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PubMed2004

From cell-ECM interactions to tissue engineering.

The extracellular matrix (ECM) consists of a complex mixture of structural and functional macromolecules and serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function. The great diversity observed in the morphology and composition of the ECM contributes enormously to the properties and function of each organ and tissue. The ECM is also important during growth, development, and wound repair: its own dynamic composition acts as a reservoir for soluble signaling molecules and mediates signals from other sources to migrating, proliferating, and differentiating cells. Approaches to tissue engineering center on the need to provide signals to cell populations to promote cell proliferation and differentiation. These "external signals" are generated from growth factors, cell-ECM, and cell-cell interactions, as well as from physical-chemical and mechanical stimuli. This review considers recent advances in knowledge about cell-ECM interactions. A description of the main ECM molecules and cellular receptors with particular care to integrins and their role in stimulation of specific types of signal transduction pathways is also explained. The general principles of biomaterial design for tissue engineering are considered, with same examples.

Animals↗

PubMed2005

A perfusion bioreactor system capable of producing clinically relevant volumes of tissue-engineered bone: in vivo bone formation showing proof of concept.

In an effort to produce clinically relevant volumes of tissue-engineered bone products, we report a direct perfusion bioreactor system. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cc) of small sized macroporous biphasic calcium phosphate scaffolds (BCP, 2-6 mm). Cell load and cell distribution were shown using methylene blue block staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate viability of the present cells. After 19 days of cultivation, the scaffolds were covered with a viable, homogeneous cell layer. The hybrid structures became interconnected and a dense layer of extracellular matrix was present as visualized by environmental scanning electron microscopy (ESEM). ESEM images showed within the extracellular matrix sphere like structures which were identified as calcium phosphate nodules by energy dispersive X-ray analysis (EDX). On line oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption can be used to estimate GBMSC population doubling times during growth in this bioreactor system. Implantation of hybrid constructs, which were proliferated dynamically, showed bone formation in nude mice after 6 weeks of implantation. On the basis of our results we conclude that a direct perfusion bioreactor system is capable of producing clinically relevant volumes of tissue-engineered bone in a bioreactor system which can be monitored on line during cultivation and show bone formation after implantation in nude mice.

Animals↗

PubMed2005

Tissue engineering of heart valves: decellularized porcine and human valve scaffolds differ importantly in residual potential to attract monocytic cells.

BACKGROUND: Tissue-engineered or decellularized heart valves have already been implanted in humans or are currently approaching the clinical setting. The aim of this study was to examine the migratory response of human monocytic cells toward decellularized porcine and human heart valves, a pivotal step in the early immunologic reaction. METHODS AND RESULTS: Porcine and human pulmonary valve conduits were decellularized, and migration of U-937 monocytic cells toward extracted heart valve proteins was examined in a transmigration chamber in vitro. Homogenized tissue specimens were size fractionated by SDS-PAGE. The decellularization procedure effectively reduced the migration of human monocytes toward all heart valve tissue. However, only the antigen reduction of human pulmonary valves abolished the monocytic response (wall, 0.88+/-0.19% versus 30.20+/-3.93% migrated cells [mean+/-SEM]; cusps, 0.10+/-0.06% versus 10.24+/-1.83%) and was significantly lower (P<0.05) than that of the decellularized porcine equivalent (wall, 5.03+/-0.14% versus 24.31+/-2.38%; cusps, 3.18+/-0.38% versus 10.24+/-1.83%). SDS-PAGE of the pulmonary heart valve tissue revealed that considerable amounts of proteins with different molecular weights that were not detected in the human equivalent remain in the decellularized porcine heart valve. CONCLUSIONS: We describe for the first time that the remaining potential of decellularized pulmonary heart valves to attract monocytic cells depends strongly on whether porcine or human scaffolds were used. These findings will have an important impact on further investigations in the field of heart valve tissue engineering.

Animals↗

PubMed2001

Critical aspects of tissue-engineered therapy for bone regeneration.

Recent advances in bone tissue engineering are established on the understanding of an engineered scaffold, the molecular milieu within the osteogenic site, and the cell(s) predisposed to an osteogenic lineage. Advances in the incorporation of a generative vehicle into a skeletal defect require temporal and spatial distribution of the scaffold, growth factor, and cell compatible with enhanced bone healing. Monitoring events culminating in osteogenesis has focused on phenotypic and intracellular indicators. Phenotypic and intracellular indicators include the presence of receptors and intracellular signals that enable cell proliferation and differentiation. Progress in the areas of scaffold design, growth factor utilization, bone cell lineage, and intracellular signaling are reviewed.

