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PubMed2004

Thermally produced biodegradable scaffolds for cartilage tissue engineering.

A novel process was developed to fabricate biodegradable polymer scaffolds for tissue engineering applications, without using organic solvents. Solvent residues in scaffolds fabricated by processes involving organic solvents may damage cells transplanted onto the scaffolds or tissue near the transplantation site. Poly(L-lactic acid) (PLLA) powder and NaCl particles in a mold were compressed and subsequently heated at 180 degrees C (near the PLLA melting temperature) for 3 min. The heat treatment caused the polymer particles to fuse and form a continuous matrix containing entrapped NaCl particles. After dissolving the NaCl salts, which served as a porogen, porous biodegradable PLLA scaffolds were formed. The scaffold porosity and pore size were controlled by adjusting the NaCl/PLLA weight ratio and the NaCl particle size. The characteristics of the scaffolds were compared to those of scaffolds fabricated using a conventional solvent casting/particulate leaching (SC/PL) process, in terms of pore structure, pore-size distribution, and mechanical properties. A scanning electron microscopic examination showed highly interconnected and open pore structures in the scaffolds fabricated using the thermal process, whereas the SC/PL process yielded scaffolds with less interconnected and closed pore structures. Mercury intrusion porosimetry revealed that the thermally produced scaffolds had a much more uniform distribution of pore sizes than the SC/PL process. The utility of the thermally produced scaffolds was demonstrated by engineering cartilaginous tissues in vivo. In summary, the thermal process developed in this study yields tissue-engineering scaffolds with more favorable characteristics, with respect to, freedom from organic solvents, pore structure, and size distribution than the SC/PL process. Moreover, the thermal process could also be used to fabricate scaffolds from polymers that are insoluble in organic solvents, such as poly(glycolic acid). Cartilage tissue regenerated from thermally produced PLLA scaffold.

Animals↗

PubMed1998

Role of morphogenetic proteins in skeletal tissue engineering and regeneration.

Morphogenesis is the developmental cascade of pattern formation and body plan establishment, culminating in the adult form. It has formed the basis for the emerging discipline of tissue engineering, which uses principles of molecular developmental biology and morphogenesis gleaned through studies on inductive signals, responding stem cells, and the extracellular matrix to design and construct spare parts that restore function to the human body. Among the many organs in the body, bone has considerable powers for regeneration and is a prototype model for tissue engineering. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and has permitted the isolation of bone morphogens, such as bone morphogenetic proteins (BMPs), from demineralized adult bone matrix. BMPs initiate, promote, and maintain chondrogenesis and osteogenesis, but are also involved in the morphogenesis of organs other than bone. The symbiosis of the mechanisms underlying bone induction and differentiation is critical for tissue engineering and is governed by both biomechanics (physical forces) and context (microenvironment/extracellular matrix), which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion glycoproteins, including fibronectins and laminin. Rules of tissue architecture elucidated in bone morphogenesis may provide insights into tissue engineering and be universally applicable for all organs/tissues, including bones and joints.

Animals↗

PubMed2003

Tissue-engineered scaffolds are effective alternatives to autografts for bridging peripheral nerve gaps.

The use of autografts for "bridging" peripheral nerve gaps is limited by lack of suitable donor nerve grafts. Using a tissue-engineering approach, we have designed a three-dimensional scaffold that presents laminin 1 (LN-1) and nerve growth factor (NGF) in vivo. Semipermeable polysulfone tubes were used as carriers to introduce the tissue-engineered scaffolds to a 10-mm sciatic nerve gap in adult rats. Two months after implantation, the gross morphology of the regenerated nerve, the success rate of regeneration, and the total number and density of myelinated axons in the tissue-engineered scaffolds matched that observed in autografts. LN-1- and NGF-containing scaffolds performed comparably to autografts when functional measures that include the relative gastrocnemius muscle weight and the sciatic functional index were quantified. Our results demonstrate that tissue-engineered scaffolds match the performance of autografts in an in vivo model of peripheral nerve regeneration, raising the possibility of the scaffolds being used clinically instead of scarce autografts.

Animals↗

PubMed2004

Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells.

