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PubMed2004

Tissue-engineered buccal mucosa for substitution urethroplasty.

OBJECTIVE: To develop tissue-engineered buccal mucosa (TEBM) for use in substitution urethroplasty, as urethral reconstruction is limited by the amount and type of tissue that is available for grafting, and BM has become the favoured tissue for use as a urethral substitute in the last decade. MATERIALS AND METHODS: After enzymatic treatment of a small (0.5 cm) BM biopsy the epidermis and dermis were mechanically separated. Oral keratinocytes were isolated from the epidermis and oral fibroblasts from the dermis. These cells were expanded and applied to sterilized de-epidermized dermis (DED) to obtain a full-thickness TE oral mucosa. Horizontal migration of keratinocytes on the DED was assessed using a tetrazolium-blue (MTT) assay. The TEBM was assessed histologically after mechanical stressing in vitro using catheterization and meshing. RESULTS: Histologically the TEBM closely resembled the native oral mucosa after culturing at an air-liquid interface for 2 weeks. The MTT assay showed good horizontal migration of keratinocytes on the DED. Serial histology revealed a gradually increasing thickness of the epidermis and remodelling of the dermis by the fibroblasts from day 1 to day 14. Despite subjecting the TEBM to mechanical stress the integrity of the epidermal-dermal junction was maintained. CONCLUSIONS: We report the successful culture of full-thickness TEBM for substitution urethroplasty, which is robust and suitable for clinical use.

Cells, Cultured↗

PubMed2001

Tissue-engineered pancreatic islets: culturing rat islets in the chitosan sponge.

Subcutaneous islet transplantation has become an attractive modality. With development of tissue-engineering techniques, it is possible to rectify the disadvantage of poor blood supply in the subcutaneous site by reconstruction of the capillary network. According to reports, the Chitosan sponge (CS) could be used for reconstruction of in vitro capillary-like network and could be used in artificial skin equivalent. In this study, we cultured the islets in CS for future application. CSs, having 200-500 microm pore size, were prepared by freeze-drying method. Rat islets were isolated from the pancreas of Lewis rats (10 weeks old, 280-300 g, male) by collagenase digestion followed by discontinuous dextran gradient centrifugation method. Each 20 islets were seeded equally into the CSs and were cultured for 62 days with various culture media such as RPMI-1640, Dulbecco's modified Eagle's medium (DMEM), and Eagle's MEM. They contained 10% fetal bovine serum (FBS) and 5 ml/L antibiotic-antimycotic mixed stock solution in the culture dishes. Insulin concentration both inside and outside of the islet-seeded CS was measured during culture. Changes in the morphology of islets were also observed in this study. Freshly isolated islets had a loose appearance with an irregular border, and most were seen as a single islet. Occasionally a cluster, consisting of 2-4 islets ranging mainly from 150 to 250 microm in diameter, was observed. Islets cultured in the CSs in different culture media retained initial morphology, which had well-delineated smooth borders for at least 53 days. The insulin release behavior of islets cultured in the CS showed constant secretory capacities for 49 days. After that they exhibited a rapid and definitive decline from the initial insulin release. Until this stage, insulin concentration in the CS was well maintained. The properties were dependent on culture medium used and insulin diffusion released from islets. This experiment is a new study model for establishment of islet culture in a three-dimensional matrix. Also extension of this observation will provide new insights for islet transplantation at the subcutaneous site by a tissue-engineering approach.

Animals↗

PubMed2005

Characterization of protein release from photocrosslinkable hyaluronic acid-polyethylene glycol hydrogel tissue engineering scaffolds.