Animals↗

PubMed2004

In vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering.

In vitro degradation behaviors of three-dimensional tissue engineering porous scaffolds made from amorphous poly(D,L-lactide-co-glycolide) with three different formulations have been systematically investigated up to 26 weeks in phosphate buffer saline solution at 37 degrees C. The following properties of the scaffolds were measured as a function of degradation time: dimensions, weight, compressive strength and modulus, polymer molecular weight and its distribution, and pore morphology. Of special interest was the determination of mechanical properties in wet environment. The pH of the PBS media was also detected. According to the characteristic changes of the various properties of porous scaffolds, the degradation process is suggested to be roughly divided into three stages tentatively named as quasi-stable stage, decrease-of-strength stage, loss-of-weight and disruption-of-scaffold stage.

Biocompatible Materials↗

PubMed2004

Preparation and characterization of a highly macroporous biodegradable composite tissue engineering scaffold.

A unique composite scaffold for bone-tissue engineering applications has been prepared by combining biodegradable poly(lactide-co-glycolide) (PLGA) with bioresorbable calcium phosphate (CaP) cement particles through the process of particle fusion and phase separation/particle leaching. The scaffold is characterized by a highly interconnected macroporosity, with macropores of 0.8-1.8 mm and porosities ranging from 81% to 91%, and improved mechanical properties with respect to the polymer alone, producing excellent dimensional stability. The scaffold properties were controlled by adjusting the processing parameters, including PLGA molar mass and concentration, CaP/PLGA ratio, and porogen size. The differences in mechanical properties between dry, wet/room temperature, and wet/37 degrees C testing conditions, of which the latter are more relevant for materials to be employed in a biological milieu, were investigated. Thus, a scaffold made from PLGA IV 1.13, PLGA concentration 12.5%, and CaP/PLGA ratio 2:1 exhibited significantly different compressive strengths of 0.16 MPa and 0.04 MPa when tested under dry and wet/37 degrees C conditions, respectively. .

Absorbable Implants↗

PubMed2006

Tissue-engineered trachea: History, problems and the future.

This review tries to summarize the efforts over the past 20 years to construct a tissue-engineered trachea. After illustrating the main technical bottlenecks faced nowadays, we discuss what might be the solutions to these bottlenecks. You may find out why the focus in this research field shifts dramatically from the construction of a tubular cartilage tissue to reepithelialization and revascularization of the prosthesis. In the end we propose a novel concept of 'in vivo bioreactor', defined as the design of a perfusion system inside the scaffold, and explain its potential application in the construction of a tissue-engineered trachea.

Animals↗

PubMed1994

Tissue engineering a blood vessel: regulation of vascular biology by mechanical stresses.

Important to the tissue engineering of a substitute blood vessel is an understanding of those factors which regulate vascular biology. A major factor in this regulation is the mechanical environment imposed by the hemodynamics of the vascular system. In this the vascular endothelium plays a critical role, and over the past two decades much has been learned about the influence of hemodynamics on vascular endothelial biology, to a large degree using cell culture to study the effects of flow and cyclic stretch. In our laboratory, such studies are now being extended through the development of a model of the arterial wall involving the co-culture of endothelial cells and smooth muscle cells. The development of such a model and its use in the study of hemodynamic effects represents necessary steps in the evolution of approaches to tissue engineering a blood vessel.

Animals↗

PubMed2003

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model.

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.

Allantois↗

PubMed2004

[Angiogenesis investigations in tissue engineering. The cylinder model on the chorioallantois membrane].

INTRODUCTION: Tissue engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold prior to in vivo transplantation. After transplantation, vascularization of the scaffold is a principal factor limiting cell viability for the first 6-8 days post transplantation. A model has been developed for systematic analysis of this process. METHODS: Fertilized white Leghorn eggs were incubated and opened at day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening onto the surface of the chorioallantoic membrane (CAM) at day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days post transplantation. RESULTS: The survival rate for embryos receiving constructs was about 90%. Histology confirmed transplant cell viability at day 4 post transplantation, and vascularization of the constructs by avian endothelial cells progressively increased thereafter. CONCLUSION: A new in vivo model to study the effect of angiogenesis in TE constructs including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties is presented. Advantages include easy access to the CAM vascular network, lack of immunocompetence, low cost, and avoidance of animal experiments.