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.

AC133 Antigen↗

PubMed2005

A collagen-glycosaminoglycan co-culture model for heart valve tissue engineering applications.

In order to develop efficient design strategies for a tissue-engineered heart valve, in vivo and in vitro models of valvular structure and cellular function require extensive characterisation. Collagen and glycosaminoglycans (GAGs) provide unique functional characteristics to the heart valve structure. In the current study, type I collagen-GAG hydrogels were investigated as biomaterials for the creation of mitral valve tissue. Porcine mitral valve interstitial cells (VICs) and endothelial cells (VECs) were isolated and co-cultured for 4 weeks in hydrogel constructs composed of type I collagen. The metabolic activity and tissue organisation of mitral valve tissue constructs was evaluated in the presence and absence of chondroitin sulphate (CS) GAG, and comparisons were made with normal mitral valve tissue. Both collagen and collagen-CS mitral valve constructs contracted to form tissue-like structures in vitro. Biochemical assay demonstrated that over 75% of CS was retained within collagen-CS constructs. Morphological examination demonstrated enhanced VEC surface coverage in collagen-CS constructs compared to collagen constructs. Ultrastructural analysis revealed basement membrane synthesis and cell junction formation by construct VECs, with an increased matrix porosity observed in collagen-CS constructs. Immunohistochemical analyses demonstrated enhanced extracellular matrix production in collagen-CS constructs, including expression of elastin and laminin by VICs. Both native valve and collagen-CS construct VECs also expressed the vasoactive molecule, eNOS, which was absent from collagen construct VECs. The present study demonstrates that collagen gels can be used as matrices for the in vitro synthesis of tissue structures resembling mitral valve tissue. Addition of CS resulting in a more porous model was shown to positively influence the bioactivity of seeded valve cells and tissue remodelling. Collagen-GAG matrices may hold promise for a potential use in heart valve tissue engineering and improved understanding of heart valve biology.

Animals↗

PubMed2004

Tissue engineering auricular reconstruction: in vitro and in vivo studies.

Although investigators have demonstrated that neocartilage can be constituted in a predetermined shape and in complex three-dimensional structures, such as a human ear, by using cell transplantation on polymer constructs, many unsolved problems still remain. The crucial issues for auricular tissue engineering consisted of optimal cell culture environment, choice of polymers, behavior of chondrocytes, study of cell-polymer constructs in an acceptable animal model, and long-term structural integrity. Here we describe our tissue engineering approaches for auricular reconstruction including auricular scaffold fabrication, in vitro chondrogenesis, in vivo immunocompromized xenograft and immunocompetent autologous animal models, and long-term follow-up. Though many current obstacles regarding auricular tissue engineering still exist, we demonstrate techniques of auricular scaffold fabrication with promising in vitro and in vivo neocartilage formation, optimal selection and application of animal models, and, to the best of our knowledge, the first report of different biodegradable biomaterial trials and the longest in vivo results (10 months) for auricular tissue engineering.

Animals↗

PubMed2003

Biologic gels in tissue engineering.

There are many different types of scaffold materials now available for tissue engineering applications. Hydrogels form one group of materials that have been used in a wide variety of applications. These hydrogels can be formed using natural materials, synthetic materials, or some combination of the two. There are advantages and disadvantages to using each type of material, and detailed investigations into the effects on various aspects of cell behavior of chemical and physical properties of the materials are needed to make an informed decision as to which material is best suited for a given application. By combining appropriate scaffold materials, such as hydrogels, with cells and proper signaling for those cells, more commercial tissue engineering products will become available for general use.

Biocompatible Materials↗

PubMedDate not supplied

Seeding techniques and scaffolding choice for tissue engineering of the temporomandibular joint disk.

