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The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review.

The construction of tissue-engineered devices for medical applications is now possible in vitro using cell culture and bioreactors. Although methods of incorporating them back into the host are available, current constructs depend purely on diffusion which limits their potential. The absence of a vascular network capable of distributing oxygen and other nutrients within the tissue-engineered device is a major limiting factor in creating vascularised artificial tissues. Though bio-hybrid prostheses such as vascular bypass grafts and skin substitutes have already been developed and are being used clinically, the absence of a capillary bed linking the two systems remains the missing link. In this review, the different approaches currently being or that have been applied to vascularise tissues are identified and discussed.

Animals↗

Engineering of volume-stable adipose tissues.

Autologous adipose tissues have been clinically used for augmentation of soft tissues lost due to mastectomy or lumpectomy in plastic and reconstructive surgery. However, this therapy has problems of absorption and subsequent volume loss of the implanted adipose tissues. In this study, volume-stable adipose tissues were engineered in vivo using mechanical support structures fabricated from biodegradable synthetic polymers. Dome-shaped mechanical support structures were fabricated by reinforcing poly(glycolic acid) fiber-based matrices with poly(L-lactic acid). The support structures were placed into subcutaneous pockets of athymic mice, and human preadipocytes suspended in fibrin matrix were injected into the space under the support structures (group I). Injection of either fibrin matrix without preadipocytes under the support structures (group II) or fibrin matrix containing preadipocytes into subcutaneous spaces with no support structures (group III) served as controls. Six weeks after implantation, the original implant volume was maintained approximately in groups I and II, whereas, group III showed significant implant shrinkage. The compressive modulus of the mechanical support structures did not change significantly over 6-week incubation in phosphate-buffered saline at 37 degrees C. Histological analyses of the implants showed regeneration of adipose tissues in group I. In contrast, groups II and III did not show extensive adipose tissue formation. This study demonstrates that volume-stable adipose tissues can be engineered in vivo using mechanical support structures. This technique offers the potential for augmentation of adipose tissues with volume conservation.

Adipocytes↗

Fabrication and biocompatibility of polypyrrole implants suitable for neural prosthetics.

Finding a conductive substrate that promotes neural interactions is an essential step for advancing neural interfaces. The biocompatibility and conductive properties of polypyrrole (PPy) make it an attractive substrate for neural scaffolds, electrodes, and devices. Stand-alone polymer implants also provide the additional advantages of flexibility and biodegradability. To examine PPy biocompatibility, dissociated primary cerebral cortical cells were cultured on PPy samples that had been doped with polystyrene-sulfonate (PSS) or sodium dodecylbenzenesulfonate (NaDBS). Various conditions were used for electrodeposition to produce different surface properties. Neural networks grew on all of the PPy surfaces. PPy implants, consisting of the same dopants and conditions, were surgically implanted in the cerebral cortex of the rat. The results were compared to stab wounds and Teflon implants of the same size. Quantification of the intensity and extent of gliosis at 3- and 6-week time points demonstrated that all versions of PPy were at least as biocompatible as Teflon and in fact performed better in most cases. In all of the PPy implant cases, neurons and glial cells enveloped the implant. In several cases, neural tissue was present in the lumen of the implants, allowing contact of the brain parenchyma through the implants.

Animals↗

EGF-grafted PDMS surfaces in artificial cornea applications.

Lack of epithelial cell coverage has remained a persistent problem in the design of an artificial cornea. In this work, polydimethylsiloxane (PDMS) surfaces were modified with epidermal growth factor (EGF) to improve the growth of corneal epithelial cells. The EGF was covalently tethered to PDMS substrates aminated by plasma polymerization of allylamine via a homobifunctional polyethylene glycol (PEG) spacer. Surface modification was confirmed by contact angle and X-ray photoelectron spectroscopy measurements. By varying the ratio of EGF to PEG from 1:50 to 1:5, EGF amounts from 40 to 90 ng/cm2 could be bound, as determined by surface plasmon resonance (SPR) and 125I radiolabelling. Human corneal epithelial cells on the various modified surfaces were cultured both in the presence and absence of EGF in the culture medium to determine the effect of covalently bound EGF on the cells. The results demonstrated that covalently bound EGF on the surfaces is active with respect to promoting epithelial cell coverage. This was significant when compared to unmodified controls.

