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PubMedDate not supplied

Increased smooth muscle actin expression from bone marrow stromal cells under retinoic acid treatment: an attempt for autologous blood vessel tissue engineering.

Vascular replacement in vital organs is sometimes necessary for human life for example because of atherosclerosis. Blood vessel tissue engineering is applied for autologous transplantations to avoid graft rejections. Stem cells are used for blood vessel tissue engineering because they are the origin of smooth muscle cells, endothelial cells and fibroblasts. This paper shows that bone marrow stromal cells (BMSCs) can be induced to differentiate into the early stage of smooth muscle cells by using 0.01 microM retinoic acid. The differentiation of BMSCs to smooth muscle cells was detected by the expression of smooth muscle alpha actin (SM alpha-actin), the earliest smooth muscle cell marker. The SM alpha-actin marker expression was demonstrated using indirect immunofluorescence technique and Western blot analysis. The induction of BMSC to form early stages of smooth muscle cells in this study is appropriate for blood vessel tissue engineering because the early stage smooth muscle cells may be stimulated to develop vascular walls with endothelial cells using a co-culture system.

Actins↗

PubMed2002

Adult mesenchymal stem cells and cell-based tissue engineering.

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.

Adult↗

PubMed2006

Dendrimer crosslinked collagen as a corneal tissue engineering scaffold: mechanical properties and corneal epithelial cell interactions.

Generation 2 polypropyleneimine octaamine dendrimers were used to generate highly crosslinked collagen with mechanical properties that would make it appropriate for use as a corneal tissue-engineering scaffold. Crosslinking of a highly concentrated collagen solution (2-4%) was effected using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC). The multifunctional dendrimers were introduced as novel multifunctional crosslinkers after the activation of the carboxylic acid groups of glutamic and aspartic acid residues in collagen. Glutaraldehyde, a common collagen crosslinker, was used as comparison, as was EDC, itself an alternative crosslinker, which forms "zero-length or self-crosslinking". The mechanical properties resultant gels were determined. Young's modulus of the dendrimer crosslinked gels was significantly higher than that observed with the other crosslinkers, increasing to 5 MPa compared with 0.1 MPa for the EDC crosslinked gels. Transmission electron microscopy (TEM) analysis of the gels demonstrated the presence of fibrils in the thermally gelled collagen controls; no fibrils were observed in the dendrimer crosslinked gels. As a result, the optical transparency of the dendrimer crosslinked collagen was significantly better than that of the collagen thermal gels. The EDC and glutaraldehyde crosslinked gels were generally less transparent than those crosslinked with the dendrimers. Glucose permeation results demonstrated that the dendrimer crosslinked collagen had higher glucose permeability than natural human cornea. Dendrimer crosslinked collagen gels supported human corneal epithelial cell growth and adhesion, with no cell toxicity. In comparison, some potentially cytotoxic effects were observed with glutaraldehyde crosslinked collagen. Overall, the dendrimer crosslinked collagen gels showed promising properties that suggest that these might be suitable scaffolds for corneal tissue engineering and potentially other tissue engineering applications.

Biocompatible Materials↗

PubMed2005

Evaluation of three growth factors for TMJ disc tissue engineering.

Arguably one of the most complex joints in the body, the temporomandibular joint (TMJ) presents one of the most difficult problems in modem medicine. Tissue engineering, for the TMJ disc in particular, has been proposed as a potential breakthrough treatment strategy for TMJ disorders. Central to tissue engineering is understanding growth factor effects on TMJ disc cells, and to the best of our knowledge, this is the first 3D growth factor study for these cells. The purpose was to examine the effects of high and low concentrations of basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF), and transforming growth factor-beta1 (TGF-beta) on porcine TMJ disc cells. Cells were seeded onto non-woven PGA scaffolds (95% porosity) in spinner flasks, then cultured with a growth factor for 6 weeks. Constructs were analyzed for mechanical and structural integrity, cell number, and matrix biosynthesis. All growth factors improved mechanical and structural integrity compared to the control. IGF and TGF-beta were most effective at promoting collagen synthesis, although there were no significant differences in glycosaminoglycan synthesis or cell number between any groups. After considering the economic advantage of IGF over TGF-beta, the conclusion of this study is to use IGF in future TMJ disc tissue engineering experiments.

