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PubMed2004

Tissue regeneration based on tissue engineering technology.

Recent development of biomedical engineering as well as basic biology and medicine has enabled us to induce cell-based regeneration of body tissue to self-repair defective tissue or substitute biological functions of damaged organs. For successful tissue regeneration, it is indispensable to give cells an environment suitable for regeneration induction. Tissue engineering is a newly emerging biomedical technology for creating an environment for tissue regeneration with various biomaterials. The paper presented here overviews recent research data on tissue regeneration based on tissue engineering, and briefly explains the key technology of tissue engineering.

Adipose Tissue↗

PubMed1999

Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

Adult↗

PubMed2006

Bone tissue engineering using foetal cell therapy.

Different cell sources for bone tissue engineering are reviewed. In particular, adult cell source strategies have been based on the implantation of unfractionated fresh bone marrow; purified, culture expanded mesenchymal stem cells, differentiated osteoblasts, or cells that have been modified genetically to express rhBMP. Several limiting factors are mentioned for these strategies such as low number of available cells or possible immunological reaction of the host. Foetal bone cells are presented as an alternative solution and review of actual treatments using these cells is presented. Finally, foetal cells used specifically for bone tissue engineering are characterised and potentially interesting therapeutic options are proposed.

Bone Regeneration↗

PubMed2004

Tissue engineering with chondrocytes and function of the extracellular matrix (Review).

Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. The destruction or malformation of organs such as nasal cartilage, pinna and trachea in otorhinology-head and neck surgery can be caused by both: primary disease or treatment modalities. A large part of modern medical practice is aimed to repair, replace, maintain or enhance the function of damaged or diseased tissues and organs. Replacement or repair is by either artificial implants or transplantation of tissues. Such interventions are hindered by factors such as rejection by the immune system, limited blood supply or morbidity of the donor site. Reconstruction of an injured face using plastic surgery is a prime example of when the limitations of materials, science and reconstructive techniques become apparent. This review aims to briefly outline the use of chondrocytes for tissue engineering with special regard to the function of the extracellular matrix for the signalling between the chondrocytes.

Animals↗

PubMed2001

Tissue-engineered skin. Current status in wound healing.

Tissue-engineered skin is a significant advance in the field of wound healing and was developed due to limitations associated with the use of autografts. These limitations include the creation of a donor site which is at risk of developing pain, scarring, infection and/or slow healing. A number of products are commercially available and many others are in development. Cultured epidermal autografts can provide permanent coverage of large area from a skin biopsy. However, 3 weeks are needed for graft cultivation. Cultured epidermal allografts are available immediately and no biopsy is necessary. They can be cryopreserved and banked, but are not currently commercially available. A nonliving allogeneic acellular dermal matrix with intact basement membrane complex (Alloderm) is immunologically inert. It prepares the wound bed for grafting allowing improved cultured allograft 'take' and provides an intact basement membrane. A nonliving extracellular matrix of collagen and chondroitin-6-sulfate with silicone backing (Integra) serves to generate neodermis. A collagen and glycosaminoglycan dermal matrix inoculated with autologous fibroblasts and keratinocytes has been investigated but is not commercially available. It requires 3 to 4 weeks for cultivation. Dermagraft consists of living allogeneic dermal fibroblasts grown on degradable scaffold. It has good resistance to tearing. An extracellular matrix generated by allogeneic human dermal fibroblasts (TransCyte) serves as a matrix for neodermis generation. Apligraf is a living allogeneic bilayered construct containing keratinocytes, fibroblasts and bovine type I collagen. It can be used on an outpatient basis and avoids the need for a donor site wound. Another living skin equivalent, composite cultured skin (OrCel), consists of allogeneic fibroblasts and keratinocytes seeded on opposite sides of bilayered matrix of bovine collagen. There are limited clinical data available for this product, but large clinical trials are ongoing. Limited data are also available for 2 types of dressing material derived from pigs: porcine small intestinal submucosa acellular collagen matrix (Oasis) and an acellular xenogeneic collagen matrix (E-Z-Derm). Both products have a long shelf life. Other novel skin substitutes are being investigated. The potential risks and benefits of using tissue-engineered skin need to be further evaluated in clinical trials but it is obvious that they offer a new option for the treatment of wounds.

Animals↗

PubMed2006

Investigation of silica-iron-phosphate glasses for tissue engineering.