Adsorption↗

Bioreactors for tissue mass culture: design, characterization, and recent advances.

This paper reviews reports on three-dimensional mammalian tissue growth in bioreactors and the corresponding mammalian tissue growth requirements. The needs for nutrient and waste removal of several mammalian tissues are reviewed and compared with the environment of many reactors currently in use such as the continuous stirred tank, the hollow fiber, the Couette-Taylor, the airlift, and the rotating-wall reactors developed by NASA. Many studies conclude that oxygen supply appears to be one of the most important factors limiting tissue growth. Various correlations to describe oxygen mass transfer are presented and discussed with the aim to provide some guidance to design, construct, and test reactors for tissue mass culture. To obtain tissue thickness clinically valuable, dimensionless and other types of analysis tend to point out that diffusive transport will have to be matched with an important convection to bring sufficient oxygen molecular flux to the growing cells located within a tissue mass. As learned from solid-state fermentation and hairy root culture, during the growth of large biomass, heterogeneity (i.e., channeling, temperature gradients, non-uniform cell growth, transfer gradients, etc.) can cause some important problems and these should be addressed in tissue engineering as well. Reactors (along with the scaffolds) should be designed to minimize these issues. The role of the uterus, the reactor built by Nature, is examined, and the environment provided to a growing embryo is reported, yielding possible paths for further reactor developments. Finally, the importance of cell seeding methods is also addressed.

Animals↗

Controlled fabrication of a biological vascular substitute.

Autologous and synthetic vessel grafts have been used as a vascular substitute for cardiovascular bypass procedures. However, these materials are limited by the availability of appropriate caliber autologous vessels, increased susceptibility to thrombosis and intimal hyperplasia following surgery. Electrospinning technology offers the potential for controlling composition, structure and mechanical properties of biomaterials. Vascular graft scaffolds have been fabricated using electrospun polymer blends of Type I collagen, elastin from ligamentum nuchae, and poly (d,l-lactide-co-glycolide). This study demonstrates improved electrospinning characteristics versus previous studies by increasing polymer concentration and adding PLGA to the polymer blend. Additionally, new in vitro biocompatibility and mechanical testing data is presented. The scaffolds possess tissue composition and mechanical properties similar to native vessels. The electrospun vessel matrix is biocompatible and does not elicit local or systemic toxic effects when implanted in vivo. This study demonstrates the promise of electrospinning as a fabrication process for a functional vascular graft for clinical use.

Animals↗

Tissue engineering and cartilage regeneration for auricular reconstruction.

OBJECTIVE: This paper will provide (a) a review on current status of auricular reconstruction (b) particularly focusing on the current data about pinna reconstruction using stem cells in combination with tissue engineering. METHODS: The paper is divided into two sections. The first section presents a brief overview of the current status of auricular reconstruction. In the second section, the authors review the aspects and the current status of stem cells and tissue engineering researches related to cartilage regeneration. CONCLUSIONS: Total auricular reconstruction represents one of the greatest challenges for the ENT and Facial Plastic surgeon. The matter of auricular cartilage reconstruction is complex, and progresses in material designs as well as in stem cells field are essential. Even if this bio-technology field is promising, the progresses still are not adequate as patient expectations remain high.

Bioartificial Organs↗

Strain distribution in an elastic substrate vibrated in a bioreactor for vocal fold tissue engineering.

A bioreactor previously described was used to quantify the shear strain along a bioengineered tissue scaffold driven at low audio frequencies (20-200 Hz). Standing wave patterns were calculated analytically by solving a classical boundary value problem for a vibrating string under tension and bending stiffness. Boundary conditions were non-traditional in that small pivot arms at the endpoints allowed neither the displacement nor the velocity to go to zero. The calculations were corroborated with stroboscopic measurement of the motion of the material in the bioreactor. Results indicate that shear strains up to 0.2 can be obtained at low frequencies (20 Hz), with a gradual decrease at higher frequencies due to the decaying amplitude response of the mechanical driver. The bioreactor may be useful for approximating the Young's modulus of the material in situ by probing for resonance frequencies in the standing wave pattern. A yet unsolved problem is a variable drag coefficient along the length of the material due to fluid turbulence in the culture medium.

Bioartificial Organs↗

Osteochondral tissue engineering.

