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PubMed2000

Tissue engineering: confronting the transplantation crisis.

Tissue engineering is the development of biological substitutes and/or the fostering of tissue regeneration/remodelling. It is emerging as a technology which has the potential to confront the crisis in transplantation caused by the shortage of donor tissues and organs. With the development of this technology, ther is emerging a new industry which is at the interface of biotechnology and the traditional medical implant field. For this technology and the associated industry to realize their full potential, there are core, enabling technologies that need to be developed. This is the focus of the Georgia Tech/Emory Center for the Engineering of Living Tissues, newly established in the United States, with an Engineering Research Center Award from the National Science Foundation. With the development of these core technologies, tissue engineering will evolve from an art form to a technology based on science and engineering.

Biomedical Engineering↗

PubMed2003

Bone formation on two-dimensional poly(DL-lactide-co-glycolide) (PLGA) films and three-dimensional PLGA tissue engineering scaffolds in vitro.

For some bone tissue engineering strategies, direct contact of newly synthesized bone with a scaffold is important for structural continuity and stability at the scaffold/bone interface. Thus, as the polymer degrades, the support function of the scaffold could be adopted by the developing bone structure. This study was designed to determine whether poly(DL-lactide-co-glycolide) with a comonomer ratio of 75:25 supports bone apposition in vitro. Osteogenic cells derived from rat bone marrow cells were cultured for 2 weeks on polymeric two-dimensional films and three-dimensional tissue engineering scaffolds. Bacteriological grade polystyrene and tissue culture polystyrene dishes served as negative and positive controls for interfacial bone deposition, respectively. The surfaces of the prepared substrates were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle, scanning electron microscopy, and atomic force microscopy. After cell culture, the elaborated matrix was examined using scanning electron microscopy, transmission electron microscopy, and energy dispersive spectroscopy. The results show that poly(DL-lactide-co-glycolide) supports appositional bone growth on both two-dimensional films and three-dimensional scaffolds, including the formation of a mineralized cement line matrix. Furthermore, surface topographical features are not required for the adherence of the cement line matrix to the polymer.

Animals↗

PubMed1996

[Tissue engineering of human cartilage tissue for reconstructive surgery using biocompatible resorbable fibrin gel and polymer carriers].

Current practical approaches in cartilage engineering still face problems with three dimensional cell distribution or require components for cell immobilization, raising biocompatibility problems. In this study, we present a new model using cells cross-linked by fibrin within biocompatible resorbable polymers. Both components have been in clinical use for a long time. Immunohistochemical procedures showed that this model provides optimal requirements for in vitro cartilage production. Immunochemically, cartilage-specific extracellular components such as proteoglycan, chondroitin sulfate and collagen II were characterized. Histomorphological methods showed a mechanically stable tissue compound that lasted for at least 5 weeks. This model may be the first to provide all biocompatible requirements for in vitro production of autologous cartilage transplants for reconstructive surgery.

Biocompatible Materials↗

PubMed2003

Cryopreservation of tissue engineered constructs for bone.

The large-scale clinical use of tissue engineered constructs will require provisions for its mass availability and accessibility. Therefore, it is imperative to understand the effects of low temperature (-196 degrees C) on the tissue engineered biological system. Initial studies used samples of the osteoblast-like cell line (SaOS-2) adhered to a two-dimensional poly(lactide-co-glycolide) thin film (2D-PLAGA) or a three-dimensional poly(lactide-co-glycolide) sintered microsphere matrix (3D-PLAGA) designed for bone tissue engineering. Experimental samples were tested for their ability to maintain cell viability, following low temperature banking for one week, in solutions of the penetrating cryoprotective agents, dimethylsulfoxide (DMSO), ethylene glycol, and glycerol. Results indicated the DMSO solution yielded the greatest percent cell survival for SaOS-2 cells adhered to both the 2D- and 3D-PLAGA scaffolds; therefore, DMSO was used to cryopreserve mineralizing primary rabbit osteoblasts cells adhered to 2D-PLAGA matrices for 35 days. Results indicated retention of the extracellular matrix architecture as no statistically significant difference in the pre- and post-thaw mineralized structures was measured. Percent cell viability of the mineralized constructs following low temperature storage was approximately 50%. These are the first studies to address the issue of preservation techniques for tissue engineered constructs. The ability to successfully cryopreserve mineralized tissue engineered matrices for bone may offer an unlimited and readily available source of bone-like materials for orthopaedic applications.