The goal of this work was to utilize the naturally derived bioactive polymer hyaluronic acid (HA) to create a combination tissue engineering scaffold and protein delivery device. HA is a non-immunogenic, non-adhesive glycosaminoglycan that plays significant roles in several cellular processes, including angiogenesis and the regulation of inflammation. In previous work, we created photopolymerizable glycidyl methacrylate-hyaluronic acid (GMHA) hydrogels that had controlled degradation rates, were cytocompatible, and were able to be modified with peptide moieties. In the present studies, we characterized the release of a model protein, bovine serum albumin (BSA), from GMHA and GMHA-polyethylene glycol (PEG) hydrogels. Although BSA could be released rapidly (> 60% within 6 h) from 1% GMHA hydrogels, we found that increasing either the GMHA or the PEG concentrations could lengthen the duration of protein delivery. Preliminary size exclusion chromatography studies indicated that the released BSA was almost entirely in its native monomeric form. Lastly, protein release was extended to several weeks by suspending BSA-poly(lactic-co-glycolic acid) microspheres within the hydrogel bulk. These initial studies indicate that the naturally derived biopolymer HA can be employed to design novel photopolymerizable composites that are suitable for delivering stable proteins from scaffolding in tissue engineering applications.

Coated Materials, Biocompatible↗

PubMed2000

Preparation of a pure autologous biodegradable fibrin matrix for tissue engineering.

Parallel to the growing role of tissue engineering, the need for cell embedding materials, which allow cells to stabilise in a three-dimensional distribution, has increased. Although several substances have been tested, fibrin is thus far the only one that permits the clinical application of cultured tissue. To date, autologous fibrinogen has usually been polymerised with bovine thrombin, which can cause severe immunological side effects. The objective of this study was to explore the practicability of obtaining autologous thrombin from a single patient in an adequate concentration and amount. Fibrinogen was cryoprecipitated from 200 ml of freshly-frozen plasma. Thrombin was isolated from the supernatant through ion-exchange chromatography. The thrombin was first bound to Sephadex A-50 and then eluated using 2 ml of a salt buffer (2.0 M NaCl in 0.015 M trisodiumcitrate, pH 7.0). The activity of the thrombin (51 NIH x ml(-1) to 414 NIH x ml(-1) reached levels comparable to those in commercially available fibrin glues (4-500 NIH x ml(-1)). The study has shown that it is possible to obtain a sufficient amount of autologous thrombin from a single donor to create a fibrin matrix of high efficiency without the risk of immunological and infectious side effects.

Absorbable Implants↗

PubMed2002

Polymer/alginate amalgam for cartilage-tissue engineering.

Marrow stroma-derived cells (MSC) are highly proliferative, multipotential cells that have been considered as ideal candidate cells for autologous tissue engineering applications. In this study, we have characterized the chondrogenic potential of human MSCs in both a PLA/alginate amalgam and pure PLA macrostructure as model three-dimensional constructs to support both chondrogenic differentiation and proliferation following TGF-beta treatment. MSCs were seeded in experimental groups that consisted of PLA-loaded constructs and PLA/alginate amalgams with and without recombinant human TGF-beta1. Chondrogenesis of the PLA and the PLA/alginate amalgam cultures was assessed at weekly intervals by histology, immunohistochemistry, scanning electron microscopy, sulfate incorporation, and RT-PCR. Chondrogenic differentiation occurs within a polymeric macrostructure with TGF-beta1 treatment as indicated by histological, immunohistochemical, sulfate incorporation, and gene expression profiles. This macrostructure can be further encased in an alginate gel/solution to optimize cell shape and to confine growth factors and cells within the polymer construct, while the polymeric scaffold provides appropriate mechanical/tissue support. The stable three-dimensional PLA/alginate amalgam represents a novel candidate system of mesenchymal chondrogenesis, which is amendable to investigation of mechanical and biological factors that normally modulate cartilage development and formation as well as a potential tissue engineering construct for cartilage repair.

Alginates↗

PubMed2004

[Experimental research using human bone marrow mesenchymal stem cells as the seed cells for bone and cartilage tissue engineering].