Osteochondral defects (i.e., defects which affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Current surgical limits in the treatment of complex joint lesions could be overcome by grafting osteochondral composite tissues, engineered by combining the patient's own cells with three-dimensional (3D) porous biomaterials of pre-defined size and shape. Various strategies have been reported for the engineering of osteochondral composites, which result from the use of one or more cell types cultured into single-component or composite scaffolds in a broad spectrum of compositions and biomechanical properties. The variety of concepts and models proposed by different groups for the generation of osteochondral grafts reflects that understanding of the requirements to restore a normal joint function is still poor. In order to introduce the use of engineered osteochondral composites in the routine clinical practice, it will be necessary to comprehensively address a number of critical issues, including those related to the size and shape of the graft to be generated, the cell type(s) and properties of the scaffold(s) to be used, the potential physical conditioning to be applied, the degree of functionality required, and the strategy for a cost-effective manufacturing. The progress made in material science, cell biology, mechanobiology and bioreactor technology will be key to support advances in this challenging field.

Bioartificial Organs↗

Development of an artificial vessel lined with human vascular cells.

OBJECTIVES: Thrombogenity of small-diameter vascular prostheses might be reduced by complete coverage of the luminal surface with vascular cells. We investigated cell seeding on polyurethane vascular prostheses. METHODS: Thirty polyurethane vascular prostheses were divided into 3 groups of 10 each: group A, diameter of 20 mm and gamma-sterilized; group B, diameter of 4 mm and gamma-sterilized; and group C, diameter of 4 mm and ethylene oxide sterilized. Human smooth muscle cells, fibroblasts, and endothelial cells were isolated from saphenous vein segments and expanded in culture. Five polyurethane vascular prostheses of each group were seeded with endothelial cells alone (mean, 4.8 +/- 1.2 x 10(6) cells), and the remaining 5 polyurethane vascular prostheses were preseeded with a mixed culture of fibroblasts and smooth muscle cells (mean, 7.7 +/- 2.3 x 10(6) cells), followed by endothelial cell seeding (mean, 4.4 +/- 0.9 x 10(6) cells). Seven days after cell seeding, the polyurethane vascular prostheses were perfused under a pulsatile flow (80 pulses/min, 140/80 mm Hg, and 120 mL/min) for 2 hours. Specimens were taken after each seeding procedure both before and after perfusion and then examined both with a scanning electron microscope and immunohistochemically. RESULTS: Isolated endothelial cell seeding revealed better initial adhesion in groups A and B than in group C (63% vs 33%). After 7 days, the cells had covered approximately 80% of the luminal surface in groups A and B, whereas group C cells rounded up and lost adhesion. After perfusion testing of group A and B prostheses, only 10% of the surface was still covered with endothelial cells. Preseeding with the mixed culture again revealed a better initial adhesion in groups A and B compared with that in group C (76% vs 41%). In groups A and B endothelial cell seeding (adhesion, 72%) resulted in a confluent endothelial cell layer. The results of immunohistochemical staining were positive for collagen IV, laminin, CD31, and Factor VIII. In group C only isolated cells were found after each seeding procedure, which rounded up and vanished during the next days. Perfusion testing of group A and B prostheses revealed that the confluent cell layer remained stable, with only small defects (<10% of the surface). The cells stained positivively for endothelial nitric oxide synthase. CONCLUSION: Seeding of a mixed culture out of fibroblasts and smooth muscle cells resulted in improved endothelial cell adhesion and resistance to shear stress. This outcome was caused by an increased synthesis of extracellular matrix proteins. Cell attachment was better on gamma-sterilized polyurethane vascular prostheses compared with on those undergoing ethylene oxide sterilization.

Bioartificial Organs↗

Injectable bioartificial myocardial tissue for large-scale intramural cell transfer and functional recovery of injured heart muscle.