Bone Substitutes↗

PubMed2005

Tissue engineering.

PURPOSE OF REVIEW: Regenerative medicine holds promise for the restoration of tissues and organs damaged by wear, trauma, neoplasm, or congenital deformity. Tissue engineering combines the disciplines of cell biology and biomedical engineering to effect the design and maturation of various tissues. Despite progress in some areas of tissue regeneration, there has not been significant translation to clinical practice. This article reviews the present understanding of and advances in regenerative medicine, as well as describing limitations in current techniques and areas that need further development. A discussion of the state of the art in the regeneration of skin, cartilage, bone, adipose tissue, and neural tissue is included. RECENT FINDINGS: Differences between extracorporeal and in-vitro tissue engineering are discussed, as well as tissue engineering principles, including the use of bioactive scaffolds, progenitor cells and stem cells, the need for cellular and tissue patterning, microcirculation development, and the use of external stimuli for differentiation. Much needs to be learned about progenitor cell biology, cell-cell interactions, cellular interactions with the extracellular matrix, and about the cues needed for differentiation of functional tissues. SUMMARY: The current limitations in regenerative medicine techniques and the gaps in current knowledge of cellular biology and tissue development represent significant research opportunities in tissue engineering.

Biomedical Engineering↗

PubMed2003

Acellularization of human placenta with preservation of the basement membrane: a potential matrix for tissue engineering.

Basement membrane directs cell migration, cell adherence, and tissue organization in vivo. These qualities would be advantageous in tissue engineering, during which repopulation of scaffolds by functional cells is a common problem. The purpose of this study was to expose human term placenta to a chemical acellularization protocol using the isolated cotyledon perfusion technique to develop an acellular scaffold rich in extracellular matrix components for tissue engineering applications. The processed blocks of tissue demonstrated no cellular material on histologic examination. Periodic acid-Schiff staining, type IV collagen labeling, and transmission electron microscopy demonstrated preservation of the extracellular matrix and basement membrane. Maintenance of the extracellular matrix architecture was seen on scanning electron microscopy. Placenta-derived acellular matrix is a potential volume scaffold for tissue engineering. It is rich in extracellular matrix, has a complex architecture of vascular channels, and has unique donor opportunities.

Basement Membrane↗

PubMed2000

Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits: in vivo restoration of valve tissue.

BACKGROUND: Tissue engineering using in vitro-cultivated autologous vascular wall cells is a new approach to biological heart valve replacement. In the present study, we analyzed a new concept to process allogenic acellular matrix scaffolds of pulmonary heart valves after in vitro seeding with the use of autologous cells in a sheep model. METHODS AND RESULTS: Allogenic heart valve conduits were acellularized by a 48-hour trypsin/EDTA incubation to extract endothelial cells and myofibroblasts. The acellularization procedure resulted in an almost complete removal of cells. After that procedure, a static reseeding of the upper surface of the valve was performed sequentially with autologous myofibroblasts for 6 days and endothelial cells for 2 days, resulting in a patchy cellular restitution on the valve surface. The in vivo function was tested in a sheep model of orthotopic pulmonary valve conduit transplantation. Three of 4 unseeded control valves and 5 of 6 tissue-engineered valves showed normal function up to 3 months. Unseeded allogenic acellular control valves showed partial degeneration (2 of 4 valves) and no interstitial valve tissue reconstitution. Tissue-engineered valves showed complete histological restitution of valve tissue and confluent endothelial surface coverage in all cases. Immunohistological analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor), myofibroblasts (alpha-actin), and matrix synthesis (procollagen I). There were histological signs of inflammatory reactions to subvalvar muscle leading to calcifications, but these were not found in valve and pulmonary artery tissue. CONCLUSIONS: The in vitro tissue-engineering approach using acellular matrix conduits leads to the in vivo reconstitution of viable heart valve tissue.

Animals↗

PubMed2004

Cells, scaffolds, and molecules for myocardial tissue engineering.