Phosphate-based glasses have previously been examined for tissue engineering applications, however they degrade rapidly in solution reducing its pH to below 5. This study presents a series of phosphate-based glass compositions that degrade at a lower rate, allowing the pH to remain close to neutral in cell culture medium. The compositions investigated were P50Ca30Na(15-x)Fe5Si(x) where x = 0, 1, 3 and 5 mol%. The dissolution and effect on pH in distilled water and cell culture medium, and ion release in distilled water were investigated over 7 days and MG63 cell attachment to glass fibres was observed after 24 hrs. Dissolution was much slower in cell culture medium (3% mass loss) compared to distilled water (50% mass loss), due to the large quantity of ions and pH buffer present. After 7 days, in cell culture medium the pH remained between 7 and 8.5, however the pH in distilled water fell to between 4 and 3, with the final pH being lower the greater the SiO2 content. Increasing the SiO2 content of the glass resulted in an increase in dissolution rate whilst the pH was maintained at 7 in cell culture medium. The attachment and spreading of MG63 cells was observed on all compositions. These glass compositions may therefore be suitable for tissue engineering applications.

Biocompatible Materials↗

PubMedDate not supplied

In vitro, in vivo, in silico: computational systems in tissue engineering and regenerative medicine.

The emergence of computational systems in tissue engineering and regenerative medicine is paralleling the rapid rise of new technology. Developments in software and hardware have allowed access to the huge data streams that are now available. The Human Genome Project led the way and opened many avenues for other branches of science and biology with extensive but contained databanks. The availability of vast amounts of data means nothing without the ability to integrate the information into a useful form. Tissue engineering has always been a practical science in which function and utility are key objectives. This review delineates key areas of this rapidly ascending branch of science and illustrates examples central to the successful integration of computational methods.

Animals↗

PubMed1997

Tissue engineering of cartilage in space.

Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth. Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10(-4)-10(-6) g) or Earth (1 g). This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue. Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior. The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.

Animals↗

PubMed2001

Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts.

Arterial vascular cells have been successfully utilized for tissue engineering in human cardiovascular structures, such as heart valves. The present study evaluates saphenous vein-derived myofibroblasts as an alternative, easy-to-access cell source for human cardiovascular tissue engineering. Biodegradable polyurethane scaffolds were seeded with human vascular myofibroblasts. Group A consisted of scaffolds seeded with cells from ascending aortic tissue; in group B, saphenous vein-derived cells were used. Analysis included histology, electron microscopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content was comparable in both groups. Collagen and stress at maximum load was significantly higher in group B. Morphology showed viable, layered cellular tissue in all samples, with collagen fibrils most pronounced in group B. In conclusion, saphenous vein myofibroblasts cultured on biodegradable scaffolds showed excellent in vitro tissue generation. Collagen formation and mechanical properties were superior to aortic tissue derived constructs. Therefore, the easy-to-access vein cells represent a promising alternative cell source for cardiovascular tissue engineering.

Aorta↗

PubMed2004

Umbilical cord blood derived endothelial progenitor cells for tissue engineering of vascular grafts.

BACKGROUND: A substantial limitation regarding present pediatric cardiac surgery is the lack of appropriate materials for the repair of congenital defects. To address this shortcoming, tissue engineering is a scientific field that aims at in vitro fabrication of living autologous grafts with the capacity of growth, repair, and regeneration. Here we focused on tissue engineered vascular grafts using human umbilical cord blood derived endothelial progenitor cells (EPCs), as a noninvasive cell source for pediatric applications. METHODS: EPCs were isolated from 20 ml fresh human umbilical cord blood by Ficoll gradient centrifugation and cultured in endothelial basal medium containing growth factors. After proliferation and differentiation cells were analyzed by immunohistochemistry and seeded onto three-dimensional (3D) biodegradable vascular scaffolds (porosity > 95%, n = 22). Twenty-four hours after seeding the vascular grafts were positioned into a pulse-duplicator-in vitro system and grown for 48 hours under biomimetic conditions. A second group was grown 6 days statically and an additional 6 days biomimetically. Controls were cultured statically. Analysis of the grafts included immunohistochemistry, histology, and scanning electron microscopy. RESULTS: Preseeding differentiated EPCs indicated constant endothelial phenotypes including acetylated low-density lipoprotein, cluster of differentiation 31, von Willebrand factor, and endothelial nitric oxide synthetase. Seeded EPCs established favorable cell-to-polymer attachment and proliferation into the 3D tubular scaffolds. Both conditioned and static cellular constructs demonstrated positive staining for cluster of differentiation 31, von Willebrand factor, and expression of endothelial nitric oxide synthase. CONCLUSIONS: Human umbilical cord derived EPCs indicated exceptional growth characteristics used for tissue engineering of vascular grafts. These cells demonstrated a constant endothelial phenotype and related functional features. Based on these results EPCs seem to be a promising autologous cell source with regard to cardiovascular tissue engineering, particularly for the repair of congenital defects.

Absorbable Implants↗

PubMed2006

Historic and current strategies in bone tissue engineering: do we have a hope in Hench?

