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PubMed2006

[Regenerative medicine in andrology: tissue engineering and gene therapy as potential treatment options for penile deformations and erectile dysfunction].

Tissue engineering and gene therapy are currently investigated in animal studies for reconstructing penile tissue or treating erectile dysfunction. This review aims to ecamine these experimental efforts from the last years and tries to give a brief introduction to the basic methodology of these new techniques from the field of regenerative medicine.

Animals↗

PubMed2004

Moral imagination in tissue engineering research on animal models.

Animal experimentation is an integrated part of tissue engineering research. This paper investigates what scientists conducting such experimentation should reasonably take into consideration from an ethical point of view. It is argued that scientists should use their moral imagination in making fundamental ethical choices, in reflecting on legal regulation, in taking public opinion seriously, and in balancing human benefit and animal harm as expected outcomes of the experiments.

Animal Experimentation↗

PubMed2006

Cartilage tissue engineering for degenerative joint disease.

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

Animals↗

PubMed2005

Histology and ultrastructure of a tissue-engineered collagen meniscus before and after implantation.

The collagen meniscus implant (CMI) is a tissue-engineering technique designed to stimulate regeneration of meniscus-like tissue in cases of irreparable tears or previous meniscectomy. CMI morphology was investigated before and after implantation by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). In a case series biopsy specimens were harvested from four patients who underwent a second arthroscopic look 6 months after placement of the CMI. CMI sections appeared composed of parallel connective laminae of 10-30 microm, connected by smaller bundles (5-10 microm). This connective network formed lacunae with diameters between 40 and 60 microm. At greater magnification, the walls of the lacunae demonstrated tightly packed and randomly distributed collagen fibrils, with diameters ranging from 73 to 439 nm. In the biopsy specimens, the lacunae were filled with connective tissue that contained newly formed vessels and fibroblast-like cells, presenting an abundant rough endoplasmic reticulum and several mitochondria. In the extracellular matrix, the collagen fibrils showed uniform diameters (126 nm +/- 32 nm). The original structure of CMI was still recognizable, and no inflammatory cells were detected within the implant. The morphological findings of this case series demonstrate that CMI provides a three-dimensional scaffold suitable for colonization by precursor cells and vessels and leading to the formation of a fully functional tissue.

Adult↗

PubMed2004

Review article: Tissue engineering of semilunar heart valves: current status and future developments.

Heart valve replacement represents the most common surgical therapy for end-stage valvular heart diseases. One major drawback that all heart valve replacements have in common is the lack of growth, repair, and remodeling capability once implanted into the body. The emerging field of tissue engineering is focusing on the in-vitro generation of functional, living semilunar heart valve replacements. This review presents a state-of-the-art overview of the physiological and biomechanical requirements of semilunar heart valves, focusing on the aortic valve. Moreover, recent heart valve tissue engineering is summarized and future options and improvements on the way towards clinical applications are discussed.

Animals↗

PubMed2001

Tissue-engineered vascular autograft: inferior vena cava replacement in a dog model.

Tissue-engineered vascular autografts (TEVAs) were made by seeding 4-6 x 10(6) of mixed cells obtained from femoral veins of mongrel dogs onto tube-shaped biodegradable polymer scaffolds composed of a polyglycolid acid (PGA) nonwoven fabric sheet and a copolymer of L-lactide and caprolactone (n = 4). After 7 days, the inferior vena cavas (IVCs) of the same dogs were replaced with TEVAs. After 3, 4, 5, and 6 months, angiographies were performed, and the dogs were sacrificed. The implanted TEVAs were examined both grossly and immunohistologically. The implanted TEVAs showed no evidence of stenosis or dilatation. No thrombus was found inside the TEVAs, even without any anticoagulation therapy. Remnants of the polymer scaffolds were not observed in all specimens, and the overall gross appearance similar to that of native IVCs. Immunohistological staining revealed the presence of factor VIII positive nucleated cells at the luminal surface of the TEVAs. In addition, lesions were observed where alpha-smooth muscle actin and desmin positive cells existed. Implanted TEVAs contained a sufficient amount of extracellular matrix, and showed neither occlusion nor aneurysmal formation. In addition, endothelial cells were found to line the luminal surface of each TEVA. These results strongly suggest that "ideal" venous grafts with antithrombogenicity can be produced.

Animals↗

PubMed2006

Experimental study of vascularized tissue-engineered bone grafts.