Adipocytes↗

PubMed2004

In vitro development of autologous tissue engineered human articular neocartilage for orthopaedic surgery.

Treatment of articular cartilage lesions remains a clinical challenge. The uses of prosthetic joint replace allograft and/or autograft transplant carry a risk of complications due to infection, loosening of its component, immunological rejection and morbidity at the donor site. There has been an increasing interest in the management of cartilage damages, owing to the introduction of new therapeutic options. Tissue engineering as a method for tissue restoration begins to provide a potential alternative therapy for autologous grafts transplantations. We aimed to evaluate how well a tissue engineered neocartilage implant, consist of human articular chondrocytes cultured with the presence of autologous serum and mixed in a fresh fibrin derived from patient, would perform in subcutaneous implantation in athymic mice.

Animals↗

PubMed2005

Tissue engineering: a challenge of today's medicine.

During the last years, tissue engineering-based therapies have been introduced in clinical practice in the head and face area. The regeneration of complex tissue structures for all sites of the body is envisioned for the future. In the present situation, specialists of the different fields publish excellent research papers in specialised journals. As a result, the scientific community, separated towards distinct sub-specialities, has difficulties in communication. To overcome this problem, the demanding, complex and interdisciplinary aspects of tissue engineering has to be approached from new ways. We have conceptualised Head & Face Medicine therefore as a thematically broad ranged journal, including all disciplines involved in the head and neck area. We hope this journal will attract basic researchers and clinicians who are involved in investigating and applying complex themes (examplified by tissue engineering) in the head and face region and will contribute to a gain in scientific information, communication, and collaboration in order to improve the outcome of patient treatments.

Biomedical Research↗

PubMed2006

Clinical application of tissue engineered human heart valves using autologous progenitor cells.

BACKGROUND: Tissue engineering (TE) of heart valves reseeded with autologous cells has been successfully performed in vitro. Here, we report our first clinical implantation of pulmonary heart valves (PV) engineered with autologous endothelial progenitor cells (EPCs) and the results of 3.5 years of follow-up. METHODS AND RESULTS: Human PV allografts were decellularized (Trypsin/EDTA) and resulting scaffolds reseeded with peripheral mononuclear cells isolated from human blood. Positive stain for von Willebrand factor, CD31, and Flk-1 was observed in monolayers of cells cultivated and differentiated on the luminal surface of the scaffolds in a dynamic bioreactor system for up to 21 days, indicating endothelial nature. PV reseeded with autologous cells were implanted into 2 pediatric patients (age 13 and 11) with congenital PV failure. Postoperatively, a mild pulmonary regurgitation was documented in both children. Based on regular echocardiographic investigations, hemodynamic parameters and cardiac morphology changed in 3.5 years as follows: increase of the PV annulus diameter (18 to 22.5 mm and 22 to 26 mm, respectively), decrease of valve regurgitation (trivial/mild and trivial, respectively), decrease (16 to 9 mm Hg) or a increase (8 to 9.5 mm Hg) of mean transvalvular gradient, remained 26 mm or decreased (32 to 28 mm) right-ventricular end-diastolic diameter. The body surface area increased (1.07 to 1.42 m2 and 1.07 to 1.46 m2, respectively). No signs of valve degeneration were observed in both patients. CONCLUSIONS: TE of human heart valves using autologous EPC is a feasible and safe method for pulmonary valve replacement. TE valves have the potential to remodel and grow accordingly to the somatic growth of the child.

Adolescent↗

PubMed2004

Mechanical stimulation toward tissue engineering of the knee meniscus.

Current clinical practices do not adequately regenerate the meniscus of the knee secondary to a tear. Complete or partial meniscus removal leads to degenerative changes within the joint. Tissue engineering of the meniscus promises a potent solution. Before embarking on tissue engineering of the meniscus, it is crucial to have a thorough comprehension of the biomechanical role that this tissue fulfills and how the structure of meniscus is uniquely suited to that purpose. To better understand this, we have examined the meniscus, as well as associated tissues, within the body. For the first time, the knee meniscus is rigorously compared to ligament, tendon, and cartilage, and inferences are drawn on how mechanical stimulation may be used to channel growth in the meniscus. We have examined in detail the loading conditions that these tissues experience in vivo and how each is uniquely adapted to its loading environment. These tissues are capable of achieving some degree of remodeling because of mechanical stimuli. By understanding the mechanisms that can stimulate and promote regeneration in related tissues, we hope to harness that knowledge to achieve the goal of meniscal regeneration.