Professors Larry Hench and Julia Polak formed the Tissue Engineering and Regenerative Medicine Centre (TERM) at Imperial College London to foster collaborations between biologists and materials scientists. Early work at the center elucidated the biomolecular interactions between primary human osteoblasts and 45S5 Bioglass . As research efforts expanded, the team discovered that the dissolution products of both 45S5 Bioglass and 58S sol-gel bioactive glasses had osteoblastic stimulatory properties. To address the shortage of appropriate cells for bone tissue engineering applications, TERM scientists also demonstrated the differentiation of embryonic stem (ES) cells to osteoblasts when treated with the dissolution products of bioactive glasses. They also found that the soluble factors ascorbic acid, beta -glycerophosphate, and dexamethasone preferentially differentiated ES cells to osteoblasts, and their combination with the dissolution products of bioactive glasses stimulated differentiation even further. Taken together, these results demonstrate the suitability of bioactive glasses as scaffolds for bone tissue engineering as they not only provide an osteoconductive and osteoproductive substrate, but also actively stimulate cells to express appropriate osteoblastic phenotypes. Professor Hench's vision to pioneer regenerative medicine research continues with the aim of developing novel therapeutics to treat musculoskeletal disability.

Animals↗

PubMed2001

Gene therapy and tissue engineering for urologic dysfunction: status and prospects.

This article reviews the recent advances in gene therapy and tissue engineering for urologic dysfunction. Although the number of gene therapy-based clinical trials has increased dramatically in the field of urologic oncology, such trials are still few within the neurourologic field. Recently, new biologic approaches employing growth factors have been utilized to treat various pathological conditions. Among them, transfer of genes such as those encoding growth factors represents a promising way to deliver therapeutic proteins to malfunctioning tissues, which leads to the improvement of organ function. Tissue engineering, which may eventually be combined with gene therapy, also offers the potential to create new functional genitourinary tissue for regeneration and replacement of tissue lost as a consequence of disease. Thus, both tissue engineering and gene therapy may hold promising new solutions in the urologic field.

Animals↗

PubMed2005

Quantitative ultrasound can assess the regeneration process of tissue-engineered cartilage using a complex between adherent bone marrow cells and a three-dimensional scaffold.

Articular cartilage (hyaline cartilage) defects resulting from traumatic injury or degenerative joint disease do not repair themselves spontaneously. Therefore, such defects may require novel regenerative strategies to restore biologically and biomechanically functional tissue. Recently, tissue engineering using a complex of cells and scaffold has emerged as a new approach for repairing cartilage defects and restoring cartilage function. With the advent of this new technology, accurate methods for evaluating articular cartilage have become important. In particular, in vivo evaluation is essential for determining the best treatment. However, without a biopsy, which causes damage, articular cartilage cannot be accurately evaluated in a clinical context. We have developed a novel system for evaluating articular cartilage, in which the acoustic properties of the cartilage are measured by introducing an ultrasonic probe during arthroscopy of the knee joint. The purpose of the current study was to determine the efficacy of this ultrasound system for evaluating tissue-engineered cartilage in an experimental model involving implantation of a cell/scaffold complex into rabbit knee joint defects. Ultrasonic echoes from the articular cartilage were converted into a wavelet map by wavelet transformation. On the wavelet map, the percentage maximum magnitude (the maximum magnitude of the measurement area of the operated knee divided by that of the intact cartilage of the opposite, nonoperated knee; %MM) was used as a quantitative index of cartilage regeneration. Using this index, the tissue-engineered cartilage was examined to elucidate the relations between ultrasonic analysis and biochemical and histological analyses. The %MM increased over the time course of the implant and all the hyaline-like cartilage samples from the histological findings had a high %MM. Correlations were observed between the %MM and the semiquantitative histologic grading scale scores from the histological findings. In the biochemical findings, the chondroitin sulfate content increased over the time course of the implant, whereas the hydroxyproline content remained constant. The chondroitin sulfate content showed a similarity to the results of the %MM values. Ultrasonic measurements were found to predict the regeneration process of the tissue-engineered cartilage as a minimally invasive method. Therefore, ultrasonic evaluation using a wavelet map can support the evaluation of tissue-engineered cartilage using cell/scaffold complexes.

Animals↗

PubMed2006

Microscale technologies for tissue engineering and biology.

Microscale technologies are emerging as powerful tools for tissue engineering and biological studies. In this review, we present an overview of these technologies in various tissue engineering applications, such as for fabricating 3D microfabricated scaffolds, as templates for cell aggregate formation, or for fabricating materials in a spatially regulated manner. In addition, we give examples of the use of microscale technologies for controlling the cellular microenvironment in vitro and for performing high-throughput assays. The use of microfluidics, surface patterning, and patterned cocultures in regulating various aspects of cellular microenvironment is discussed, as well as the application of these technologies in directing cell fate and elucidating the underlying biology. Throughout this review, we will use specific examples where available and will provide trends and future directions in the field.

