudin activity were evaluated in vitro. Five types of prostheses were compared in vivo: (1) plain and (2) CVD- and hirudin-coated stents; (3) plain, (4) CVD-coated, and (5) CVD- and hirudin-coated PTFE-stentgrafts. In 20 sheep, 16 protheses of each type were inserted in arteries pretreated with a Fogarty maneuver. The animals were followed for either 1 (n = 10) or 6 (n = 10) months. Immediately after implantation and after 1, 3, and 6 months, intravascular ultrasound (IVUS) and angiography were performed. The vascular specimens were analyzed histologically. RESULTS: Within 10 weeks, the hirudin activity of coated stents dropped 60 % due to external factors; the activity of coated PTFE stentgrafts dropped 20 %. After 1, 3, and 6 months, IVUS and histology revealed a significantly reduced patency of the hirudin-coated stentgrafts compared to the other prostheses. Only IVUS showed a significantly reduced patency of hirudin coated stents after 1 and 3 months compared to plain and CVD-coated PTFE-stentgrafts. The reduced patency was caused by neointimal hyperplasia. CONCLUSIONS: In an experimental setting, hirudin coating did not improve the patency of vascular endoprostheses.
Assuntos
Prótese Vascular , Materiais Revestidos Biocompatíveis , Hirudinas , Politetrafluoretileno , Stents , Angiografia , Animais , Seguimentos , Humanos , Masculino , Microscopia Eletrônica de Varredura , Desenho de Prótese , Ovinos , Ultrassonografia de Intervenção , Grau de Desobstrução Vascular/fisiologiaRESUMO
The poor biocompatibility of metallic coronary stents which leads to un-satisfying restenosis rates is mainly caused by contact activation of blood cells, smooth muscle cells and endothelial cells. Mimicking a metal surface with a biocompatible coating that actively suppresses mechanisms leading to restenosis may overcome today's limitations regarding the complications of metal stents. Nitinol coronary stents were coated by CVD polymerization of functionalized [2.2]paracyclophanes. The monomers 4-amino [2.2]paracyclophane, 4-hydroxy methyl [2.2]paracyclophane and [2.2]paracyclophane-4,5,12,13-tetracarboxylic acid dianhydride were previously synthesized. A suitable installation for the CVD polymerization procedure was designed and used for the polymerization procedures. Physical and chemical properties of the polymers were shown to fulfill the requirements regarding the application as a stent coating material. The functional groups of the polymer coatings were used for the immobilization of the thrombin inhibitor r-hirudin. In vitro results indicate that the bioactively coated stents are less thrombogenic than virgin metallic stents. Surface-bound r-hirudin decreases platelet adhesion drastically due to interactions between platelets and r-hirudin.
Assuntos
Materiais Biocompatíveis , Hirudinas , Próteses e Implantes , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Doença das Coronárias/terapia , Hirudinas/administração & dosagem , Hirudinas/metabolismo , Humanos , Técnicas In Vitro , Teste de Materiais , Metais , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Adesividade Plaquetária , Polímeros/síntese química , Polímeros/química , Stents , Propriedades de Superfície , Trombina/metabolismo , VolatilizaçãoRESUMO
Surface immobilization of the thrombin inhibitor r-hirudin was carried out on two different polymers. Linkage to poly(urethane-graft-acrylic acid) (PAC/PU) was done via carboxylic acid groups, using a water soluble carbodimide, while the immobilization on a modified poly[(ethene-co-vinyl acetate)-graft-vinyl chloride] (PVC/EVA) was achieved via the alcohol groups of the polymer using HDI as spacer. Direct immobilization of r-hirudin leaded to a remarkable loss of thrombin activity. As proved by means of protein chemical analysis, loss of activity was due to a selective coupling via the N-terminal amino group of r-hirudin, which is essential for its thrombin activity. Based on these results we developed an immobilization method via an epsilon-amino group of r-hirudin preserving full biological activity of the r-hirudin coated surface.
RESUMO
An alternative to open heart surgery in treating arterial diseases causing restricted blood flow is the implantation of intracoronary metallic stents. In spite of the advances in implantation and in spite of the excellent mechanical properties of metallic stents, there are still limitations because of the thrombogenicity of the metal. We have, hence, directed our attention to the coating of metallic stents with an ultrathin polymer layer by chemical vapor deposition (CVD) polymerization of 2-chloroparacyclophan. In a second step of surface modification the poly(2-chloroparaxylylene) layer is modified by treatment with a sulfur dioxide plasma in order to obtain a more hydrophilic surface with new functional groups. The results demonstrate the stable polymer coating of the stents and the improvement of haemocompatibility after treatment with sulfur dioxide plasma. Platelet adhesion is decreased from 85% for the metal surface to 20% for the CVD-coated and sulfur-dioxide-plasma treated surface.