Unlike heart valves or blood vessels, heart muscle has no replacement alternatives. The most challenging goal in the field of cardiovascular tissue engineering is the creation/ regeneration of an engineered heart muscle. Recent advances in methods of stem cell isolation, culture in bioreactors, and the synthesis of bioactive materials promise to create engineered cardiac tissue ex vivo. At the same time, new approaches are conceived that explore ways to induce tissue regeneration after injury. The purpose of our review is to describe the principles, status, and challenges of myocardial tissue engineering with emphasize on the concept of in situ cardiac tissue engineering and regeneration.

Biocompatible Materials↗

PubMed2005

Optical transillumination tomography for imaging of tissue-engineered blood vessels.

Recent progress in tissue engineering led to the development of completely biological human vessels grown from the patient's own cells. Those tissue-engineered blood vessels (TEBV) are grown on an individual basis at high costs per item, and therefore require close growth monitoring and quality control. We designed and tested an optical transillumination tomography system using red laser light to image weakly scattering specimens, such as TEBV. Fixated TEBV were imaged and the results compared to optical coherence tomography. This preliminary scanner prototype had an in-plane resolution of 50 microm and allowedto see small inhomogeneities and defects in the samples. Tissue attenuation was found to be 70 cm(-1). Main advantages of the transillumination tomography scanner over optical coherence tomography were the inexpensive instrumentation and the potential to rapidly acquire complete 3D sections with a CCD camera. The prototype presented in this study provides a basis to further improve image quality and acquisition speed.

Animals↗

PubMed2006

An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct.

A major obstacle to 3-dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the model's vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.20+/-3.14% compared with 3.59+/-2.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.

Animals↗

PubMed1998

Creation of viable pulmonary artery autografts through tissue engineering.

BACKGROUND: "Repair" of many congenital cardiac defects requires the use of conduits to establish right ventricle to pulmonary artery continuity. At present, available homografts or prosthetic conduits lack growth potential and can become obstructed by tissue ingrowth or calcification leading to the need for multiple conduit replacements. Tissue engineering is an approach by which cells are grown in vitro onto biodegradable polymers to construct "tissues" for implantation. A tissue engineering approach has recently been used to construct living cardiac valve leaflets from autologous cells in our laboratory. This study assesses the feasibility of a tissue engineering approach to constructing tissue-engineered "living" pulmonary artery conduits. MATERIALS AND METHODS: Ovine artery (group A, n = 4) or vein (group V, n = 3) segments were harvested, separated into individual cells, expanded in tissue culture, and seeded onto synthetic biodegradable (polyglactin/polyglycolic acid) tubular scaffolds (20 mm long x 15 mm diameter). After 7 days of in vitro culture, the autologous cell/polymer vascular constructs were used to replace a 2 cm segment of pulmonary artery in lambs (age 68.4 +/- 15.5 days, weight 18.7 +/- 2.0 kg). One other control animal received an acellular polymer tube sealed with fibrin glue without autologous cells. Animals were sacrificed at intervals of 11 to 24 weeks (mean follow-up 130.3 +/- 30.8 days, mean weight 38.9 +/- 13.0 kg) after echocardiographic and angiographic studies. Explanted tissue-engineered conduits were assayed for collagen (4-hydroxyproline) and calcium content, and a tissue deoxyribonucleic acid assay (bis-benzimide dye) was used to estimate number of cell nuclei as an index of tissue maturity. RESULTS: The acellular control graft developed progressive obstruction and thrombosis. All seven tissue-engineered grafts were patent and demonstrated a nonaneurysmal increase in diameter (group A = 18.3 +/- 1.3 mm = 95.3% of native pulmonary artery; group V = 17.1 +/- 1.2 mm = 86.8% of native pulmonary artery). Histologically, none of the biodegradable polymer scaffold remained in any tissue-engineered graft by 11 weeks. Collagen content in tissue-engineered grafts was 73.9% +/- 8.0% of adjacent native pulmonary artery. Histologically, elastic fibers were present in the media layer of tissue-engineered vessel wall and endothelial specific factor VIII was identified on the luminal surface. Deoxyribonucleic acid assay showed a progressive decrease in numbers of cell nuclei over 11 and 24 weeks, suggesting an ongoing tissue remodeling. Calcium content of tissue-engineered grafts was elevated (group A = 7.95 +/- 5.09; group V = 13.2 +/- 5.48; native pulmonary artery = 1.2 +/- 0.8 mg/gm dry weight), but no macroscopic calcification was found. CONCLUSIONS: Living vascular grafts engineered from autologous cells and biodegradable polymers functioned well in the pulmonary circulation as a pulmonary artery replacement. They demonstrated an increase in diameter suggesting growth and development of endothelial lining and extracellular matrix, including collagen and elastic fibers. This tissue-engineering approach may ultimately allow the development of viable autologous vascular grafts for clinical use.

