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PubMed1996

Tissue engineering of a bioartificial kidney: a universal donor organ.

Cell therapy and tissue engineering may well likely dominate medical therapeutics in the next century. Growing a functional glomerular filter and tubule reabsorber from a combination of cells, biomaterials, and synthetic polymers to replace renal excretory and regulatory functions is a specific example of these evolving technologies. The kidney was the first organ whose function was substituted by an artificial device. The kidney was also the first organ to be successfully transplanted. The ability to replace renal function with these revolutionary technologies in the past was due to the fact that renal excretory function is based on natural physical forces which govern solute and fluid movement from the body compartment to the external environment. The need for coordinated mechanical or electrical activities got renal substitution was not required. Accordingly, the kidney may well be the first organ to be available as a tissue-engineered implantable device as a fully functional replacement part for the human body. The prospects of a "universal donor" bioartificial kidney for the treatment of end-stage renal disease are clearly achievable as we approach the next millennium.

Artificial Organs↗

PubMed1996

Leucocyte adhesion under flow conditions: principles important in tissue engineering.

An understanding of inflammatory responses is important in a wide variety of tissue engineering applications. This review describes the current understanding of a central aspect of inflammatory responses, the adhesion of leucocytes to blood vessel walls prior to their emigration into tissues. These highly specific adhesive interactions are mediated by three main families of receptors: the selectins, integrins, and members of the immunoglobulin superfamily. Under flow conditions, the various receptors make distinct contributions to a multistep process of adhesion in which leucocytes roll, adhere firmly, and eventually transmigrate. Two examples in which these principles are important in tissue engineering research, lymphocyte adherence in transplant rejection and monocyte adherence in atherosclerosis, are discussed in the last part of the paper.

Animals↗

PubMed1996

Tissue-engineered skeletal muscle organoids for reversible gene therapy.

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postmitotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

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↗

PubMed1998

Tissue engineering: a new approach in cardiovascular surgery--seeding of human fibroblasts on resorbable mesh.

INTRODUCTION: In tissue engineering the material properties of synthetic compounds are manipulated to enable delivery of dissociated cells onto a scaffold in a manner that will result in in vitro formation of new functional tissue. The seeding of human fibroblasts on resorbable mesh is a precondition of a successful creation of human tissue such as autologous cardiac valves. MATERIAL AND METHODS: Polymeric scaffolds (n = 12) composed of polyglycolic acid (PGA) with a fiber diameter of 12-15 mm and a polymer density of 70 mg/ml were used as square sheets of 0.3 x 1 x 1 cm. Fibroblasts (passage 5), harvested from human foreskin, were seeded (3.4 x 10(6)) and cultured over a 3 week period on a PGA mesh. RESULTS: Microscopic examination of the seeded mesh demonstrated that the human fibroblasts were attached to the polymeric fibers and had begun to spread out and to divide. Electron microscopy showed a continuous distribution and formation of the cells throughout the "polymeric architecture". Spotlike hydrolysis of PGA fibers was observed. After 3 weeks the seeded scaffolds resembled a solid sheet of tissue. CONCLUSION: These preliminary results, successful seeding of human fibroblasts on a PGA mesh, represent a first basic step on the way to construct human tissue such as autologous cardiac valves and demonstrate that tissue engineering might be a promising new device in therapy of cardiovascular disease.

Cells, Cultured↗

PubMed1995

Biomaterials in tissue engineering.

Biomaterials play a pivotal role in field of tissue engineering. Biomimetic synthetic polymers have been created to elicit specific cellular functions and to direct cell-cell interactions both in implants that are initially cell-free, which may serve as matrices to conduct tissue regeneration, and in implants to support cell transplantation. Biomimetic approaches have been based on polymers endowed with bioadhesive receptor-binding peptides and mono- and oligosaccharides. These materials have been patterned in two- and three-dimensions to generate model multicellular tissue architectures, and this approach may be useful in future efforts to generate complex organizations of multiple cell types. Natural polymers have also played an important role in these efforts, and recombinant polymers that combine the beneficial aspects of natural polymers with many of the desirable features of synthetic polymers have been designed and produced. Biomaterials have been employed to conduct and accelerate otherwise naturally occurring phenomena, such as tissue regeneration in wound healing in the otherwise healthy subject; to induce cellular responses that might not be normally present, such as healing in a diseased subject or the generation of a new vascular bed to receive a subsequent cell transplant; and to block natural phenomena, such as the immune rejection of cell transplants from other species or the transmission of growth factor signals that stimulate scar formation. This review introduces the biomaterials and describes their application in the engineering of new tissues and the manipulation of tissue responses.

