Functional Tissue Replacements Research Articles

Regenerative Medicine: Applications and Development in Urology

Purpose Congenital abnormalities and acquired disorders can lead to organ damage and loss. Nowadays, transplantation represents the only effective treatment option. However, there is a marked decrease in the number of organ donors, which is even yearly worsening due to the population aging. The regenerative medicine represents a realistic option that allows to restore and maintain the normal functions of tissues and organs. This article reviews the principles of regenerative medicine and the recent advances with regard to its application to the genitourinary tract. Recent findings The field of regenerative medicine involves different areas of technology, such as tissue engineering, stem cells and cloning. Tissue engineering involves the field of cell transplantation, materials science and engineering in order to create functional replacement tissues. Stem cells and cloning permit the extraction of pluripotent, embryonic stem cells offering a potentially limitless source of cells for tissue engineering applications. Most current strategies for tissue engineering depend upon a sample of autologous cells from the patient's diseased organ. Biopsies from patients with extensive end-stage organ failure, however, may not yield enough normal cells. In these situations, stem cells are envisaged as being an alternative source. Stem cells can be derived from discarded human embryos (human embryonic stem cells), from fetal tissue or from adult sources (bone marrow, fat, skin). Therapeutic cloning offers a potentially limitless source of cells for tissue engineering applications. Regenerative medicine and tissue engineering scientists have increasingly applied the principles of cell transplantation, materials science and bioengineering to construct biological substitutes that will restore and maintain normal function in urological diseased and injured tissues such as kidney, ureter, bladder, urethra and penis. Conclusions Regenerative medicine offers several applications in acquired and congenital genitourinary diseases. Tissue engineering, stem cells and, mostly, cloning have been applied in experimental studies with excellent results. Few preliminary human applications have been developed with promising results.

In Vivo Chondrogenesis of Mesenchymal Stem Cells in a Photopolymerized Hydrogel

Surgical options for cartilage reconstruction can be significantly improved through advances in cartilage tissue engineering, whereby functional tissue replacements are created by growing cells on polymer scaffolds. The objective of this study was to use a photopolymerizable hydrogel to implant bone marrow-derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote cartilage tissue formation by the cells in vivo. Athymic nude mice were injected subcutaneously with polymer solutions of poly(ethylene) oxide diacrylate containing mesenchymal stem cells and placed under a UVA lamp to transdermally photopolymerize (solidify) the injected liquid. Experimental groups included polymer solutions with hyaluronic acid (HA), transforming growth factor (TGF)-beta3, or both. After 3 weeks of implantation, cartilage formation was evaluated by gene expression analysis and histologic techniques. Hyaluronic acid increased the viscosity of the polymer solutions, which helped maintain the injections at the desired site during photopolymerization. Mesenchymal stem cells in hydrogels containing both HA and TGF-beta3 produced the highest quality cartilage, based on expression of the cartilage-specific genes and production of proteoglycan and collagen II. When used independently, TGF-beta3 and HA alone induced cartilage-specific gene expression and collagen type II production; however, TGF-beta3 was essential for proteoglycan production. HA enhanced proteoglycan production when combined with TGF-beta3 and reduced expression and production of collagen I. This study is the first to demonstrate the minimally invasive implantation and subsequent chondrogenic differentiation of mesenchymal stem cells in the subcutaneous environment. This lays the foundation for further optimization of a novel and practical technology for cartilage reconstruction.

Regenerative medicine: applications and development in urology

PURPOSE. Congenital abnormalities and acquired disorders can lead to organ damage and loss. Nowadays, transplantation represents the only effective treatment option. However, there is a marked decrease in the number of organ donors, which is even yearly worsening due to the population aging. The regenerative medicine represents a realistic option that allows to restore and maintain the normal functions of tissues and organs. This article reviews the principles of regenerative medicine and the recent advances with regard to its application to the genitourinary tract. RECENT FINDINGS. The field of regenerative medicine involves different areas of technology, such as tissue engineering, stem cells and cloning. Tissue engineering involves the field of cell transplantation, materials science and engineering in order to create functional replacement tissues. Stem cells and cloning permit the extraction of pluripotent, embryonic stem cells offering a potentially limitless source of cells for tissue engineering applications. Most current strategies for tissue engineering depend upon a sample of autologous cells from the patient's diseased organ. Biopsies from patients with extensive end-stage organ failure, however, may not yield enough normal cells. In these situations, stem cells are envisaged as being an alternative source. Stem cells can be derived from discarded human embryos (human embryonic stem cells), from fetal tissue or from adult sources (bone marrow, fat, skin). Therapeutic cloning offers a potentially limitless source of cells for tissue engineering applications. Regenerative medicine and tissue engineering scientists have increasingly applied the principles of cell transplantation, materials science and bioengineering to construct biological substitutes that will restore and maintain normal function in urological diseased and injured tissues such as kidney, ureter, bladder, urethra and penis. CONCLUSIONS. Regenerative medicine offers several applications in acquired and congenital genito-urinary diseases. Tissue engineering, stem cells and, mostly, cloning have been applied in experimental studies with excellent results. Few preliminary human applications have been developed with promising results.