AIM: To investigate the feasibility of human bone marrow mesenchymal stem cells(hMSCs) as the seed cells for bone and cartilage tissue engineering. METHODS: Purified hMSCs were cultured in-vitro and induced to differentiate into osteoblasts and chondrocytes. Cellular morphologies were observed under inverted and electron microscopes. The specific markers of the osteoblasts and chondrocytes were detected by histochemical staining, immunohistochemical staining, and RT-PCR. RESULTS: After the hMSCs were passaged for 15 generations, the choractenistic morphology and cell surface antigens of hMSCs remained unchanged. The level of alkaline phosphatase(ALP) in the culture supernatant of the osteoinduction groups was higher than those in the control groups (P<0.05). The morphology of the cells in the osteoinduction and chondroinduction groups changed from spindle-shaped cells into polygon-shaped cells. A large number of the dilated rough endoplasmic reticulua, Golgi apparatus and mitochondria could be seen under transmission electron microscope. Calcium deposition was detected on the surfaces of the hMSCs after osteoinduction. Collagen(COL)-like processes were detected under scanning electron microscope. The staining of the ALP, calcium nudis, COL-I and osteocalcin(OC) were positive, and expressions of the COL-I and OC mRNAs were detected after osteoinduction. The expression of COL-II was detected by immunohistochemical staining and RT-PCR and a lot of the metachromatic-staining matrix around the cells was observed with toluidine blue staining after chondroinduction. CONCLUSION: hMSCs from human bone marrow can be purified, expanded and differentiated into osteoblasts and chondrocytes in-vitro, providing an alternative source for bone and cartilage tissue engineering.

Alkaline Phosphatase↗

PubMed2005

The morphology and biomechanical characteristics of subcutaneously implanted tissue-engineered human septal cartilage.

The purpose of the study was to examine the morphology and biomechanical characteristics of in vivo cultured tissue-engineered human septal cartilage as a prospective autogenous transplant material for subcutaneous implantation in reconstructive procedures. Chondrocytes were enzymatically isolated from human septal cartilage biopsies. The cell number was expanded in monolayer culture. Chondrocytes were then fixed on a non-woven poly-lactide-poly-glycolide (PGLA) polymer scaffold by means of fibrin glue. The PGLA-polymer construct was implanted subcutaneously on the back of athymic mice and allowed to mature for 6 or 12 weeks. After killing the mice, the formed cartilage was tested on a material testing machine with a highly standardized reproducible setting. Biomechanical testing consisted of an indentation test, which revealed the failure load and compressive modulus of the neocartilage. The failure load shows the upper limit of supported stress. The compressive modulus is a measure of the templates' stiffness. After testing, the templates were histologically stained. Native human septal cartilage served as a control group. Histological and macroscopic examination showed cartilage formation of a hyaline-like morphology. Histological staining revealed the synthesis of abundant mucopolysaccharid matrix. The biomechanical characteristics of neocartilage proved to be of no statistical difference compared to native human septal cartilage. The failure load and compressive modulus were initially somewhat lower and reached the control group's results after 12 weeks in-vivo. Summarizing, tissue engineered nasal cartilage matches typical mechanical characteristics of native hyaline cartilage. Its elasticity and failure load are of sufficient quality to meet the clinical requirements for reconstructive surgery.

Animals↗

PubMed2003

[Tissue engineering used in andrology].

Trauma, tumor, disease and congenital abnormalities may lead to genital organ damage or function failure, and consequently the requirement of its reconstruction. Tissue engineering follows the principles of cell transplantation, materials science and engineering toward the development of biological substitutes that would restore and maintain normal function. These new techniques have been recently introduced into the field of andrology. Based on the latest advances, the present paper afferds a general prospect of the future direction of the development of tissue engineering in andrology.

Genitalia, Male↗

PubMed2006

Influence of a superficial tangential zone over repairing cartilage defects: implications for tissue engineering.