Amino Acid Sequence↗

PubMed1994

Temporomandibular joint disc replacement made by tissue-engineered growth of cartilage.

OBJECTIVE: To test the effectiveness of the new technique of tissue-engineered growth of cartilage, temporomandibular joint (TMJ) disc replacements were created by seeding dissociated chondrocytes on synthetic, three-dimensional, bioresorbable polymer constructs of a predetermined anatomic shape, incubating the cell-polymer constructs in vitro, and transplanting them into test animals. MATERIALS AND METHODS: Twelve highly porous and bioresorbable cell-transplantation devices in the shape of TMJ discs were created using biodegradable polylactid and polyglycolic acid fibers. Bovine articular cartilage was dissociated into chondrocytes and the cells were allowed to attach to the three-dimensional polymer scaffolds and multiply in vitro. After 1 week, the cell-polymer constructs were implanted subcutaneously into nude mice. The neocartilage was assessed by magnetic resonance imaging (MRI) techniques, gross inspection, histology, and biomechanical and biochemical analysis after 12 weeks. RESULTS: All implants seeded with chondrocytes showed gross evidence of histologically organized hyaline cartilage. The scaffolds maintained their specific shape. They not only showed appropriate intrinsic stability during neomorphogenesis of cartilage in vitro and in vivo, but also seemed to guide the growth of cartilage. The presence of sulfated glycosaminoglycans was shown by aldehyde fuchsin alcian blue staining of the specimens. Type II collagen, considered to be indicative of cartilage formation, was found in the specimens tested. MRI showed signal characteristics similar to those of hyaline cartilage. Analysis of neocartilage force/displacement curves and aqueous phase compliance using a closed compression chamber suggested that the ability of the constructs to resist deformation was similar to that of native donor cartilage. CONCLUSION: The technology of tissue-engineered growth of cartilage on individually designed scaffolds may have many applications not only in reconstructive surgery of the TMJ, but also in craniomaxillofacial, plastic, and orthopedic surgery.

Animals↗

PubMed1995

Tissue engineering heart valves: valve leaflet replacement study in a lamb model.

BACKGROUND: Valve replacements using either bioprosthetic or mechanical valves have the disadvantage that these structures are unable to grow, repair, or remodel and are both thrombogenic and susceptible to infection. These characteristics have significantly limited their durability and longevity. In an attempt to begin to overcome these shortcomings, we have tested the feasibility of constructing heart valve leaflets in lambs by seeding a synthetic polyglycolic acid fiber matrix in vitro with fibroblasts and endothelial cells. METHODS: Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low-density lipoprotein marker and separated from the fibroblasts using a fluorescent activated cell sorter. A synthetic biodegradable scaffold constructed from polyglycolic acid fibers was seeded with fibroblasts, which grew to form a tissue-like sheet. This tissue was subsequently seeded with endothelial cells, which formed a cellular monolayer coating around the leaflet. Using these constructs, autologous (n = 3) and allogenic (n = 4) tissue engineered leaflets were implanted in 7 animals. In each animal the right posterior leaflet of the pulmonary valve was resected and replaced with an engineered valve leaflet. RESULTS: All animals survived the procedure. Postoperative echocardiography demonstrated no evidence of stenosis and trivial pulmonary regurgitation in the autografts and moderate regurgitation in the allogenic valves. Collagen analysis of the constructs showed development of an extracellular matrix. Histologic evaluation of the constructs demonstrated appropriate cellular architecture. CONCLUSIONS: This preliminary experiment showed that a tissue engineered valve leaflet constructed from its cellular components can function in the pulmonary valve position. Tissue engineering of a heart valve leaflet is feasible, and these preliminary studies suggest that autograft tissue will probably be superior to allogenic tissue.

Animals↗

PubMed1993

Tissue engineering.