Gastrointestinal tissue engineering

Tissue engineering is an emerging discipline that combines engineering principles and the biological sciences toward the development of functional replacement tissue. Virtually every tissue in the body has been investigated and tremendous advances have been made in many areas. This article focuses on the gastrointestinal tract and reviews the current status of bioengineering gastrointestinal tissues, including the esophagus, stomach, small intestine and colon. Although progress has been achieved, there continues to be significant challenges that need to be addressed.

Scaffolds for stem cells

Today, most people know at least something about stem cells. Embryonic stem cells enjoy regular mentions in news programs and magazines, probably because of the controversial way in which they are generated but also because of their huge potential in medicine. Here we distinguish between and define types of stem cells, discuss techniques used so far to create various cells and tissues from stem cells, and discuss how three-dimensional supports and stem cells have been and should be used to encourage the development of functional replacement tissue.

Review: Tissue Engineering of the Urinary Bladder: Considering Structure-Function Relationships and the Role of Mechanotransduction

A variety of conditions encountered in urology result in bladder dysfunction and the need for bioengineered tissue substitutes. Traditionally, a number of synthetic materials and natural matrices have been used in experimental and clinical settings. However, the production of functional bladder tissue replacements remains elusive. The urinary bladder sustains considerable structural deformation during its normal function and represents an ideal model tissue in which to study the effects of biomechanical simulation on tissue morphogenesis, differentiation, and function. However, the actual role of mechanical forces within the bladder has received little attention. A strategy in which in vitro-generated tissue constructs are conditioned by exposure to the same mechanical forces as they would encounter in vivo could potentially be used both in the development of functional tissue replacements and to further study the role of biomechanical signalling. The purpose of this review is to examine the role and structure-function relationship of the urinary bladder and, through consultation of the literature available on mechanotransduction and tissue engineering of alternative tissues, to determine the factors that need to be considered when biomechanically engineering a functional bladder.

Biomaterials as Scaffold for Bone Tissue Engineering

Almost 20 years after the invention of tissue engineering, autogenous bone grafting has remained the favored strategy for the treatment of bone defects. As an alternative, a vast variety of bone substitutes has been developed and is available for clinical use. The ongoing search for bone substitutes, however, reflects the limitations imposed to both autogenous and allogenous bone grafts as well as to bone substitute materials. The concept of tissue engineering holds great promise for the future treatment of osseous defects. Research in this interdisciplinary field is carried out to find a way of producing biologic substitutes as functional tissue replacement. For this, functionally active cells are applied on supporting scaffolds under controlled stimulation with growth factors. Scaffolds are temporary matrices for bone growth and provide a specific environment and architecture for tissue development. Ideally, scaffolds favor cellular attachment, growth and differentiation in vitro and in vivo. Especially ceramics and biodegradable polymers are widely used and have been tested in various animal studies. Yet, to allow for precise production of specific custom-made scaffolds, rapid prototyping (RP) techniques have recently drawn a lot of attention. Using these methods scaffolds with a predefined, well-controlled internal and external architecture mimicking the structure of natural bone can be generated. Although biocompatibility of the materials used in the process and the structural resolution that can be technically achieved so far limit the range of use, rapid manufacturing techniques do offer great opportunities to generate suitable scaffolds for bone tissue engineering in the near future.