Temporomandibular joint (TMJ) disk removal, or diskectomy, is a detrimental yet necessary surgery for patients with extremely displaced disks. Tissue engineering is an enticing methodology for improvement of the postoperative outcome of diskectomy. Unfortunately, the field of tissue engineering of the TMJ disk is only in the early stages of development. The initial objective of this investigation was to study the cellular response of TMJ disk cells in alginate culture. However, a marked decrease in cell population and the lack of detection of extracellular matrix (ECM) products did not support the use of alginate culture. The second objective was then to attempt TMJ disk cell culture in polyglycolic acid (PGA) nonwoven meshes. However, as suitable seeding methods for TMJ disk cells on PGA had not been determined, three techniques were selected for study: spinner flask, orbital shaker, and a novel pelleting technique. PGA constructs maintained cellularity throughout the culture period, and scaffolds seeded with the spinner flask produced about 35 microg of collagen per construct. Thus, as evidenced by the production of a major extracellular component, PGA nonwoven meshes seeded with TMJ disk cells, using a spinner flask, may be a first positive culturing step in tissue engineering the TMJ disk.

Alginates↗

PubMed2001

Conjugate addition reactions combined with free-radical cross-linking for the design of materials for tissue engineering.

PEG-diacrylamide was synthesized and utilized to make materials for tissue engineering. Acrylamide groups readily react with thiol groups, and peptides containing a single thiol group were coupled to the PEG-diacrylamide in aqueous solution at room temperature in about 2 h. If only a portion of the acrylamide groups were targeted for reaction with peptide, sufficient amounts of PEG-diacrylamide remained to be polymerized by free-radical mechanisms via photoinitiation. The photopolymerization step can be performed in contact with cells, providing a means to produce bioactive scaffolds for tissue engineering. The photopolymerization conditions and precursor composition greatly affect the stiffness of the materials, which subsequently affected cell spreading. The kinetics and extent of cell spreading on the bioactive materials were measured and compared to cell spreading on tissue culture polystyrene. Although the PEG materials resist protein adsorption, the experiments suggest that the cells can secrete extracellular matrix that can adhere to the gels.

Acrylamides↗

PubMed2004

Translational research for injectable tissue-engineered bone regeneration using mesenchymal stem cells and platelet-rich plasma: from basic research to clinical case study.

Translational research involves application of basic scientific discoveries into clinically germane findings and, simultaneously, the generation of scientific questions based on clinical observations. At first, as basic research we investigated tissue-engineered bone regeneration using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) in a dog mandible model. We also confirmed the correlation between osseointegration in dental implants and the injectable bone. Bone defects made with a trephine bar were implanted with graft materials as follows: PRP, dog MSCs (dMSCs) and PRP, autogenous particulate cancellous bone and marrow (PCBM), and control (defect only). Two months later, dental implants were installed. According to the histological and histomorphometric observations at 2 months after implants, the amount of bone-implant contact at the bone-implant interface was significantly different between the PRP, PCBM, dMSCs/ PRP, native bone, and control groups. Significant differences were also found between the dMSCs/PRP, native bone, and control groups in bone density. These findings indicate that the use of a mixture of dMSCs/ PRP will provide good results in implant treatment compared with that achieved by autogenous PCBM. We then applied this injectable tissue-engineered bone to onlay plasty in the posterior maxilla or mandible in three human patients. Injectable tissue-engineered bone was grafted and, simultaneously, 2-3 threaded titanium implants were inserted into the defect area. The results of this investigation indicated that injectable tissue-engineered bone used for the plasty area with simultaneous implant placement provided stable and predictable results in terms of implant success. We regenerated bone with minimal invasiveness and good plasticity, which could provide a clinical alternative to autogenous bone grafts. This might be a good case of translational research from basic research to clinical application.

Aged↗

PubMed2004

Flap prefabrication - the bridge between conventional flaps and tissue-engineered flaps.