The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care. A new field, tissue engineering, applies the principles of biology and engineering to the development of functional substitutes for damaged tissue. This article discusses the foundations and challenges of this interdisciplinary field and its attempts to provide solutions to tissue creation and repair.

Animals↗

PubMed1996

Tissue-engineered heart valves. Autologous valve leaflet replacement study in a lamb model.

BACKGROUND: We have previously reported the successful creation of tissue-engineered valve leaflets and the implantation of these autologous tissue leaflets in the pulmonary valve position. This study was designed to trace cultured cells that were seeded onto a biodegradable polymer with the use of a 1,1'-dioctadecyl-3,3,3' 3'-tetramethylindo-carbocyanine perchlorate (Di-1) cell-labeling method. We also examined the time-related biochemical, biomechanical, and histological characteristics and evolution of these tissue constructs. METHODS AND RESULTS: Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low density lipoprotein marker and separated from fibroblasts with the use of a fluorescence-activated cell sorter. A synthetic biodegradable scaffold consisting of polyglycolic acid fibers was seeded first with fibroblasts, then coated with endothelial cells. Using these methods, we implanted autologous cell/polymer constructs in six animals. In two additional control animals, a leaflet of polymer was implanted without prior cell seeding. In each animal, cardiopulmonary bypass was used to completely resect the right posterior leaflet of the pulmonary valve and replace it with an engineered valve leaflet with (n = 6) or without (n = 2) prior cultured cell seeding. The animals were killed either after 6 hours or after 1, 6, 7, 9, or 11 weeks, and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. 4-Hydroxyproline assays were performed to determine collagen content. Leaflet strength was evaluated in vitro with a mechanical tester Factor VIII and elastin stains were done to verify histologically that endothelial cells and elastin, respectively, were present. Animals receiving leaflets made from polymers without cell seeding were killed and examined in a similar fashion after 8 weeks. In the control animals, the acellular polymer leaflets were completely degraded, with no residual leaflet tissue at 8 weeks. The tissue-engineered valve leaflet persisted in each animal in the experimental group. 4-Hydroxyproline analysis of the constructs showed a progressive increase in collagen content. Immunohistochemical staining demonstrated elastin fibers in the matrix and factor VIII on the surface of the leaflet. The cell-labeling experiments demonstrated that the cells on the leaflets had persisted from the in vitro seeding of the leaflets. CONCLUSIONS: In the tissue-engineered heart valve leaflet, transplanted autologous cells generated a proper matrix on the polymer scaffold in a physiological environment at a period of 8 weeks after implantation.

Animals↗

PubMedDate not supplied

Muscular tissue engineering: capillary-incorporated hybrid muscular tissues in vivo tissue culture.

Requirements for a functional hybrid muscular tissue are 1) a high density of multinucleated cells, 2) a high degree of cellular orientation, and 3) the presence of a capillary network in the hybrid tissue. Rod-shaped hybrid muscular tissues composed of C2C12 cells (skeletal muscle myoblast cell line) and type I collagen, which were prepared using the centrifugal cell-packing method reported in our previous article, were implanted into nude mice. The grafts, comprised three hybrid tissues (each dimension, diameter, approximately 0.3 mm, length, approximately 1 mm, respectively), were inserted into the subcutaneous spaces on the backs of nude mice. All nude mice that survived the implantation were sacrificed at 1, 2, and 4 wk after the implantation. The grafts were easily distinguishable from the subcutaneous tissues of host mice with implantation time. The grafts increased in size with time after implantation, and capillary networks were formed in the vicinities and on the surfaces of the grafts. One week after implantation, many capillaries formed in the vicinities of the grafts. In the central portion of the graft, few capillaries and necrotic cells were observed. Mononucleated myoblasts were densely distributed and a low number of multinucleated myotubes were scattered. Two weeks after implantation, the formation of a capillary network was induced, resulting in the surfaces of the grafts being covered by capillaries. Numerous elongated multinucleated myotubes and mononucleated myoblasts were densely distributed and numerous capillaries were observed throughout the grafts. Four weeks after implantation a dense capillary network was formed in the vicinities and on the surfaces of the grafts. In the peripheral portion of the graft, multinucleated myotubes in the vicinities of the rich capillaries were observed. Thus, hybrid muscular tissues in vitro preconstructed was remodeled in vivo, which resulted in facilitating the incorporation of capillary networks into the tissues.