OBJECTIVES: Most tissue-engineering approaches to restore injured heart muscle result in distortion of left ventricular geometry. In the present study we suggest seeding embryonic stem cells in a liquid matrix for myocardial restoration. METHODS: Undifferentiated green fluorescent protein-labeled mouse embryonic stem cells (2 x 10 6 ) were seeded in Matrigel (B&D, Bedford, Mass). In a Lewis rat heterotopic heart transplant model an intramural left ventricular pouch was fashioned after ligation of the left anterior descending coronary artery. The liquid mixture (0.125 mL) was injected in the resulting infarcted area within the pouch and solidified within a few minutes after transplantation (37 degrees C). Five recipient groups were formed: transplanted healthy hearts (group I), infarcted control hearts (group II), matrix recipients alone (group III), the study group that received matrix plus cells (group IV), and a group that received embryonic stem cells alone (group V). After echocardiography 2 weeks later, the hearts were harvested and stained for green fluorescent protein and cardiac muscle markers (connexin 43 and alpha-sarcomeric actin). RESULTS: The graft formed a sustained structure within the injured area and prevented ventricular wall thinning. The inoculated cells remained viable and expressed connexin 43 and alpha-sarcomeric actin. Fractional shortening and regional contractility were better in animals that received bioartificial tissue grafts compared with control animals (infarcted, matrix only, and embryonic stem cells only: group I, 17.0% +/- 3.5%; group II, 6.6% +/- 2.1%; group III, 10.3% +/- 2.2%; group IV, 14.5% +/- 2.5%; and group V, 7.8% +/- 1.8%). CONCLUSIONS: Liquid bioartificial tissue containing embryonic stem cells constitutes a powerful new approach to restoring injured heart muscle without distorting its geometry and structure.

Animals↗

Experimental generation of a tissue-engineered functional and vascularized trachea.

OBJECTIVE: We sought to grow in vitro functional smooth muscle cells, chondrocytes, and respiratory epithelium on a biologic, directly vascularized matrix as a scaffold for tracheal tissue engineering. METHODS: Ten- to 15-cm-long free jejunal segments with their own vascular pedicle were harvested and acellularized from donor pigs (n = 10) and used as a vascular matrix. Autologous costal chondrocytes, smooth muscle cells, and respiratory epithelium and endothelial progenitor cells were first cultured in vitro and then disseminated on the previously acellularized vascular matrix. Histologic, immunohistologic, molecular imaging, and Western blotting studies were then performed to assess cell viability. RESULTS: The endothelial progenitor cells re-endothelialized the matrix to such an extent that endothelial cell viability was uniformly documented through 2-(18F)-fluoro-2'-deoxyglucose positron emission tomography. This vascularized scaffold was seeded with functional (according to Western blot analysis) smooth muscle cells and successfully reseeded with viable ciliated respiratory epithelium. Chondrocyte growth and production of extracellular cartilaginous matrix was observed as soon as 2 weeks after their culture. CONCLUSIONS: The fundamental elements for a bioartificial trachea were successfully engineered in vitro in a direct vascularized 10- to 15-cm-long bioartificial matrix. Future experimental work will be directed to give them a 3-dimensional aspect and a biomechanical profile of a functioning trachea.

Animals↗

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.

Animals↗

Distinct cell-to-fiber junctions are critical for the establishment of cardiotypical phenotype in a 3D bioartificial environment.

The first step toward improving the cell-matrix interactions that occur in bioartificial myocardial tissue is an understanding of the ultrastructural links between cells and host fibers. Here, we identify a distinct type of junction that helps the cells to find anchorage in the three-dimensional environment, and we evaluate the phenotype of the resulting tissue. Neonatal rat cardiomyocytes were seeded in two different collagen scaffolds after pre-hydration of the scaffold. Conventional and electron microscopy were used to analyze the tissue microstructure. Viability was assessed by life/dead assay and physical properties of the resulting tissue were evaluated. The resulting tissue displayed high cellular viability, spontaneous contractions over 12 weeks, and responded to passive stretch similar to native rat myocardium. Contractile force responded physiologically to calcium (Ca), adrenaline, and stretch administration. Ultrastructural studies revealed a cell-to-fiber junction, as well as a background matrix configuration, which has not been described before in this context. The cells aligned along collagen fibers and engaged in complex intercalations. The cell-to-fiber affinity is essential for the phenotypical performance of bioartificial myocardial tissue equivalents. Moreover, given the appropriate porosity of the scaffold, pre-hydration promotes migration and affinity of cells to host structures.

Animals↗

Stem cell research.

One of the most active areas of research in medicine today is stem cell biology. This review introduces the reader to the field of stem cell biology and its therapeutic potential. More importantly, the potential application of stem cell therapy in acute lung injury will be explored.

Animals↗