Animals↗

PubMed2004

Expansion of chondrocytes in a three-dimensional matrix for tracheal tissue engineering.

BACKGROUND: The generation of autologous tracheal implants by tissue-engineering techniques is a promising concept for otherwise untreatable patients. A functional cartilaginous backbone represents a prerequisite for any bioartificial tracheal graft. The aim of this study was to define suitable cell types and culture conditions for the generation of tracheal cartilage. METHODS: We obtained tracheal, costal, and auricular cartilage from porcine donor animals (n = 10). The chondrocytes were cultured two-dimensionally in cell flasks or mixed with a liquid collagen solution forming a three-dimensional culture system. Labeling with carboxy fluorescein diacetate succinimidyl ester (CFDA SE) and biochemical reduction of formazan served to determine cell viability and proliferation. The extracellular matrix produced by the chondrocytes was characterized by Western blot. RESULTS: The CFDA SE labeling proved viability and the MTT assays documented a proliferation of the chondrocytes over time in vitro. While the chondrocytes in the three-dimensional cell culture system produced hyaline cartilage composed of collagen II, the two-dimensional culture conditions resulted in nonspecific collagen synthesis. CONCLUSIONS: Chondrocytes grown in a three-dimensional matrix can effectively proliferate and produce cartilage and are viable for more than 2 weeks. Costal chondrocytes are suitable for tracheal cartilage tissue engineering.

Animals↗

PubMed2004

Novel porous aortic elastin and collagen scaffolds for tissue engineering.

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.

Animals↗

PubMed2003

Influence of donor age and culture conditions on tissue engineering of mucosa autografts.

In oral surgery the transplantation of tissue engineered mucosa is used more frequently. The conventional single cell suspension culture method (SCSM) involves murine feeder cells and foetal calf serum. The explant technique (ET) has been used as alternative culture procedure. Aim was to study the efficacy of the ET and the SCSM without feeder cells to grow primary cultures and to test the effects of donor age, of extracellular matrix proteins (ECMP), and of autogenous serum on cell growth in explant cultures. These factors were assessed in cultures of 58 patients overall. In 48 cultures of 12 patients primary cell growth was compared between the ET and the SCSM. Eighteen of 24 cultures were established with the ET whereas only 3 of 24 were established with the SCSM. To test the influence of donor age on cell multiplication, the proliferation rate (DNA synthesis measured by bromodeoxyuridine uptake) and the overall growth (DNA content) was determined in cultures of five young and five old donors. In cultures from old donors (mean age 56 years) proliferation was lower but more sustained relative to the cultures from the young donors (mean age 25 years). In old donors overall in vitro cell growth was only 2/3 of that in young donors. In cultures of 20 donors the influence on cell adhesion and growth of the ECMP fibronectin and laminin was assessed by planimetry. While ECMP augmented explant adhesion, these substances did not enhance keratinocyte growth significantly. Comparing the influence of autogenous and foetal calf serum on cell growth no differences were observed in all cultures of the six donors. In conclusion, the ET without additional ECMP coating and with autogenous instead of foetal calf serum are now used to culture gingival keratinocytes for tissue engineering mucosa grafts. Consequently xenogenous components are avoided, being a considerable advantage.

Adolescent↗

PubMedDate not supplied

Mastoid obliteration by BMP-2/collagen composites: an experimental study using tissue engineering.

PURPOSE: Several materials have been used in the application of mastoid cavity obliteration during surgery for cholesteatoma; however, nothing has won universal acceptance. Through the advancement of tissue engineering, bone morphogenetic protein-2 (BMP-2)/collagen composites have been elucidated as inducers of heterogenic bone formation. This study was performed to investigate whether these composites are potentially obliteration materials for use in the mastoid cavity by using an animal experimental study. MATERIALS AND METHODS: The composites were implanted in the rat mastoid to investigate whether new bone would be tissue engineered in the mastoid and, if so, whether the newly formed bone was stable. The composites were examined histologically over a 24-week period. RESULTS: The composites implanted in the rat mastoid were able to tissue engineer new bone, and the newly formed bone was stable as assessed histologically, with almost normal bone structure, that was not resorbed during the 24-week period. Adverse immunological reactions were not found during our observation. CONCLUSIONS: Bone that was tissue engineered by the BMP-2/collagen composites was stable as assessed by histological examination and persisted in the rat mastoid. The present study shows that the composites have the potential to become real materials for use in mastoid obliteration.

Animals↗