Flap prefabrication is one of the most exciting areas in Plastic Surgery because of its bridging role between conventional reconstructive surgery and tissue engineering. Using this technique,tissues such as bone, cartilage, skin and muscle can be preassembled to form precise composites that will fit any defect. In pre-lamination, for example, an ear may be created by burying cartilage underneath forearm skin and later harvested as a skin-cartilage composite free flap to replace the missing part. Vascular induction is yet another means of customising flaps where new blood supply is introduced to create transplantable tissue. For example, bone chips wrapped in avascular carrier such as muscle can become vascularised grafts. Our experiment describes jejunal prefabrication in a rat model using the same technique. Intestinal segments wrapped in muscle flaps become independent of their mesenteric blood supply by "parasitising" on the muscle's blood supply. This idea arose from our initial observations that intestinal segments transferred to the neck to reconstruct the oesophagus could survive accidental disruption of the pedicle if sufficient time had elapsed. Clearly, the bowel had picked up new blood supply from its bed. Subsequently, jejunal prefabrication was used to reconstruct the oesophagus in a patient in whom there were no recipient vessels for free jejunal transfer. The pedicled latissimus dorsi muscle flap was used as a carrier for the jejunum. Another application of this idea could be in the area of allogeneic trachea or pancreatic transplantation, since present-day techniques have yet to overcome problems such as insufficient vascularity and unpredictable transplant survival. Future applications incorporating biomaterials and cultured cells will usher in the era of tissue-engineered flaps.

Animals↗

PubMed2001

[Reconstruction of tissue engineered vascular model in vitro].

OBJECTIVE: To explore the feasibility of reconstructing tissue engineered vessel in vitro. METHODS: Bovine endothelial cells were isolated from calf thoracic aorta by enzyme digestion methods and subcultured and purified. The endothelial cells of the 3rd to 7th passages were seeded into the inner surface of tubular scaffold material by polyglycolic acid(PGA) coated with cross-linked collagen, and cultured in vitro for 10 days using dynamic rotation culture technique. Scanning electron microscopy was used to analyse the morphological characteristics, and prostacyclin released by endothelial cells was measured by radioimmunoassay of 6-keto-prostaglandin F1 alpha. RESULTS: The VIII factor staining of cultured endothelial cells was positive. The endothelial cells adhered well on the inner surface of tubular scaffold material with confluent monolayer covering(91.2 +/- 1.5)%. The endothelialized model released prostacyclin at a rate of (4.6 +/- 0.5) micrograms/cm2.min. There was significant difference to control group (P < 0.05). CONCLUSION: The PGA coating with collagen is an ideal scaffold for endothelial cells, the coverage rate is increased through dynamic rotation culture technique. It will lay a good foundation for architecture of a laminated structure of tissue engineered vessel.

Animals↗

PubMed2003

Tissue engineering of bone and cartilage. From the preclinical model to the patient.

Tissue engineering studies are complex in nature and involve many stages of testing before an experimental construct is ripe for human clinical trials. Biocompatibility questions, toxicological problems, pharmacological aspects, safety issues and proof of the principle itself represent but a few of the concerns that must be addressed. This contribution deals with the different types of animal experiment that are usually undertaken in tissue engineering studies. During the initial phase, small animal models are used to screen all potential components of the proposed construct, such as matrix, cells and signalling substances. In the second, the principle itself is put to the test using an appropriate defect model in a suitable animal species. Careful consideration of these latter two aspects is of paramount importance, many factors having to be considered and special requirements satisfied for meaningful experimentation. Finally, clinically relevant models are set up in animals to furnish basic information respecting the construct's suitability and ripeness for testing in human clinical trials.

Animals↗

PubMed2005

Preparation and evaluation of ammonia-treated collagen/chitosan matrices for liver tissue engineering.

To further enhance the properties of existing collagen/chitosan scaffolds for liver tissue engineering, a very simple method was developed to form noncovalently linked mimic of the liver extracellular matrices. Collagen/chitosan mixtures in various proportions (i.e., 1:0, 3:2, 1:1, 2:3, and 0:1 v/v) were lyophilized or evaporated to form sponges or flat films before they were gelled using an aqueous 25% ammonia solution. The porosities of the obtained sponges were above 90% with various pore sizes. The highest mechanical strength (1.9+/-0.7 MPa) and the lowest degradation time (65+/-1.7 days) were achieved by the collagen/chitosan (1:1) matrices. Hepatocytes cultured on the collagen/chitosan (1:1) matrices exhibited relatively high glutamate-oxaloacetate transaminase and glucose secretion functions 25 days post-seeding. Nuclues of the hepatocytes were more elongated and arranged in certain directions on the 1:1 matrices. The cytocompatibility and enhanced biostability of our new ammonia-treated collagen/chitosan matrices suggest that they could be used as scaffolds for liver tissue engineering.