Animals↗

PubMed2006

Nano-featured scaffolds for tissue engineering: a review of spinning methodologies.

Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising new approach in the restoration and reconstruction of imperfect tissues. In this approach, scaffolds play a pivotal role in supporting the cells to accommodate and guide their growth into a specific tissue; therefore, designing scaffolds that are favorable to cellular growth is of great importance. Electrospinning is a straightforward, cost-effective, and versatile technique that has been applied recently for the fabrication of nano-featured scaffolds suitable for tissue engineering. By mimicking a natural extracellular matrix, it offers many advantages over conventional scaffold methodologies. This paper reviews the current state of art of designing nanostructure scaffolds by using the electrospinning technique. Furthermore, an overview of this technique and its spinning mechanism is described, with special attention to areas of interest to the readers.

Animals↗

PubMed2003

Bone tissue engineering by primary osteoblast-like cells in a monolayer system and 3-dimensional collagen gel.

PURPOSE: To engineer living bone tissue in vitro, bone cells must be multiplied and differentiated in cell culture. Osteoblasts are known to be the crucial cells responsible for the bone modeling process. Periosteal-derived osteoblasts were therefore cultured for up to 3 weeks in Petri dishes as well as in a 3-dimensional collagen gel. METHODS: Proliferation, migration, and differentiation of cells as well as the synthesis of extracellular matrix proteins were monitored during the culture period by histology, electron microscopy, and immunohistochemistry. Mineral formation was investigated by electron diffraction studies and element analysis. RESULTS: Osteoblasts proliferated and migrated in Petri dishes as well as in the collagen gel without loss of viability during the whole experimental period. They demonstrated a mature osteoblast phenotype as indicated by the synthesis of a bone-like extracellular matrix. They formed an extracellular matrix containing osteocalcin, osteonectin, and newly synthesized collagen type I in both environments. Mineral formation was seen in colocalization with the bone-like extracellular matrix proteins in Petri dishes. Microanalytical investigations revealed a matrix vesicle-mediated mineral formation at early stages of culture. CONCLUSIONS: Our cell culture confirmed the ability to multiplicate differentiated and viable osteoblast-like cells in 2- and 3-dimensional space. Additionally, bone-like mineralization can be induced by primary osteoblasts in monolayer culture. The data suggest that this approach can be used as a tool in bone tissue engineering.

Animals↗

PubMed2003

Gene therapy and tissue engineering for sports medicine.

Sports injuries usually involve tissues that display a limited capacity for healing. The treatment of sports injuries has improved over the past 10 to 20 years through sophisticated rehabilitation programs, novel operative techniques, and advances in the field of biomechanical research. Despite this considerable progress, no optimal solution has been found for treatment of various sports-related injuries, including muscle injuries, ligament and tendon ruptures, central meniscal tears, cartilage lesions, and delayed bone fracture healing. New biological approaches focus on the treatment of these injuries with growth factors to stimulate and hasten the healing process. Gene therapy using the transfer of defined genes encoding therapeutic proteins represents a promising way to efficiently deliver suitable growth factors into the injured tissue. Tissue engineering, which may eventually be combined with gene therapy, may potentially result in the creation of tissues or scaffolds for regeneration of tissue defects following trauma. In this article we will discuss why gene therapy and tissue engineering are becoming increasingly important in modern orthopaedic sports medicine practice. We then will review recent research achievements in the area of gene therapy and tissue engineering for sports-related injuries, and highlight the potential clinical applications of this technology in the treatment of patients with musculoskeletal problems following sports-related injuries.

Athletic Injuries↗

PubMedDate not supplied

In situ crosslinkable hyaluronan hydrogels for tissue engineering.