Animals↗

PubMed1996

Tissue engineering.

The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in health care. Current treatment modalities include transplantation of organs, surgical reconstruction, use of mechanical devices, or supplementation of metabolic products. Tissue engineering is the application of the principles and methods of engineering and the life sciences to the fundamental understanding of structure/function relationships in normal and pathological tissues and the development of biological substitutes to restore, maintain, or improve function. This article discusses the challenges of this interdisciplinary field and its attempts to provide solutions to tissue creation and repair.

Artificial Organs↗

PubMed1998

Tissue engineering in urologic surgery.

The use of nonurologic tissues in the genitourinary tract is common (owing to a lack of a better alternative) despite the known possible adverse effects. Selective cell transplantation is providing a means to engineer genitourinary tissues that may be used for reconstruction. This novel technology involves an interdisciplinary approach, combining techniques of cell biology and materials sciences towards the development of functional tissues or organs. Tissues associated with urology, such as clitoral, cavernosal, urethral, vesical, ureteral, and renal have been created in the laboratory, with varying degrees of function. Cells have also been recently used in patients as bulking agents for the treatment of vesicoureteral reflux and urinary incontinence. As the science of tissue engineering evolves, one can expect a wider application of this technology to the armamentarium of urologic surgery.

Cell Transplantation↗

PubMed1995

Tissue-engineered cartilage for implantation and grafting.

Facial plastic and reconstructive surgeons are continually faced with the dilemma of what material to use for implantation and grafting. Tissue-engineered cartilage is a relatively new and exciting concept which utilizes chondrocytes and cultures them on a three-dimensional biodegradable template. This template/cell complex is first briefly incubated in vitro, then implanted into a recipient host. In situ the template resorbs and is replaced with new cartilage that is viable, compatible, and mature. This paper discusses the biochemical composition of cartilage, the concept of tissue engineering, advances in template quality, and cartilage immunogenicity. Future clinical applications of this type of graft research include microtia repair, facial reconstruction, rhinoplasty, and other facial cosmetic procedures.

Animals↗

PubMed1997

Tissue-engineered heart valve leaflets: does cell origin affect outcome?

BACKGROUND: We previously reported the successful creation of tissue-engineered valve leaflet constructs and the implantation of these autologous tissue leaflets in the pulmonary valve position in a lamb model. The optimal cell origin for creating these valve leaflets remains unclear. This study was designed to compare dermal with arterial wall myofibroblasts as the cells of origin for the leaflet constructs. METHODS AND RESULTS: Mixed cell populations of endothelial cells and fibroblasts were isolated from ovine femoral arteries or subdermis and then expanded in vitro. A synthetic biodegradable polymer scaffold was then seeded with the cultured cells. The tissue scaffold was composed of a polyglactin woven mesh sandwiched between two nonwoven polyglycolic acid mesh sheets, which measured 3x3 cm in size and 3.2 mm in thickness. The cell-seeded polymer construct was implanted to replace one pulmonary valve leaflet in the same juvenile animal from which the cells had originally been obtained. Using cardiopulmonary bypass, the right posterior leaflet of the pulmonary valve was completely resected and replaced with an autologous engineered valve leaflet. In group D (n=5), the cells were obtained from subdermis, and in group A (n=4), they were obtained from the arterial wall. Eight to 10 weeks after leaflet implantation, the animals were killed, and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. The dimensions of each tissue-engineered leaflet (TEL) were compared with those of the two remaining native valve leaflets to obtain a growth index. A 4-hydroxyproline assay was performed to evaluate collagen content. Leaflet tensile strength was evaluated in vitro by using a Vitrodyne V-1000 mechanical tester. Factor VIII and elastin stains were performed to histologically assess the presence of endothelial cells and elastin, respectively. In all animals, the TEL persisted in the pulmonary valve position after 8 to 10 weeks, and all polyglycolic acid polymer had been degraded. Group A leaflets had a higher growth index (0.86+/-0.11) than group D (0.41+/-0.08) (P<.05). Macroscopically, the group D leaflets appeared thicker and contracted. Histologically, elastic fibers were more abundant in group A than in group D. Total collagen content and biomechanical testing showed no differences between groups. Leaflets from both groups had positive staining for factor VIII on the surface, confirming growth of endothelial cells to cover the TEL. CONCLUSIONS: Autologous TEL derived from vascular fibroblasts seem to develop functionally and morphologically like the native valve leaflets in the pulmonary circulation. Use of arterial myofibroblasts for the creation of TEL seems preferable to dermal fibroblasts with current tissue culture conditions.