Animals↗

PubMed2002

Design of segmented poly(ether ester) materials and structures for the tissue engineering of bone.

In this study, PEOT/PBT segmented copolymers of different compositions have been evaluated as possible scaffold materials for the tissue engineering of bone. By changing the composition of PEOT/PBT copolymers, very different mechanical and swelling behaviors are observed. Tensile strengths vary from 8 to 23 MPa and elongations at break from 500 to 1300%. Water-uptake ranges from 4 up to as high as 210%. The in vitro degradation of PEOT/PBT copolymers occurs both by hydrolysis and oxidation. In both cases degradation is more rapid for copolymers with high PEO content. PEOT/PBT scaffolds with varying porosities and pore sizes have been prepared by molding and freeze-drying techniques in combination with particulate-leaching. The most hydrophilic PEOT/PBT copolymers did not sustain goat bone marrow cell adhesion and growth. However, surface modification by gas plasma treatment showed a very much improved polymer-cell interaction for all PEOT/PBT copolymer compositions. Their mechanical properties, degradability and ability to sustain bone marrow cell growth make PEOT/PBT copolymers excellent materials for bone tissue engineering.

Animals↗

PubMed2005

The effects of bone marrow-derived mesenchymal stem cells and fascia wrap application to anterior cruciate ligament tissue engineering.

After an anterior cruciate ligament (ACL) injury, surgical reconstructions are necessary in most cases, either with autografts, allografts, or artificial ligaments. Potential tissue-engineered ligaments would circumvent the disadvantages apparent in these methods. While seeding of mesenchymal stem cells (MSCs) and fascia wrap could potentially improve tissue regeneration and mechanical properties, their exact roles were evaluated in the current study. Knitted biodegradable scaffolds of poly-L-lactic acid (PLLA) and poly-glycolic-lactic acid (PGLA) yarns were used to reconstruct ACL in 48 rabbits. These were divided into four equal groups: only knitted scaffolds were used in group I; knitted scaffolds and mesenchymal stem cells were used in group II; knitted scaffolds, MSCs, and fascia lata were used in group III; knitted scaffolds and fascia lata were used in group IV. Carboxyfluorescein diacetate (CFDA)-labeled MSCs were used to trace the fate of seeded cells in groups II and III. Histology, Western blot analysis, and mechanical properties of reconstructed ACL were analyzed after 20 weeks. Fibroblast ingrowths were seen in all four groups while CFDA-labeled MSCs could be found after 8 weeks of implantation in groups II and III. Both the amount of collagen type I and collagen type III in groups III and IV were significantly higher than in group II, which was much higher than in group I. Both maximal tensile loads and stiffness of the reconstructed ACLs in groups I, II, III, and IV were significantly lower than normal controls after 20 weeks of implantation. It is concluded that MSCs could promote synthesis of collagen type I and collagen type III in tissue-engineered ligaments, while fascia wraps have stronger effects. Both MSC seeding and fascia wrap could not enhance ultimate tensile load and stiffness.

Animals↗

PubMed2006

Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma.

Human intervertebral disc (IVD) degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor-beta1 (TGF-beta1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In this study, we examine the role of TGF-beta1 in platelet-rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue-engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was 1 ng/ml TGF-beta1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time-dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT-PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p-Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue-engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue-engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration.

Adult↗

PubMed2000

Tensile properties of bioactive fibers for tissue engineering applications.

Cell transplantation using biocompatible, biodegradable scaffolds offers the possibility of creating or regenerating tissue to replace organ function when deficiency arises. The role of these temporary substrates is to support and guide the expanding cell culture until it becomes structurally integrated with the host tissue. 45S5 Bioglass(R) is a 4-component, melt-derived bioactive glass, which has been approved for human clinical use by the Food and Drug Administration. The biocompatibility and biodegradability of 45S5 Bioglass(R) are long established, whereas research into its performance as an extracellular scaffold is currently underway. In this study the tensile strengths (93 +/- 8 and 82 +/- 14 MPa), elongation to fracture (0.7 +/- 0.05%) and Weibull's moduli (3.0 and 3.5) of 45S5 Bioglass(R) fibers (mean diameters 193 and 280 microm) for tissue engineering applications are reported. The tensile strengths of the fibers are compared with those of bulk 45S5 Bioglass(R) and a range of biodegradable polymer materials currently used in the field of tissue engineering. Aspects of glass and fiber technology relevant to the design and manufacture of extracellular ceramic scaffolds are also discussed.

Animals↗