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PubMed2001

Tissue engineering: current state and prospects.

Organ shortage and suboptimal prosthetic or biological materials for repair or replacement of diseased or destroyed human organs and tissues are the main motivation for increasing research in the emerging field of tissue engineering. No organ or tissue is excluded from this multidisciplinary research field, which aims to provide vital tissues with the abilities to function, grow, repair, and remodel. There are several approaches to tissue engineering, including the use of cells, scaffolds, and the combination of the two. The most common approach is biodegradable or resorbable scaffolds configured to the shape of the new tissue (e.g. a heart valve). This scaffold is seeded with cells, potentially derived from either biopsies or stem cells. The seeded cells proliferate, organize, and produce cellular and extracellular matrix. During this matrix formation, the starter matrix is degraded, resorbed, or metabolized. First clinical trials using skin or cartilage substitutes are currently under way. Both the current state of the field and future prospects are discussed.

Culture Techniques↗

PubMed1998

Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering.

As the field of dental implants continues to grow at a rapid rate so does our quest to find new techniques to enhance bone grafting. Tissue engineering is an exciting new technique in bone grafting. Therefore, the purposes of this study were to develop a simple, reproducible method to isolate human osteoblast-like cells (HOBs) and to evaluate in vitro cell proliferation within 2 different 3-dimensional (3-D) constructs targeted for tissue engineering applications. Ultimately, HOBs that have been amplified within 3-D constructs may be employed for bone regeneration techniques, such as onlay and sinus grafting prior to implant placement. Our cell isolation protocol employed human fetal calvaria tissue sequentially digested with trypsin and collagenase. The HOB cells from only the third and fourth digests were obtained, cultured and evaluated within the constructs. An osteoblast-like phenotype was in part verified for these HOB cells by demonstrating a significantly higher alkaline phosphatase activity than for human gingival fibroblasts, and a comparable level to the osteoblast cell line MG-63. The HOB cells were cultured within either poly (D,L-lactide) (PLA) or a fused fiber ceramic and evaluated for the ability to support in vitro HOB amplification. HOB proliferation was validated by scanning electron microscopy, identifying cells throughout the 3-D constructs. Continuous cell viability was demonstrated for the duration of the 33-day evaluation period and the extent of cell amplification reached approximately 20 times the seeding density. The in vitro amplification results further indicate that tissue engineering strategies with either the PLA or fused fiber construct may be suitable for bone regeneration therapy for dental implants.

Biomedical Engineering↗

PubMed1998

[The recent advances in the study of tissue engineering tendon].

This paper reviewed the main achievements in the research on tissue engineering tendon, focusing on major problems concerning the substitute for extracellular matrix (ECM) of tendon, biological characteristics of tendon cells, and tendon cells compounding with ECM substitute. It was concluded the important problems in the study of the tissue engineering having specific reparative functions could be: to prepare the ECM materials suitable for the tendon cells to attach, grow, and function; to establish the tendon cell line whose growth, proliferation, and immunological antigenicity could be modulated and controlled, and simulating the mechanical environment of tendon in vivo, to adopt three-dimensional tendon cell culture method.

Animals↗

PubMed2006

Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering.

The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissue-engineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (+/-0.1) microg/mg compared to 0.1 (+/-0.1) microg/mg in decellularised bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility. Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery and tissue-engineering applications.

Animals↗

PubMed2007

Human mesenchymal progenitor cell responses to a novel textured poly(L-lactide) scaffold for ligament tissue engineering.

Biocompatibility and cell seeding capability of a new cell scaffold made of textured polylactic acid (PLA) fibers was investigated as a new material for tissue engineering of anterior cruciate ligaments (ACL). Adhesion and proliferation of human mesenchymal progenitor cells (MPC) was investigated after 15 days by scanning electron microscopy and standard histology. Expression of collagen type I and III, fibronectin, tenascin C, decorin, smooth muscle actin, and the matrix metalloproteinases MMP-1 and MMP-2, as well as their tissue inhibitors TIMP-1 and TIMP-2 was analyzed using real-time PCR. Protein expression of collagen I and III, tenascin C, and proliferating nuclear antigen (PCNA) was determined by immunohistology. Apoptosis was analyzed by detection of p53 expression and TUNEL staining. MPC seeded the scaffold homogeneously and showed good cell growth and no increased rate of apoptosis. After 15 days, the matrix forming genes collagen type I, tenascin C, and decorin were upregulated, indicating the formation of a ligament-like matrix. MMP-1 and TIMP-1 were also significantly increased, suggesting initial matrix remodeling. It was concluded that the new porous PLA scaffold allowed homogeneous cell seeding, a fibroblastic phenotype and the production of a ligament-like matrix and, therefore, might be a suitable cell carrier for ACL tissue engineering.

Apoptosis↗

PubMed2006

Extracellular matrix dynamics associated with tissue-engineered intravascular sclerotherapy.

BACKGROUND: The extracellular dynamics after intravascular sclerotherapy with an injectable, fibroblast-based engineered construct is unknown. METHODS: Rabbits underwent ethanol sclerotherapy of a jugular vein segment. Control animals (n = 40) underwent no further treatment or an acellular collagen hydrogel was injected. Experimental animals (n = 20) received a tissue-engineered construct. After 1, 2, 4, and 20 to 24 weeks, segments were evaluated for collagen, glycosaminoglycan (GAG), matrix metalloproteinase (MMP) 2 and 9, and tissue inhibitors of MMP (TIMPs) 1 and 2 and scored on a scale of 0 to 3. Groups and time points were compared using nonparametric statistical analysis. RESULTS: Collagen content was higher in animals that received fibroblasts (P < .05). Glycosaminoglycan analysis showed a higher grade only at 1 week (P < .05). Collagen and GAG deposition were prominent at weeks 1 through 4, and decreased over time. Both MMP-2 and MMP-9 and TIMP-1 and TIMP-2 grade decreased with time (P < .01) in all groups, with no differences between groups. CONCLUSION: Enhancement of intravascular sclerotherapy by tissue engineering stems, at least in part, from increased local deposition of collagen and GAG. MMP and TIMPs may play a role in recanalization after experimental sclerotherapy. Tissue engineering may be a valuable adjunct for the treatment of vascular malformations.