Animals↗

PubMedDate not supplied

Tissue engineering of autologous cartilage transplants for rhinology.

In reconstructive surgery there is increasing demand for cartilage transplants to fill defects, especially nose and/or outer ear defects. Tissue engineering is one of the most modern pathways to generate autologous cartilage transplants. Isolated chondrocytes obtained from a tiny patient's biopsy were seeded on bioresorbable preshaped cell carriers to provide a 3-dimensional cell arrangement as in vivo. The combined use of these cell carriers in form of a non-woven mesh and a constant medium perfusion was performed to generate a cartilage-like cell-polymer-construct, which was finally subcutanously implanted in nude mice for full maturation. After explantation of 6 months, expression of cartilage specific extracellular matrix molecules was obvious by using histochemical and immunohistochemical methods. These data show that tissue engineering with isolated multiplied human chondrocytes from a tiny biopsy seeded on bioresorbable polymer is a promising system to generate autologous cartilage transplants for replacements in reconstructive surgery.

Animals↗

PubMed1998

Polymer concepts in tissue engineering.

Traumatic injuries, cancer treatment, and congenital abnormalities are often associated with abnormal bone shape or segmental bone loss. Restoration of normal structure and function in these cases requires replacement of the missing bone that may be accomplished by surgical transfer of natural tissue from an uninjured location elsewhere in the body. However, this procedure is limited by availability, adequate blood supply, and secondary deformities at the donor site. One strategy to overcome these problems is to develop living tissue substitutes based on synthetic biodegradable polymers. Three methods of bone regeneration using biodegradable polymers are being studied in our laboratory: tissue induction, cell transplantation, and fabrication of vascularized bone flaps. Injectable polymers are used for filling skeletal defects and guiding bone tissue growth. Their main advantage is minimizing the surgical intervention or the severity of the surgery. Polymer-cell constructs also hold great promise in the field of tissue engineering. They provide a scaffold on which cells grow and organize themselves. As the cells begin to secrete their own extracellular matrix, the polymer degrades and is eventually eliminated from the body, resulting in completely natural tissue replacement. Bone flaps can be fabricated ectopically into precise shapes and sizes. With an attached vascular supply, these flaps can be transferred into areas deficient in vascularity. This article discusses polymer concepts regarding bone tissue engineering and reviews recent advances of our laboratory on guided bone regeneration using biodegradable polymer scaffolds.

Animals↗

PubMed1998

Surface characteristics and biocompatibility of lactide-based poly(ethylene glycol) scaffolds for tissue engineering.

Novel lactide-based poly(ethylene glycol) (PEG) polymer networks (GL9-PEGs) were prepared by UV copolymerization of a glycerol-lactide triacrylate (GL9-Ac) with PEG monoacrylate (PEG-Ac) to use as scaffolds in tissue engineering, and the surface properties and biocompatibility of these networks were investigated as a function of PEG molecular weight and content. Analysis by ATR-FTIR and ESCA revealed that PEG was incorporated well within the GL9-PEG polymer networks and was enriched at the surfaces. From the results of SEM, AFM, and contact angle analyses, GL9-PEG networks showed relatively rough and irregular surfaces compared to GL9 network, but the mobile PEG chains coupled at their termini were readily exposed toward the aqueous environment when contacting water such that the surfaces became smoother and more hydrophilic. This reorientation and increase in hydrophilicity were more extensive with increasing PEG molecular weight and content. As compared to GL9 network lacking PEG, protein adsorption as well as platelet and S. epidermidis adhesion to GL9-PEG networks were significantly reduced as the molecular weight and content of PEG was increased, indicating that GL9-PEG networks are more biocompatible than the GL9 network due to PEG's passivity. Based on the physical and biological characterization reported, the GL9-PEG materials would appear to be interesting candidates as matrices for tissue engineering.

Bacterial Adhesion↗