BACKGROUND: Vascularized bone grafting has become a useful method for treatment of large bone defects and infected nonunions that lack adequate blood supply. This method has disadvantages, however, such as donor-site complications. To overcome these disadvantages, the authors developed a new method for creating vascularized tissue-engineered bone grafts in an experimental model. METHODS: Fisher rat bone marrow mesenchymal stem cells were cultured for 2 weeks in fully opened, interconnected, porous hydroxyapatite ceramic. The composites of mesenchymal stem cells and hydroxyapatite were implanted in the medial calf region together with the saphenous vascular bundle in syngeneic rats. Two weeks after implantation, the vascular pedicle with the implants was exposed and covered with silicone rubber to prevent vascular invasion through surrounding tissues (vascularized mesenchymal stem cell/hydroxyapatite implants). In addition, nonvascularized mesenchymal stem cell/hydroxyapatite implants were created with a ligated vascular pedicle. Implants of vascularized hydroxyapatite alone were prepared as a control. All implants were retrieved 4 weeks after surgery. RESULTS: Histologically, de novo bone formations were observed in the vascularized implants. This was in contrast with only necrotic tissue observed in the nonvascularized implants. Bone formation was not observed entirely in the vascularized hydroxyapatite-alone implants. For biochemical analysis, alkaline phosphatase activity and osteocalcin content of the vascularized mesenchymal stem cell/hydroxyapatite implants were significantly higher than those of the nonvascularized mesenchymal stem cell/hydroxyapatite and vascularized hydroxyapatite-alone implants. CONCLUSION: The results of this study indicated that the vascularized tissue-engineered bone grafts could be transferred as free vascularized grafts to lesions that lack adequate blood supply.

Alkaline Phosphatase↗

PubMed2003

A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells.

This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Ham's F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell-polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudo-stratified columnar epithelium, with a distinct interface between tissue-engineered cartilage and epithelium. Safranin-O staining shows that tissue-engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.

Animals↗

PubMed2005

In vitro characterization of chitosan-gelatin scaffolds for tissue engineering.

Recently, chitosan-gelatin scaffolds have gained much attention in various tissue engineering applications. However, the underlying cell-matrix interactions remain unclear in addition to the scaffold degradation and mechanical characteristics. In this study, we evaluated (i) the degradation kinetics of chitosan and chitosan-gelatin scaffolds in the presence of 10mg/L of lysozyme for dimensional stability, weight loss, and pH changes for a period of 2 months, (ii) tensile and compressive properties of films and scaffolds in wet state at 37 degrees C, (iii) viability of fibroblasts and human umbilical vein endothelial cells (HUVECs) on scaffolds, and (iv) the alteration in cell spreading characteristics, cytoskeletal actin distribution, focal adhesion kinase (FAK) distribution and PECAM-1 expression of HUVECs under static and 4.5, 8.5, 13 and 18 dyn/cm2 shear stress conditions. Degradation results showed that gelatin-containing chitosan scaffolds had faster degradation rate and significant loss of material than chitosan. Mechanical properties of chitosan are affected by the addition of gelatin although there was no clear trend. Three-dimensional chitosan and chitosan-gelatin scaffolds supported fibroblast viability equally. However, chitosan membranes decreased cell-spreading area, disrupted F-actin and localized FAK in the nucleus of HUVECs. Importantly, the lowest shear stress tested (4.5 dyn/cm2) for 3 h washed away cells on chitosan suggesting weak cell adhesion. In the blends, effect of gelatin was dominant; actin and FAK distribution were comparable to gelatin in static culture. However, at higher shear stresses, presence of chitosan inhibited shear-induced increase in cell spreading and weakened cell adhesive strength. No significant differences were observed in PECAM-1 expression. In summary, these results showed significant influence of blending gelatin with chitosan on scaffold properties and cellular behavior.

Actins↗

PubMed2006

Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering.

Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber mesh, although the success rate of tooth production is low. The present study was designed to compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks. After 24h, there were a significantly higher number of cells attached to the collagen sponge scaffold than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively that a collagen sponge scaffold allows tooth production with a higher degree of success than polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.

Alkaline Phosphatase↗

PubMed2001

Donor cell fate in tissue engineering for articular cartilage repair.

Articular cartilage repair is a clinical challenge because of its limited intrinsic healing potential. Considerable research has focused on tissue engineering and transplantation of viable chondrogenic cells to enhance cartilage regeneration. However, the question remains: do transplanted allogenic cells survive in the repair with time? This study assessed donor cell fate after transplantation of male New Zealand White rabbit perichondrium cell and polylactic acid constructs into osteochondral defects created in the medial femoral condyles of female New Zealand White rabbits. Repair tissue was harvested at 0, 1, 2, 3, 7, and 28 days after implantation and was evaluated for cell viability and total cell number using confocal microscopic analysis. The number of donor cells in each sample was estimated using quantitative polymerase chain reaction targeting a gender-specific gene present on the Y-chromosome, the sex-determining region Y gene, and a control deoxyribonucleic acid present in male and female cell deoxyribonucleic acid, the matrix metalloproteinase-1 gene promoter. Average cell viability was found to be 87% or more at all times. Donor cells were present in repair tissue for 28 days after implantation. However, the number of donor cells declined from approximately 1 million at Time 0 to approximately 140,000 at 28 days. This decline in donor cells was accompanied by a significant influx of host cells into the repair tissue. This study shows that the sex-determining region Y gene is a valuable marker for tracking the fate of transplanted allogenic cells in tissue engineering.