The superficial tangential zone (STZ) plays a critical role in normal cartilage function but is not yet a focus for designing tissue-engineered constructs for cartilage repair. Without material properties of sufficient quality in this zone, transplanted constructs in vivo may have little chance of survival. This finite element study investigates the impact of the superficial tangential zone on the mechanical function of normal articular surfaces as well as those with transplanted constructs. The zone is modeled as a thin transversely isotropic material with strain dependent permeability. The analyses predict that a normal transversely isotropic STZ placed over a repair region reduces the axial compression (55-68%) of, and the rate of fluid loss (45-82%) from the articular surface. A reduction was also found in von Mises stress (26-57%), axial strain (22-56%), and radial strain (69-73%), and an increase in fluid pressure (19-45%) in repair tissue under the STZ. Incorporating a quality superficial tangential zone in tissue-engineered constructs may be a critical factor in achieving mechanical environments conducive for successful cartilage repairs.

Animals↗

PubMed1998

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

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

Cells, Cultured↗

PubMed2001

A human tissue-engineered vascular media: a new model for pharmacological studies of contractile responses.

Our method for producing tissue-engineered blood vessels based exclusively on the use of human cells, i.e., without artificial scaffolding, has previously been described (1). In this report, a tissue-engineered vascular media (TEVM) was specifically produced for pharmacological studies from cultured human vascular smooth muscle cells (VSMC). The VSMC displayed a differentiated phenotype as demonstrated by the re-expression of VSMC-specific markers and actual tissue contraction in response to physiological stimuli. Because of their physiological shape and mechanical strength, rings of human TEVM could be mounted on force transducers in organ baths to perform standard pharmacological experiments. Concentration-response curves to vasoconstrictor agonists (histamine, bradykinin, ATP, and UTP) were established, with or without selective antagonists, allowing pharmacological characterization of receptors (H1, B2, and P2Y1, and pyrimidinoceptors). Sustained agonist-induced contractions were associated with transient increases in cytosolic Ca2+ concentration, suggesting sensitization of the contractile machinery to Ca2+. ATP caused both Ca2+ entry and Ca2+ release from a ryanodine- and caffeine-sensitive store. Increased cyclic AMP or cyclic GMP levels caused relaxation. This human TEVM displays many of functional characters of the normal vessel from which the cells were originally isolated, including contractile/relaxation responses, cyclic nucleotide sensitivity, and Ca2+ handling mechanisms comparable to those of the normal vessel from which the cells were originally isolated. These results demonstrate the potential of this human model as a versatile new tool for pharmacological research.

Adenosine Triphosphate↗

PubMedDate not supplied

The mechanical influence of tissue engineering techniques on tracheal strength: an experimental study on sheep trachea.

Tissue engineering is an attractive concept for facilitating the transplantation of different tissue types with a low immunogeneity and a well-preserved tissue structure. We examined the influence of treatment with trypsin/ethylenediamine tetraacetic acid (EDTA) on the mechanical properties of tracheae. Fresh sheep tracheae were stored in a trypsin/EDTA solution for various periods of time (24, 46, and 72 h) and their breaking strength was subsequently examined. The results were compared with native trachea. The treatment with trypsin/EDTA results in a considerable reduction of the mechanical stability under load, in this case compared with the tensile strength, that does not correlate with the results obtained from light-optical microscopy. The results from this study reveal that tissue preparation with trypsin/EDTA does not seem to be a suitable method of preparatory treatment of tracheae intended for transplantation.

Animals↗

PubMed1995

Biomaterials in tissue engineering.

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

Amino Acid Sequence↗

PubMed2003

Tissue-engineered versus native cartilage: linkage between cellular mechano-transduction and biomechanical properties.

Recent studies demonstrate that chondrocytes in native articular cartilage and in tissue-engineered constructs respond to mechanical stimuli through multiple regulatory pathways. Responses of the cells are manifested by intra- and intercellular signalling, alterations in transcription level, protein translation, post-translational modifications, and synthesis of intracellular and extracellular macromolecules. In addition, mechanical stimuli can alter the balance between anabolic and catabolic processes that are critically important to cell-mediated extracellular assembly and degradation of the tissue matrix and, therefore, to the survival of tissue engineered constructs. Chondrocyte mechanotransduction is therefore a critically important link to the biomechanical properties of native cartilage and to developing constructs. Since implanted cartilage repair tissue will be subjected to mechanical loads throughout its lifetime, it is essential that the resident cells respond appropriately to the range of static and dynamic compressive and shear deformations in vivo in a manner that enables adaptive remodelling and minimizes catabolic degradation. The in vivo environment should thereby signal tissue-specific maturation and integration processes in order to achieve the most appropriate tissue morphology and biomechanical function.