We describe the development of an injectable, cell-containing hydrogel that supports cell proliferation and growth to permit in vivo engineering of new tissues. Two thiolated hyaluronan (HA) derivatives were coupled to four alpha,beta-unsaturated ester and amide derivatives of poly(ethylene glycol) (PEG) 3400. The relative chemical reactivity with cysteine decreased in the order PEG-diacrylate (PEGDA)>>PEG-dimethacrylate>PEG-diacrylamide>PEG-dimethacrylamide. The 3-thiopropanoyl hydrazide derivative (HA-DTPH) was more reactive than the 4-thiobutanoyl hydrazide, HA-DTBH. The crosslinking of HA-DTPH with PEGDA in a molar ratio of 2:1 occurred in approximately 9 min, suitable for an in situ crosslinking applications. The in vitro cytocompatibility and in vivo biocompatibility were evaluated using T31 human tracheal scar fibroblasts, which were suspended in medium in HA-DTPH prior to addition of the PEGDA solution. The majority of cells survived crosslinking and the cell density increased tenfold during the 4-week culture period in vitro. Cell-loaded hydrogels were also implanted subcutaneously in the flanks of nude mice, and after immunohistochemistry showed that the encapsulated cells retained the fibroblast phenotype and secreted extracellular matrix in vivo. These results confirm the potential utility of the HA-DTPH-PEGDA hydrogel as an in situ crosslinkable, injectable material for tissue engineering.

Animals↗

PubMed2002

Proliferating cells versus differentiated cells in tissue engineering.

The efficiency of cell or tissue cultures is usually judged by how quickly confluence is reached within a Petri dish or on a scaffold. Growth factors and fetal bovine serum are employed to drive cultured cells from one mitosis to the next as quickly as possible. The tissue specific interphase is extremely short under these conditions, so that the degree of differentiation desired in tissue engineering cannot be achieved. To reach the goal of functional differentiation in vitro mitosis and interphase must be separated experimentally and tailored to the specific requirements of the cell-type used. This could be achieved by a three step concept for tissue-engineering in vitro as we present here. The expansion phase is followed by a phase in which tissue differentiation is initiated. The final phase serves to express and maintain histotypical differentiation of the generated tissue.

Animals↗

PubMed1999

Future perspectives in reconstructive surgery using tissue engineering.

Whenever there is a lack of native urologic tissue, reconstruction usually is performed with native nonurologic tissues, such as gastrointestinal segments, skin, or mucosa from multiple body sites. The use of native nonurologic tissues in the genitourinary tract is associated with adverse effects. Tissue engineering efforts currently are underway for almost every type of tissue and organ within the urinary system including bladder, ureter, urethra, and genitalia. Most of the efforts expended to engineer genitourinary tissues have occurred within the last decade. Tissue engineering techniques require a cell culture facility designed for human application. Personnel who have mastered the techniques of cell harvest, culture and expansion, and polymer design are essential for the successful application of this technology. The first human application of cell-based tissue engineering technology for urologic applications recently occurred with the injection of autologous cells for the correction of vesicoureteral reflux in children and urinary incontinence in adults. Trials involving bladder replacement using tissue engineering techniques currently are being arranged. Recent progress suggests that engineered urologic tissues may have clinical applicability.

Adult↗

PubMed2003

Tissue-engineered esophagus: experimental substitution by onlay patch or interposition.

OBJECTIVES: We proposed to fabricate a tissue-engineered esophagus and to use it for replacement of the abdominal esophagus. METHODS: Esophagus organoid units, mesenchymal cores surrounded by epithelial cells, were isolated from neonatal or adult rats and paratopically transplanted on biodegradable polymer tubes, which were implanted in syngeneic hosts. Four weeks later, the tissue-engineered esophagus was either harvested or anastomosed as an onlay patch or total interposition graft. Green Fluorescent Protein labeling by means of viral infection of the organoid units was performed before implantation. Histology and immunohistochemical detection of the antigen alpha-actin smooth muscle were performed. RESULTS: Tissue-engineered esophagus grows in sufficient quantity for interposition grafting. Histology reveals a complete esophageal wall, including mucosa, submucosa, and muscularis propria, which was confirmed by means of immunohistochemical staining for alpha-actin smooth muscle. Tissue-engineered esophagus architecture was maintained after interposition or use as a patch, and animals gained weight on a normal diet. Green Fluorescent Protein-labeled tissue-engineered esophagus preserved its fluorescent label, proving the donor origin of the tissue-engineered esophagus. CONCLUSIONS: Tissue-engineered esophagus resembles the native esophagus and maintains normal histology in anastomosis, with implications for therapy of long-segment esophageal tissue loss caused by congenital absence, surgical excision, or trauma.

Actins↗