Amines↗

PubMed2005

Selective laser sintering of biocompatible polymers for applications in tissue engineering.

The ability to use biological substitutes to repair or replace damaged tissues lead to the development of Tissue Engineering (TE), a field that is growing in scope and importance within biomedical engineering. Anchorage dependent cell types often rely on the use of temporary three-dimensional scaffolds to guide cell proliferation. Computer-controlled fabrication techniques such as Rapid Prototyping (RP) processes have been recognised to have an edge over conventional manual-based scaffold fabrication techniques due to their ability to create structures with complex macro- and micro-architectures. Despite the immense capabilities of RP fabrication for scaffold production, commercial available RP modelling materials are not biocompatible and are not suitable for direct use in the fabrication of scaffolds. Work is carried out with several biocompatible polymers such as Polyetheretherketone (PEEK), Poly(vinyl alcohol) (PVA), Polycaprolactone (PCL) and Poly(L-lactic acid) (PLLA) and a bioceramic namely, Hydroxyapatite (HA). The parameters of the selective laser sintering (SLS) process are optimised to cater to the processing of these materials. SLS-fabricated scaffold specimens are examined using a Scanning Electron Microscope (SEM). Results observed from the micrographs indicate the viability of them being used for building TE scaffolds and ascertain the capabilities of the SLS process for creating highly porous scaffolds for Tissue Engineering applications.

Biocompatible Materials↗

PubMed2006

In vitro evaluation of potential calcium phosphate scaffolds for tissue engineering.

Nanocrystalline calcium phosphates are very interesting candidates as scaffolds for bone tissue engineering. These materials show excellent in vivo biocompatibility, cell proliferation, and resorption. In this work we have studied the osteoblast-like cell behavior seeded onto HA and BCP synthesized by controlled crystallization method and treated at different temperatures. In vitro cell attachment, proliferation, differentiation, spreading, and cytotoxicity tests have been carried out. The results can be explained as a function of the phase composition and microstructure. Under in vitro closed conditions, nanocrystalline HA depletes the calcium of the medium avoiding cell proliferation, whereas well-crystallized HA enhances high cell proliferation. On the other hand, nanocrystalline BCPs supply Ca(2+) to the medium due to the higher solubility of the beta-TCP component, allowing an excellent in vitro cellular response when osteoblast-like cells are seeded on it. These features make BCPs excellent candidates as scaffolds for bone tissue engineering.

Alkaline Phosphatase↗

PubMed2004

Therapeutic cloning and tissue engineering.

A severe shortage of donor organs available for transplantation in the United States leaves patients suffering from diseased and injured organs with few treatment options. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for tissue engineering applications. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.

Animals↗

PubMed2004

Molecular diffusion in tissue-engineered cartilage constructs: effects of scaffold material, time, and culture conditions.

Diffusion is likely to be the primary mechanism for macromolecular transport in tissue-engineered cartilage, and providing an adequate nutrient supply via diffusion may be necessary for cell proliferation and extracellular matrix production. The goal of this study was to measure the diffusivity of tissue-engineered cartilage constructs as a function of scaffold material, culture conditions, and time in culture. Diffusion coefficients of four different-sized fluorescent dextrans were measured by fluorescence recovery after photobleaching in tissue-engineered cartilage constructs seeded with human adipose-derived stem cells or acellular constructs on scaffolds of alginate, agarose, gelatin, or fibrin that were cultured for 1 or 28 days in either chondrogenic or control conditions. Diffusivities in the constructs were much greater than those of native cartilage. The diffusivity of acellular constructs increased 62% from Day 1 to Day 28, whereas diffusivity of cellular constructs decreased 42% and 27% in chondrogenic and control cultures, respectively. The decrease in diffusivity in cellular constructs is likely due to new matrix synthesis, which may be enhanced with chondrogenic media, and matrix contraction by the cells in the fibrin and gelatin scaffolds. The increase in diffusivity in the acellular constructs is probably due to scaffold degradation and swelling.

Adipocytes↗