Animals↗

PubMed2003

In vitro tissue engineering to generate a human-sized auricle and nasal tip.

OBJECTIVES/HYPOTHESIS: Tissue engineering has successfully generated cartilage in a xenograft and an autograft model. However, challenges remain with both of these in vivo techniques before clinical application can be realized. We hypothesized that a human-sized cartilaginous structure could be generated completely in vitro as a complementary or an alternative technique. METHODS: Scaffolds were created in the shape of five full-sized human auricles and five nasal tip cartilaginous skeletons. Bovine shoulder chondrocytes at a concentration of 50 million cells/mL were seeded onto the scaffolds and were grown for 12 weeks in vitro. Two of the auricular scaffolds had internal support provided by soft acrylic sheets and were later implanted into nude rats. RESULTS: All of the scaffolds maintained shape and size through 12 weeks of in vitro culture. On gross examination the scaffolds were progressively replaced by cartilage, which was confirmed by histological and biochemical analysis. The auricular scaffolds with the acrylic internal support had the most natural rigidity, which was observed by gentle palpation. The nasal scaffolds maintained excellent definition even without internal support. CONCLUSION: An adult human-sized auricle and nasal tip cartilaginous structure can be grown entirely in vitro using principles of tissue engineering.

Animals↗

PubMedDate not supplied

Novel methodology for fabrication of tissue-engineered tubular constructs using magnetite nanoparticles and magnetic force.

Novel technologies for creating three-dimensional constructs with complex shapes would be highly useful in tissue engineering. In the present study, tubular structures were constructed using magnetic force. Magnetite nanoparticles in cationic liposomes were taken up by target cells. The magnetically labeled cells were seeded onto ultralow-attachment plates, and a magnet was placed under the wells. After 24 h of culture, the magnetically labeled cells formed a cell sheet. Subsequently, when a cylindrical magnet was rolled onto the cell sheet, the cell sheet was attracted to the magnet and formed a tube around it. The magnet was then removed, leaving behind a tubular structure. Two types of tissue were used to create tubular structures: urinary tissue, consisting of a monotypic urothelial cell layer; and vascular tissue, consisting of heterotypic layers of endothelial cells, smooth muscle cells, and fibroblasts. The present results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach to constructing tissue-engineered tubular structures.

Animals↗

PubMed2006

Tissue-engineered axially vascularized contractile skeletal muscle.

BACKGROUND: As tissue-engineered muscle constructs increase in scale, their size is limited by the need for a vascular supply. In this work, the authors demonstrate a method of producing three-dimensional contractile skeletal muscles in vivo by incorporating an axial vascular pedicle. METHODS: Primary myoblast cultures were generated from adult F344 rat soleus muscle. The cells were suspended in a fibrinogen hydrogel contained within cylindrical silicone chambers, and situated around the femoral vessels in isogeneic adult recipient rats. The constructs were allowed to incubate in vivo for 3 weeks, at which point they were explanted and subjected to isometric force measurements and histologic evaluation. RESULTS: The resulting three-dimensional engineered skeletal muscle constructs produced longitudinal contractile force when electrically stimulated. Length-tension, force-voltage, and force-frequency relationships were similar to those found in developing skeletal muscle. Desmin staining demonstrated that individual myoblasts had undergone fusion to form multinucleated myotubes. Von Willebrand staining showed that the local environment within the chamber was richly angiogenic, and capillaries had grown into and throughout the constructs from the femoral artery and vein. CONCLUSIONS: Three-dimensional, vascularized skeletal muscle can be engineered in vivo. The resulting tissues have histologic and functional properties consistent with native skeletal muscle.

Animals↗

PubMed2006

Growth factor-delivery systems for tissue engineering: a materials perspective.