Animals↗

PubMed2005

Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering.

OBJECTIVE: To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering. DESIGN: Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the delta-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: Significant differences were found in cell proliferation and extracellular matrix accumulation among the five scaffold groups. Significant cell proliferation was observed for all scaffold types but occurred later (20-40 days) in PGA constructs compared to the other groups (0-20 days). By 40 days, agarose constructs had the highest sGAG to DNA ratio, while alginate and collagen I had the lowest levels. Quantitative differences in the Delta-disaccharide composition of the GAGs accumulated in the different scaffolds were also found, with the most striking variations in unsulfated and disulfated delta-disaccharides. Agarose constructs had the highest fraction of disulfated residues and the lowest fraction of unsulfated residues, with a 6-sulfated/4-sulfated disaccharide ratio most similar to that of native articular cartilage. CONCLUSIONS: The similarities and differences among scaffolds in proteoglycan accumulation and GAG composition suggest that the scaffold material directly or indirectly influences chondrocyte proteoglycan metabolism and may have an influence on the quality of tissue engineered cartilage.

Animals↗

PubMed2006

Simultaneous implant placement and bone regeneration around dental implants using tissue-engineered bone with fibrin glue, mesenchymal stem cells and platelet-rich plasma.

This study was undertaken to evaluate the use of tissue-engineered bone as grafting material for alveolar augmentation with simultaneous implant placement. Twelve adult hybrid dogs were used in this study. One month after the extraction of teeth in the mandible region, bone defects on both sides of the mandible were induced using a trephine bar with a diameter of 10 mm. Dog mesenchymal stem cells (dMSCs) were obtained via iliac bone biopsy and cultured for 4 weeks before implantation. After installing the dental implants, the defects were simultaneously implanted with the following graft materials: (i) fibrin, (ii) dMSCs and fibrin (dMSCs/fibrin), (iii) dMSCs, platelet-rich plasma (PRP) and fibrin (dMSCs/PRP/fibrin) and (iv) control (defect only). The implants were assessed by histological and histomorphometric analysis, 2, 4 and 8 weeks after implantation. The implants exhibited varying degrees of bone-implant contact (BIC). The BIC was 17%, 19% and 29% (control), 20%, 22% and 25% (fibrin), 22%, 32% and 42% (dMSCs/fibrin) and 25%, 49% and 53% (dMSCs/PRP/fibrin) after 2, 4 and 8 weeks, respectively. This study suggests that tissue-engineered bone may be of sufficient quality for predictable enhancement of bone regeneration around dental implants when used simultaneous by with implant placement.

Alkaline Phosphatase↗

PubMed1994

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

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

Animals↗

PubMed2006

Chemical characteristics and cytocompatibility of collagen-based scaffold reinforced by chitin fibers for bone tissue engineering.

Chitin is a kind of seemly material to match PLLA for a scaffold, which may create an appropriate environment for the regeneration of tissues. In this study, we prepared and evaluated a new nano-hydroxyapatite/collagen/PLLA (nHACP) scaffold reinforced by chitin fibers for bone-tissue engineering. The chitin fibers were crosslinked with PLLA by dicyclohexylcarbodimide (DCC). The chemical characteristics were evaluated by Fourier transformed infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The mechanical strength was measured by compressive tests. The fibers, crosslinked with PLLA, could enhance the compressive strength of the scaffold about four times. Human marrow mesenchymal stem cells (MSCs) culture showed that the reinforced nHACP scaffolds were more cytocompatible than that without reinforcement. The crosslinks hardly affected the cytocompatibility of the reinforced scaffolds. The results suggested that the reinforced scaffolds (DCC crosslinked) might be a promising candidate for bone-tissue engineering.

Animals↗