Animals↗

PubMed2005

Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor.

A previously reported "grafting and coating" method (J. Biomed. Mater. Res. (Appl. Biomater.) 63 (2002) 838) was modified and used to introduce stable collagen layer and incorporate basic fibroblast growth factor (bFGF) on PLLA scaffold surface to prepare tissue engineering scaffold with improved biocompatibility. To make the modification of the 3-D porous PLLA scaffold possible, grafting of polymethacrylic acid (PMAA) onto the PLLA surface was initiated by the -OOH/Fe2+ system instead of the UV light used in the former method. Water soluble carbodimmide chemistry was applied to graft collagen onto the PLLA scaffold surface, followed by physical coating of the collagen solution with or without basic fibroblast growth factor (bFGF). Surface modification of 2-D PLLA membrane was also done for fundamental understanding of the modification. The -COOH density on/in the PMAA grafted PLLA membrane/scaffold was measured by colorimetric method and the collagen content on/in the collagen immobilized PLLA membrane/scaffold was measured by ninhydrin method. Chondrocyte culturing on the collagen immobilized PLLA surfaces showed significantly improved cell spreading and growth. Incorporation of fibroblast growth factors in the collagen layer further enhanced the cell growth. This convenient and effective method can be used to prepare bioactive scaffolds with extra cellular matrix (ECM)-mimic composition for tissue engineering.

Animals↗

PubMed2000

Influence of growth factors on tissue-engineered pediatric elastic cartilage.

OBJECTIVE: To investigate the influence of growth factors on tissue-engineered pediatric human elastic cartilage relative to potential clinical application. DESIGN: Controlled study. SUBJECTS: Eleven children ranging in age from 5 to 15 years provided auricular elastic cartilage specimens measuring approximately 1 x 1 x 0.2 cm and weighing approximately 100 mg. INTERVENTIONS: Three million chondrocytes were plated into 4 groups of Ham F-12 culture medium: group 1, Ham F-12 culture medium only; no growth factors (control group); group 2, Ham F-12 culture medium and basic fibroblast growth factor; group 3, Ham F-12 culture medium and transforming growth actor beta; and group 4, Ham F-12 culture medium and a combination of both growth factors. At 3 weeks, the cells were harvested and mixed with a copolymer gel of polyethylene glycol and polypropylene oxide (Pluronic F-127). The cell solution was injected subcutaneously into athymic mice. The constructs were harvested at up to 22 weeks of in vivo culture and histologically analyzed. RESULTS: The average number of cells generated in vitro was as follows: group 1, 12 million; group 2, 40 million; group 3, 7 million; and group 4, 35 million. Group 2 in vivo gross specimens were the largest and heaviest. Histologically, the control group and the basic fibroblast growth factor group (groups 1 and 2) exhibited characteristics compatible with normal auricular cartilage; groups 3 and 4 demonstrated cellular disorganization and moderate to severe fibrous tissue infiltration. CONCLUSIONS: Basic fibroblast growth factor demonstrates the greatest positive influence on the in vitro and in vivo growth of engineered pediatric human auricular cartilage. The results suggest that basic fibroblast growth factor has the potential for clinical application in which a goal will be to generate a large volume of tissue-engineered cartilage from a small donor specimen in a short period of time and of a quality similar to native human elastic cartilage.

Adolescent↗

PubMed2003

Gelatin-chondroitin-hyaluronan tri-copolymer scaffold for cartilage tissue engineering.

The mechanism by which the cell synthesizes and secretes extracellular matrix (ECM) and is, in turn, regulated by the ECM is termed dynamic reciprocity. The aim of the present work was to produce a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer to mimic natural cartilage matrix for use as a scaffold for cartilage tissue engineering. The scaffold produced had a uniform pore size of about 180 microm and adequate porosity of 75%. Porcine chondrocytes were seeded onto the tri-copolymer scaffold and cultured in Petri dishes or spinner flasks for 2, 3, 4, or 5 weeks. Chondrocytes were uniformly distributed in the scaffold in the spinner flask cultures, but less so in the Petri dish cultures. Secretion of ECM was found under histology examination. In spinner flask cultures, chondrocytes retained their phenotype for at least 5 weeks, as shown immunohistochemically, and synthesized type II collagen. These results show that gelatin/chondroitin sulfate/hyaluronan tri-copolymer has potential for use as a cartilage tissue engineering scaffold.