The transplantation of organs, their surgical reconstruction or implantation of synthetic devices that can perform the function of organs, are the currently available methods for treating loss of tissue/organs in humans. However, the limitations associated with these techniques have led to the development of tissue engineering. One of the primary goals of tissue engineering is to provide growth factor delivery systems that can induce desired cell responses both in vitro and in vivo, in order to cause accelerated tissue regeneration. To make growth factors a more therapeutically viable alternative for the treatment of chronic degenerative diseases, a wide range of natural and synthetic materials have been employed as vehicles for their controlled delivery. The choice of material and design of the carrier device influence the mode of immobilization of growth factors on the scaffolds and their local/systemic administration. From a tissue engineer's perspective, materials could be used for designing scaffolds as well as for delivering single or multiple growth factors. Therefore, this review discusses growth factor delivery systems, with particular reference to carrier-based growth factor delivery systems with a focus on materials.

Animals↗

PubMed2004

Endothelium properties of a tissue-engineered blood vessel for small-diameter vascular reconstruction.

PURPOSE: A tissue-engineered blood vessel (TEBV) produced in vitro by the self-assembly method was developed in our laboratory for the replacement of small-diameter blood vessels. The interior of this vessel is covered by an endothelium. The aim of the present study was to evaluate whether the endothelial layer would make a favorable contribution at the time of implantation of the TEBV by investigating in vitro the hemocompatible properties of the endothelial cells covering its interior. METHODS: The secretion of the von Willebrand factor (vWF) and expression of thrombomodulin by the endothelium were assessed, and the adhesive molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) were quantified as a function of maturation time. To evaluate the functional response of the endothelium on injury, the cellular response to physiological stimulatory factors (thrombin and lipopolysaccharide [LPS]) was analyzed. RESULTS: The endothelial cells formed a confluent monolayer displaying favorable hemocompatible properties (78% +/- 10% of cells expressing thrombomodulin with only 12 +/- 3 mU/10(6) cells of vWF secreted over a 2-hour period), which acquired their full expression after a culture period of 4 days. Moreover, pro-adhesive properties toward inflammatory cells were not observed. The cells were also able to respond to physiological-stimulating agents (thrombin and LPS) and demonstrated a statistically significant overexpression of the corresponding molecules under the conditions tested. CONCLUSIONS: These results indicate that the endothelium of the tissue-engineered blood vessel produced by the self-assembly approach displays advantageous qualities with regard to the vessel's future implantation as a small-diameter vascular prosthesis.

Blood Vessels↗

PubMedDate not supplied

Tissue engineering using magnetite nanoparticles and magnetic force: heterotypic layers of cocultured hepatocytes and endothelial cells.

Novel technologies to establish three-dimensional, in vivo-like tissue consisting of various types of cells are required for tissue engineering. We applied magnetic force to construct a heterotypic, layered coculture system of rat hepatocytes and human aortic endothelial cells (HAECs) that was not limited by cell type. Magnetite cationic liposomes carrying a positive surface charge to improve adsorption accumulated in HAECs at a concentration of 38 pg of magnetite per cell. Magnetically labeled HAECs specifically accumulated onto hepatocyte monolayers at sites where a magnet (4000 G) was positioned, and then adhered to form a heterotypic, layered construct with tight and close contact. This cocultured construct significantly (p < 0.05) enhanced albumin secretion by hepatocytes compared with that in homotypic cultures of hepatocytes or heterotypic cocultures of hepatocytes and HAECs without magnets. These results suggest that this novel use of magnetite nanoparticles and magnetic force, which we refer to as "magnetic force-based tissue engineering" (Mag-TE), offers a major advancement in tissue engineering.

Animals↗

PubMed2006

Combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering.

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.

Aggrecans↗

PubMed2005

Bone reconstruction: from bioceramics to tissue engineering.