Computer-Aided Tissue Engineering of a Human Vertebral Body

Tissue engineering is developing into a less speculative science involving the careful interplay of numerous design parameters and multidisciplinary professionals. Problem solving abilities and state of the art research tools are required to develop solutions for a wide variety of clinical issues. One area of particular interest is orthopedic biomechanics, a field that is responsible for the treatment of over 700,000 vertebral fractures in the United States alone last year. Engineers are currently lacking the technology and knowledge required to govern the subsistence of cells in vivo, let alone the knowledge to create a functional tissue replacement for a whole organ. Despite this, advances in computer-aided tissue engineering are continually growing. Using a combinatory approach to scaffold design, patient-specific implants may be constructed. Computer-aided design, optimization of geometry using voxel finite element models or other optimization routines, creation of a library of architectures with specific material properties, rapid prototyping, and determination of a defect site using imaging modalities highlight the current availability of design resources. This study proposes a novel methodology from start to finish which could, in the future, be used to design a tissue-engineered construct for the replacement of an entire vertebral body.

Challenges of Primate Embryonic Stem Cell Research

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.

Applications of tissue engineering in the genitourinary tract

Congenital abnormalities and acquired disorders can lead to organ damage or loss of tissue within the genitourinary tract. For reconstructive purposes, tissue-engineering efforts are currently underway for virtually every type of tissue and organ within the urinary tract. Tissue engineering incorporates the fields of cell transplantation, materials science and engineering for the purpose of creating functional replacement tissue. This article reviews some of the principles of tissue engineering and some of the applications of these principles to the genitourinary tract.

Extracellular matrix (ECM) microstructural composition regulates local cell-ECM biomechanics and fundamental fibroblast behavior: a multidimensional perspective

The extracellular matrix (ECM) provides the principal means by which mechanical information is communicated between tissue and cellular levels of function. These mechanical signals play a central role in controlling cell fate and establishing tissue structure and function. However, little is known regarding the mechanisms by which specific structural and mechanical properties of the ECM influence its interaction with cells, especially within a tissuelike context. This lack of knowledge precludes formulation of biomimetic microenvironments for effective tissue repair and replacement. The present study determined the role of collagen fibril density in regulating local cell-ECM biomechanics and fundamental fibroblast behavior. The model system consisted of fibroblasts seeded within collagen ECMs with controlled microstructure. Confocal microscopy was used to collect multidimensional images of both ECM microstructure and specific cellular characteristics. From these images temporal changes in three-dimensional cell morphology, time- and space-dependent changes in the three-dimensional local strain state of a cell and its ECM, and spatial distribution of beta1-integrin were quantified. Results showed that fibroblasts grown within high-fibril-density ECMs had decreased length-to-height ratios, increased surface areas, and a greater number of projections. Furthermore, fibroblasts within low-fibril-density ECMs reorganized their ECM to a greater extent, and it appeared that beta1-integrin localization was related to local strain and ECM remodeling events. Finally, fibroblast proliferation was enhanced in low-fibril-density ECMs. Collectively, these results are significant because they provide new insight into how specific physical properties of a cell's ECM microenvironment contribute to tissue remodeling events in vivo and to the design and engineering of functional tissue replacements.

First human transplantation of a bioengineered airway tissue

First human transplantation of a bioengineered airway tissue

Mechanical Properties and Cellular Proliferation of Electrospun Collagen Type II

A suitable technique for articular cartilage repair and replacement is necessitated by inadequacies of current methods. Electrospinning has potential in cartilage repair by producing scaffolds with fiber diameters in the range of native extracellular matrix. Chondrocytes seeded onto such scaffolds may prefer this environment for differentiation and proliferation, thus approaching functional cartilage replacement tissue. Scaffolds of collagen type II were created by an electrospinning technique. Individual scaffold specimens were prepared and evaluated as uncross-linked, cross-linked, or crosslinked/seeded. Uncross-linked scaffolds contained a minimum and average fiber diameter of 70 and 496 nm, respectively, whereas cross-linked scaffolds possessed diameters of 140 nm and 1.46 microm. The average thickness for uncross-linked scaffolds was 0.20 +/- 0.02 mm and 0.52 +/- 0.07 mm for cross-linked scaffolds. Uniaxial tensile tests of uncross-linked scaffolds revealed an average tangent modulus, ultimate tensile strength, and ultimate strain of 172.5 +/- 36.1 MPa, 3.3 +/- 0.3 MPa, and 0.026 +/- 0.005 mm/mm, respectively. Scanning electron microscopy of cross-linked scaffolds cultured with chondrocytes demonstrated the ability of the cells to infiltrate the scaffold surface and interior. Electrospun collagen type II scaffolds produce a suitable environment for chondrocyte growth, which potentially establishes the foundation for the development of articular cartilage repair.