Animals↗

PubMed2002

Tissue engineering of human salivary gland organoids.

Radiation therapy for malignant head and neck tumours is mainly responsible for inadvertent damage of the salivary glands. Xerostomia is the major symptom of this condition, with consequent mucositis, dental caries, dysphagia and nutritional deficits. At present there is no routine treatment for radiation-induced salivary dysfunction. Based on the principles of tissue engineering, this study presents a new experimental concept for reconstituting salivary gland function after radiation therapy for head and neck cancer. Human parotid cells were cultured with two different types of commercially available microcarriers-Cytodex 3 and Cytopore 1-for up to 3 weeks in vitro. Cultures were controlled daily by means of inverted microscopy. Medium samples were tested for alpha-amylase, tissue polypeptide antigen (TPA) and S100 in order to control parotid cell function in vitro. The vitality of the cells was investigated by in vitro staining with erythrosine. Immunocytochemical analysis for amylase and cytokeratin was performed in order to confirm epithelial character and maintain acinar cell type. Parotid gland cells could be cultured in a differentiated and vital state on both types of microcarriers for up to 3 weeks. Almost all of the cultured cells exhibited immunoreactivity for cytokeratin. High concentrations of TPA, a specific marker for salivary duct epithelium, indicated persistent differentiation of this cell type in vitro. Positivity for amylase was detectable in 20-45%, of cells growing on the microcarriers, and especially on Cytodex 3. Decreasing amylase levels in the culture medium indicated functional deficiencies of the remaining acinar cells. Tissue engineering of human salivary gland organoids on microcarriers is a new approach for potential causative treatment of radiation-induced xerostomia. Before clinical application can be considered significant improvements in the in vitro cultivation of salivary gland tissue and scaffold design have to be realized.

Dextrans↗

PubMed1998

Fluorescence activated cell sorting: a reliable method in tissue engineering of a bioprosthetic heart valve.

BACKGROUND: Techniques of tissue engineering are used to seed human autologous cells in vitro on degradable mesh to create new functional tissue like a bioprosthetic heart valve. A precondition is subsequent seeding of native-valve-analogous pure endothelial and myofibroblast cell lines. The aim of this study is to find a safe method of isolating viable cell lines out of tissues from the operating room. METHODS: Mixed cells from ascending aorta obtained from the operating room were incubated with an endothelial-specific fluorescent marker. The labeled cells were activated and sorted by flow cytometry. Isolated cell lines were cultured and thereafter square sheets of polymeric scaffold were seeded with myofibroblasts, followed by endothelial cells. The created tissue was stained with hematoxylin and eosin, van Gieson stain, and stains for factor VIII and CD34. RESULTS: Control culture samples (n = 25) revealed vital uncontaminated endothelial and myofibroblast cell lines. Microscopy of the seeded meshes (n = 16) demonstrated a tissue-like structure. Van Gieson stain showed production of collagen. Endothelial cells formed a superficial monolayer, demonstrated by factor VIII and CD34; no invasive formation of capillaries was detectable. CONCLUSIONS: These results demonstrate that fluorescence activated cell sorting is a reliable and safe method to gain pure vital autologous cell lines out of human mixed cells for subsequent seeding on degradable mesh and that those cells are active to form new tissue.

Aorta↗

PubMed2005

Tissue engineering of vascular conduits: fabrication of custom-made scaffolds using rapid prototyping techniques.

BACKGROUND: The technique of stereolithography, which automatically fabricates models from X-ray computed tomography or magnetic resonance imaging (MRI) data linked to computer-aided design programs, has been applied to the fabrication of scaffolds for tissue engineering. We previously reported on the application of stereolithography in scaffold fabrication of a trileaflet heart valve. In our current experiment we demonstrate a new technique for the fabrication of custom-made conduits for the potential replacement of a coarcted aortic segment. METHODS AND RESULTS: In this experiment the image data derived from a 12-year-old male patient with aortic coarctation scanned by MRI were processed by a computer-aided design program to reconstruct the aortic arch with isthmus stenosis three dimensionally. By defining the stenotic section and the adjacent normal vessel a custom-made nonstenotic descending aorta was reconstructed to replace the stenosed part. The rapid prototyping technique was used to establish stereolithographic models for fabricating biocompatible and biodegradable vascular scaffolds with the anatomic structure of the recalculated human descending aorta through a thermal processing technique. CONCLUSION: Our results suggest that the re-creation and reproduction of complex vascular structures by computer-aided design techniques may be useful to fabricate custom-made polymeric scaffolds for the tissue engineering of living vascular prostheses.

Aortic Coarctation↗

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↗