Over the past 30 years, an enormous array of biomaterials proposed as ideal scaffolds for cell growth have emerged, yet few have demonstrated clinical efficacy. Biomaterials, regardless of whether they are permanent or biodegradable, naturally occurring or synthetic, need to be biocompatible, ideally osteoinductive, osteoconductive, integrative, porous and mechanically compatible with native bone to fulfill their desired role in bone tissue engineering. These materials provide cell anchorage sites, mechanical stability and structural guidance and in vivo, provide the interface to respond to physiologic and biologic changes as well as to remodel the extracellular matrix in order to integrate with the surrounding native tissue. Calcium phosphate ceramics and bioactive glasses were introduced more than 30 years ago as bone substitutes. These materials are considered bioactive as they bond to bone and enhance bone tissue formation. The bioactivity property has been attributed to the similarity between the surface composition and structure of bioactive materials, and the mineral phase of bone. The drawback in using bioactive glasses and calcium phosphate ceramics is that close proximity to the host bone is necessary to achieve osteoconduction. Even when this is achieved, new bone growth is often strictly limited because these materials are not osteoinductive in nature. Bone has a vast capacity for regeneration from cells with stem cell characteristics. Moreover, a number of different growth factors including bone morphogenetic proteins, have been demonstrated to stimulate bone growth, collagen synthesis and fracture repair both in vitro and in vivo. Attempts to develop a tissue-engineering scaffold with both osteoconductivity and osteoinductivity have included loading osteoinductive proteins and/or osteogenic cells on the traditional bioactive materials. Yet issues that must be considered for the effective application of bioceramics in the field of tissue engineering are the degree of bioresorption and the poor mechanical strength. The synthesis of a new generation of biomaterials that can specifically serve as tissue engineering scaffolds for drug and cell delivery is needed. Nanotechnology can provide an alternative way of processing porous bioceramics with high mechanical strength and enhanced bioactivity and resorbability.

Bone Diseases↗

PubMed2001

Born again bone: tissue engineering for bone repair.

Destruction of bone tissue due to disease and inefficient bone healing after traumatic injury may be addressed by tissue engineering techniques. Growth factor, cytokine protein, and gene therapies will be developed, which, in conjunction with suitable carriers, will regenerate missing bone or help in cases of defective healing.

Animals↗

PubMed2003

Comparison of tracheal and nasal chondrocytes for tissue engineering of the trachea.

BACKGROUND: This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage-derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced. METHODS: Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter x 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea. RESULTS: The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content. CONCLUSIONS: This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.

Animals↗

PubMed2006

Thermoreversible laminin-functionalized hydrogel for neural tissue engineering.

Traumatic injury to the central nervous system (CNS) triggers cell death and deafferentation, which may activate a cascade of cellular and network disturbances. These events often result in the formation of irregularly shaped lesions comprised of necrotic tissue and/or a fluid-filled cavity. Tissue engineering represents a promising treatment strategy for the injured neural tissue. To facilitate minimally invasive delivery of a tissue engineered system, a thermoreversible polymer is an attractive scaffold candidate. We have developed a bioactive scaffold for neural tissue engineering by tethering laminin-1 (LN) to methylcellulose (MC), a thermoresponsive hydrogel. The base MC chain was oxidized via sodium m-periodate to increase MC tethering capacity. Protein immobilization was facilitated by a Schiff base reaction between primary amine groups on LN and the carbonyl groups of the oxidized MC chain. Immunoassays demonstrated tethering of LN at 1.6 +/- 0.5 ng of LN per milligram of MC. Rheological measurements for different MC-LN constructs indicated MC composition- and MC treatment-dependent effects on solution-gelation transition temperature. Cellular assays with primary rat cortical neurons demonstrated enhanced cell adhesion and viability on LN-functionalized MC when compared with base and oxidized MC. This bioadhesive thermoresponsive scaffold may provide a robust delivery vehicle to injured CNS tissue for neural cell transplantation strategies.

Animals↗

PubMed2005

Histological and immunohistochemical studies of tissue engineered odontogenesis.

