RESUMO
BACKGROUND: Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue. METHODOLOGY/PRINCIPAL FINDINGS: This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks. CONCLUSIONS/SIGNIFICANCE: The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs.
Assuntos
Cartilagem Articular/metabolismo , Articulações/patologia , Engenharia Tecidual/métodos , Animais , Bovinos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Imuno-Histoquímica/métodos , Masculino , Modelos Biológicos , Sefarose/química , Estresse Mecânico , Resistência à Tração , Fatores de TempoRESUMO
Hyaluronan is a ubiquitous component of the extracellular matrix with important roles in cell and tissue functions. Hyaluronan content is often elevated in cardiovascular diseases, such as mitral valve disease and atherosclerosis. The objective of this study was to determine the impact of endogenously produced hyaluronan dynamically cultured three-dimensional model of collagenous tissues. Collagen gels containing excess HA and hyaluronan synthase (has) overexpressing cells were grown in a cyclic strain environment to simulate cell-mediated matrix organization. Cyclic strain caused a significant elevation in the collagen fibril density, cell number, and hyaluronan content of the resulting collagen gels compared to those grown under a static strain regimen. The material behavior of collagen gels containing has overexpressing cells was also notably weakened compared to controls. Transmission electron microscopy and immunohistochemistry showed that proteoglycan distribution was influenced by both strain and has overexpression. The results were also dependent on the specific has isozyme overexpressed. This investigation helps to identify the mechanism by which hyaluronan acts in vivo to alter tissue material behavior in cardiovascular diseases such as myxomatous mitral valve disease and atherosclerosis.
Assuntos
Colágeno/metabolismo , Géis , Glucuronosiltransferase/metabolismo , Doenças das Valvas Cardíacas , Doenças Vasculares , Animais , Hialuronan Sintases , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Ratos Endogâmicos F344RESUMO
Hyaluronan is an essential component of the native extracellular matrix that has often been added exogenously to biomaterials. The role of endogenously produced hyaluronan on soft tensile tissue mechanics, however, has been largely overlooked. To investigate this aspect of hyaluronan using a cell-mediated approach, cells overexpressing the hyaluronan synthases (has), namely has-1, has-2, has-3 or the empty vector control LXSN, were seeded within collagen gel scaffolds. The resulting engineered tissues were grown under static tension for 6 weeks. Following 6 weeks of culture, the samples were characterized to assess collagen gel contraction, matrix organization, production of hyaluronan, and tissue material properties. The engineered tissues containing cells transfected to overexpress one of the has isozymes had significantly increased retention of hyaluronan within the scaffold; elevated hyaluronan secretion into the culture medium (all but has-2); reduced contraction; reduced collagen density; and significantly altered material properties compared to the LXSN controls. These results indicate that the cell-mediated endogenous overproduction of hyaluronan within biomaterials alters their material, morphological and biochemical characteristics. This investigation, the first to examine the role of endogenously produced hyaluronan in engineered tissue mechanics, suggests that overproduction of hyaluronan in soft connective tissues can transform their biological and biomechanical functionality.
Assuntos
Colágeno/metabolismo , Glucuronosiltransferase/metabolismo , Animais , Células Cultivadas , Colágeno/química , Colágeno/ultraestrutura , Reagentes de Ligações Cruzadas/química , Meios de Cultura , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/genética , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Resistência à Tração , Engenharia Tecidual , Água/metabolismoRESUMO
Hyaluronan (HA) is a versatile molecular tool with considerable potential for tissue engineering applications. The inclusion of HA has created biocompatible biomaterials and engineered tissues that can be crosslinked or degraded controllably and can facilitate angiogenesis, osteointegration, and cell phenotype preservation. The utility of HA in tissue engineering has been broadened further by the recently identified HA synthases, which can be manipulated to stimulate the endogenous production of HA by cells seeded within biomaterial scaffolds. Overall, HA shows great promise in the development of engineered tissues and biomaterials for a variety of biomedical needs including orthopedic, cardiovascular, pharmacologic, and oncologic applications.
Assuntos
Ácido Hialurônico , Engenharia Tecidual , Animais , HumanosRESUMO
BACKGROUND AND AIM OF THE STUDY: Organ cultures maintain cells within their native microstructural environment, and thus offer greater potential for studying tissue disease and remodeling than do monolayer cell cultures or pathological examinations of diseased tissue. To validate an in-vitro heart valve organ culture model, cell viability was examined within valve tissues over sustained culture periods. METHODS: Following culture of blocks of valve tissue for 1 to 49 days, cross-sections were cut with a vibratome, stained with a LIVE/DEAD kit, and imaged with confocal microscopy to quantify the number of live and dead cells present. RESULTS: In numerous organ cultures, valvular interstitial cells were found to be viable beyond 30 days. Live cells were abundant in the central region of the valve, but more sparse in the deepest central regions. Dead cells were found mainly on the surface of both fresh tissues and tissues after prolonged culture, with few dead cells occurring centrally. CONCLUSION: This is the first reported mapping of cell viability within heart valve organ cultures, and results suggest that extended organ culture of valve leaflets is indeed possible. The derived viability staining methods have wide applicability for organ cultures of other tissues as well as tissue-engineered matrices.