Global gene expression of cells attached to a tissue engineering scaffold

A goal of tissue engineering is to produce a scaffold material that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Little is known about how cells respond on a molecular level to tissue engineering scaffold materials. In this work we used oligonucleotide microarrays to interrogate gene expression profiles associated with cell–biomaterial interactions. We seeded collagen–glycosaminoglycan meshes, a widely used tissue engineering scaffold material, with human IMR-90 fibroblasts and compared transcript levels with control cells grown on tissue culture polystyrene. Genes involved in cell signaling, extracellular matrix remodeling, inflammation, angiogenesis and hypoxia were all activated in cells on the collagen–GAG mesh. Understanding the impact of a scaffold on attached cells will facilitate the design of improved tissue engineering materials.

Capsule induction technique in a rat model for bladder wall replacement: an overview

The search for a reliable technique for functional genitourinary tissue replacement remains a challenging task. The most recent advances in cell biology and tissue engineering have utilized various avascular and acellular collagen scaffolds with or without seeded cells. These techniques, however, are frequently complicated by tissue necrosis, contracture and resorption due to limited vascularization. We employed a new three-stage, evolving animal model with stage I optimizing the culture delivery vehicle, stage II employing a seeded vascularized capsule flap, and stage III adding a contractile matrix in the form of pedicled gracilis muscle prelaminated with autologous, in vitro-expanded urothelial cells to reconstruct an entire supratrigonal bladder-wall defect in rats. Specimens stained with hematoxylin and eosin (H&E), α 1-actin staining, and a specific immunohistochemical staining (AE 1&AE 3-anticytoceratin monoclonal antibody stain) showed a continuous, multilayered, functioning urothelial lining along the transposed prelaminated gracilis flap in the animals of the final-stage experiment. Successful urinary reconstruction requires a contractile neoreservoir resistant to resorption over time and a stable, protective urothelial lining. We demonstrated that a gracilis muscle flap can be seeded with autologous cultured urothelial cells suspended in fibrin glue. This prelaminated flap can be safely transposed onto its pedicle and become successfully integrated into the remaining bladder wall, demonstrating urothelial lining and the potential to contract. Further studies in larger animals with urodynamic assessment is warranted to determine if this type of bladder-wall replacement technique is suitable for urinary reconstruction in humans.

Anatomically shaped osteochondral constructs for articular cartilage repair

Few successful treatment modalities exist for surface-wide, full-thickness lesions of articular cartilage. Functional tissue engineering offers a great potential for the clinical management of such lesions. Our long-term hypothesis is that anatomically shaped tissue constructs of entire articular layers can be engineered in vitro on a bony substrate, for subsequent implantation. To determine the feasibility, this study investigated the development of bilayered scaffolds of chondrocyte-seeded agarose on natural trabecular bone. In a series of three experiments, bovine chondrocytes were seeded in (1) cylindrical bilayered constructs of agarose and bovine trabecular bone, 0.53 cm 2 in surface area and 3.2 mm thick, and were cultured for up to 6 weeks; (2) chondrocyte-seeded anatomically shaped agarose constructs reproducing the human patellar articular layer (area=11.7 cm 2, mean thickness=3.4 mm), cultured for up to 6 weeks; and (3) chondrocyte-seeded anatomically shaped agarose constructs of the patella (same as above) integrated into a corresponding anatomically shaped trabecular bone substrate, cultured for up to 2 weeks. Articular layer geometry, previously acquired from human cadaver joints, was used in conjunction with computer-aided design and manufacturing technology to create these anatomically accurate molds. In all experiments, chondrocytes remained viable over the entire culture period, with the agarose maintaining its shape while remaining firmly attached to the underlying bony substrate (when present). With culture time, the constructs exhibited positive type II collagen staining as well as increased matrix elaboration (Safranin O staining for glycosaminoglycans) and material properties (Young's modulus and aggregate modulus). Despite the use of relatively large agarose constructs partially integrated with trabecular bone, no adverse diffusion limitation effects were observed. Anatomically shaped constructs on a bony substrate may represent a new paradigm in the design of a functional articular cartilage tissue replacement.

Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs

Most tissue engineering (TE) strategies for creating functional replacement tissues or organs rely on the application of temporary three-dimensional scaffolds to guide the proliferation and spread of seeded cells in vitro and in vivo. The characteristics of TE scaffolds are major concerns in the quest to fabricate ideal scaffolds. This paper identifies essential structural characteristics and the pre-requisites for fabrication techniques that can yield scaffolds that are capable of directing healthy and homogeneous tissue development. Emphasis is given to solid freeform (SFF), also known as rapid prototyping, technologies which are fast becoming the techniques of choice for scaffold fabrication with the potential to overcome the limitations of conventional manual-based fabrication techniques. SFF-fabricated scaffolds have been found to be able to address most, if not all the macro- and micro-architectural requirements for TE applications. This paper reviews the application/potential application of state-of-the-art SFF fabrication techniques in creating TE scaffolds. The advantages and limitations of the SFF techniques are compared. Related research carried out worldwide by different institutions, including the authors’ research are discussed.

Permeability of musculoskeletal tissues and scaffolding materials: experimental results and theoretical predictions.

Fluid movement produces a wide range of phenomena in musculoskeletal tissues, including streaming potentials, hydrodynamic lubrication, nutrient transport, and mechanical signaling, all of which are governed by tissue permeability. Permeability is a measurement of the ease with which a fluid passes through a material and is described by Darcy's law. An appreciation of the flow-related structure-function relationships that have been found for musculoskeletal tissues as well as the materials used to engineer substitutes is important for clinicians and engineers alike. In addition, fluid transport phenomena is one of the most often neglected, but important, aspects of developing functional musculoskeletal tissue replacements. Mathematical models provide the means to relate permeability to microstructure and enable one to span multiple hierarchical length scales. In this article, we have summarized the experimentally determined permeability for a range of musculoskeletal tissues. In addition, we have included a summary of the microstructural models that are available to relate bulk permeability to microstructural flow profiles. These models have the potential to predict cellular level fluid shear stresses, nutrient and drug transport, degradation kinetics, and the fluid-solid interactions that govern mechanical response.

The use of cells in reparative medicine.

Cells are the functional elements of reparative medicine and tissue engineering. The use of living cells as a therapy presents several challenges. These include identification of a suitable source, development of adequate methods, and proof of safety and efficacy. We are now well aware that stem or pluripotent cells offer an exciting potential source for a host of functional cell types. Their true potential will only be realized through continued effort to increase basic scientific understanding at all levels, the development of adequate methods to achieve a functional phenotype, and attention to safety issues associated with adequate control of cell localization, proliferation, and differentiation. There is also new understanding regarding the immunology of parenchymal cells and new promising approaches to immune modulation, which will open the door to broader therapies using allogeneic cell sources without prohibitive immune suppression. Control of cell growth and phenotypic expression does not end in the culture vessel, but goes beyond to the patient. A living therapy is not static but dynamic, as is the host response. The cells or tissue construct in most cases will not behave as a whole-organ transplant. It is therefore important that we understand a cell or tissue therapy's ability to react and interact within the host since clinical effectiveness has proven to be one of the most difficult milestones to achieve. A living cell therapy offers great potential to alter the human condition, encompassing alteration of the current biological state of a targeted tissue or organ, augmentation of depleted or lost function, or absolute functional tissue replacement. The extent to which we are able to achieve effective cell therapies will depend on assimilating a rapidly developing base of scientific knowledge with the practical considerations of design, delivery, and host response.

Reproduction of functional smooth muscle tissue and partial bladder replacement

To find a means of bladder augmentation that would avoid the complications encountered with the use of bowel segments, using a newly developed acellular biomaterial, the bladder acellular matrix graft (BAMG), as a homologous graft. Thirty-four rats underwent a partial cystectomy (40-50%) and grafting with a BAMG of equal size. Eleven rats died within the first 72 h. probably from urinary leakage caused by obstruction of the bladder neck with stones or coagula; the surviving 23 were killed at varying intervals after cystectomy and examined. After providing initial bladder enlargement, the graft was progressively infiltrated by the vessels and smooth muscle cells of the host: furthermore, the mucosal lining was complete within 10 days. After 4 weeks, all bladder wall components were evident histologically in the graft. The ingrowth was complete after 8 weeks, except for neural regeneration, which was only partial. At 12 weeks, the bladder wall muscle structure in the graft was so well developed that it was difficult to delineate the junction between host bladder and BAMG. Neural regeneration continued to improve. Normal bladder capacities were maintained throughout the study. The BAMG appears to serve, without rejection, as a framework of collagen and elastin for the ingrowth of all bladder wall components. The reason for the better acceptance of the BAMG than of other bladder augmentation grafts requires further investigation.