The successful regeneration of complex tooth structures based on tissue-engineering principles was recently reported. The process of this regeneration, however, remains poorly characterized. In this study, we have used histochemistry to examine the regeneration process of tissue engineered teeth in order to determine the cell types that give rise to these engineered tooth structures. Porcine third molar tooth buds were dissociated into single-cell suspensions and seeded onto a biodegradable polyglycolic acid polymer scaffold. Following varying periods of growth in rat hosts, the specimens were evaluated by histology and immunohistochemistry. Aggregates of epithelial cells were first observed 4-6 weeks after implantation. These aggregates assumed three different shapes: a natural tooth germ-like shape, a circular shape, or a bilayer-bundle. Based on the structure of the stellate reticulum in the dental epithelium, the circular and bilayer-bundle aggregates could be clearly classified into two types: one with extensively developed stellate reticulum, and the other with negligible stellate reticulum. The epithelial cells in the circular aggregates differentiated into ameloblasts. The continuous bilayer bundles eventually formed the epithelial sheath, and dentin tissue was evident at the apex of these bundles. Finally, enamel-covered dentin and cementum-covered dentin formed, a process most likely mediated by epithelial-mesenchymal interaction. These results suggest that the development of these engineered teeth closely parallels that of natural odontogenesis derived from the immature epithelial and mesenchymal cells.

Ameloblasts↗

PubMedDate not supplied

New multi-cue bioreactor for tissue engineering of tubular cardiovascular samples under physiological conditions.

In the present study we have developed a multi-cue bioreactor (MCB) that is capable of delivering a range of stimuli to assist the development of a tissue-engineered construct. The MCB provides an accurate and utilizable computer-controlled pulsatile pump and strain induction mechanism and it has the capability of applying physiological conditions to samples. The device described here emulates the pressure and straining environment found at the aortic root. This function, along with an integral perfusion and sterile containment system, allows for long-term culture and whole-tissue testing capability. Aortic and pulmonary arteries were obtained from freshly isolated porcine hearts and subjected to various loading regimens (Deltapressure/flow/force). Through analyzing data acquired by the MCB transducer array it was possible to differentiate the dynamic mechanical properties of the tissue types tested. In addition, the MCB illustrates a novel concept in cardiovascular tissue engineering: being able to support long-term tissue culture of cell-seeded substrates while they are under the influence of mechanical cues. After 7 days of pulsation in the MCB cell alignment was observed. The MCB represents a versatile model that will enable the development of tissue engineering not only for cardiovascular tissue, but for all tubular tissues such as esophageal, tracheal, and bronchial systems.

Bioreactors↗

PubMed2004

[Allogenic transplantation of human mesenchymal stem cells for tissue engineering purposes: an in vitro study].

Due to their plasticity and high proliferation capacity in vitro, human mesenchymal stem cells (MSC) are promising candidates for tissue engineering approaches of mesenchymal tissues like bone, cartilage, or tendon. Undifferentiated MSC do not express immunologically relevant cell surface markers. They inhibit the proliferation of allogeneic T-cells in vitro and elicit no immune response after allogeneic or xenogenic transplantation. Thus, MSC ought to be seen as immunoprivileged or immunomodulating cells. Here, we characterize the immune status and -behavior of MSC and MSC-derived osteogenic precursors in order to evaluate the usefulness of allogeneic MSC for tissue engineering of bone. Human MSC were isolated from bone marrow of hematologically normal voluntary donors. Osteogenic differentiation was induced by adding dexamethasone, ascorbic acid and beta-glycerophosphate. After 0, 8, 16 and 24 days, MSC were co-cultivated with allogeneic mononuclear cells. In parallel, the expression of immunologically relevant cell surface markers was monitored by flow cytometry. Undifferentiated and differentiated MSC did not stimulate allogeneic lymphocytes. MSC were negative for MHC-II, CD40, CD40L, CD80 (B7-1) and CD86 (B7-2), positive for MHC-I, and kept this expression pattern during osteogenic differentiation. Our results support the hypothesis that MSC are immunoprivileged cells which are potentially at disposal for HLA-incompatible cell replacement therapies.

Bone Regeneration↗