Animals↗

PubMed2006

[Experimental study on cryopreservation of seeding cells of tissue engineered tendons].

This study sought to find out a good way for the cryopreservation of tendon seeding cells so as to facilitate the preparation of tissue engineering tendons as products. The related questions are how different factors affect cell survival rate at the procedure of preservation and whether cryopreservation affects seeding cells' biological characters as well as collagen secretive function. The results of experiment indicate that DMSO is a more effective cryoprotectant in cryopreservation of tissue engineered tendon seeding cells. Blood serum nourishment is very important in cell culture, preservation and treatment. The same sustenance after cryopreservation increases cell survival rate. In the process of cryopreservation, the concentration of cells is important to cell survival rate; cell survival rate will decrease when it is less than 1.0 x 10(6)/ml. In the process of cryopreservation, the cooling speed is also important to cell survival rate, slow cooling method achieves higher cell survival rate than does the rapid cooling method. Cryopreservation by use of 10%DMSO+15%FCS+75%DMEM does not affect seeding cells' collagen secretive function greatly and does not affect seeding cells' growth curve, cell cycle and chromosome mode obviously. The prescription of 10%DMSO +15%FCS+75%DMEM is suited for the cryopreservation of tendon seeding cells.

Cell Count↗

PubMed2004

PDLLA/Bioglass composites for soft-tissue and hard-tissue engineering: an in vitro cell biology assessment.

The aim of this study was to examine the effect of increased content of 45S5 Bioglass (0-40 wt%) in poly(dl-lactic acid) (PDLLA) porous foams on the behaviour of MG-63 (human osteosarcoma cell line) and A549 cells (human lung carcinoma cell line). The ability of these cell lines to grow on bioactive composites was quantitatively investigated in order to assess the potentiality for their use in hard and soft-tissue engineering. Two hours after cell seeding, an increase of cell adhesion according to the increased content of Bioglass((R)) present in the foams for both cell types was observed. Cell proliferation studies performed over a period of 4 weeks showed a better aptitude of the A549 cells to proliferate on PDLLA foams containing 5 wt% Bioglass when compared to the proliferation on foams with 40 wt% Bioglass. A lower proliferation rate was obtained for cells on pure PDLLA. Scanning electron microscopy analysis showed for both cell types the presence of cells inside the porous structure of the foams. These results confirmed the biocompatibility of PDLLA/Bioglass composite foams and the positive effect of Bioglass on MG-63 cell behaviour and also showed for the first time the possibility for human lung epithelial type II cells to adhere and proliferate on these porous scaffolds. In addition, we describe a positive effect of 45S5 Bioglass on A549 cell behaviour in a dose-dependent manner, indicating the potential of using PDLLA/Bioglass composites with an optimal concentration of 45S5 Bioglass not only in bone tissue engineering but also in lung tissue engineering.

Biocompatible Materials↗

PubMed2001

Tissue-engineered bone repair of sheep cranial defects with autologous bone marrow stromal cells.

Cranial bone defect remains a major challenge to craniofacial surgeons because of limited availability of autologous bone graft to repair the defects and the donor site defects secondary to tissue harvesting. In contrast, tissue-engineering technique can generate a large bone tissue using small amount of autologous cells and therefore avoid these problems. Bone Marrow Stromal Cells (MSCs) have the potential of multi-lineage (including osteogenic) differentiation. The objective of this study was to investigate the potential of using autologous MSCs to repair cranial bone defects by a tissue-engineering approach. Autologous MSCs were isolated from eight adult sheep respectively and were in vitro expanded and induced to become osteogenic cells. Bilateral full-thickness defects (20 mm in diameter) of parietal bones were created in animals and the bone defects were either repaired with the bone implants constituted with MSCs and calcium alginate at the experimental side (n = 8) or treated with calcium alginate only without MSCs (n = 4) or left unrepaired (n = 4) at the control side. New bone tissues were observed either grossly or histologically at the defects of experimental group as early as 6 weeks post-repairing, but not in control groups. The engineered bone tissue became more mature at 18 weeks post-repairing. Three-dimensional computerized tomography (CT) scan revealed an almost complete repair of the defect of experimental group at 18 weeks. This study may provide insight for future clinical repair of cranial defect.

Alginates↗

PubMed2006

Zonal release of proteins within tissue engineering scaffolds.

The manufacture of a scaffold for tissue engineering applications that can control the location and timing of growth factor release is described. The scaffold is formed by the sintering of poly(DL-lactic acid) (P(DL)LA) microparticles, plasticized with poly(ethylene glycol) (PEG), although the method can be used for many other polymer types. The microparticles were loaded with model proteins, trypsin and horseradish peroxidase (HRP), or recombinant human bone morphogenetic protein-2 (rhBMP-2). Entrapment efficiencies above 75% were achieved using a solid-in-oil-in-water system. Controlled release of active protein was achieved for at least 30 days. Microparticles were built into protein-loaded or protein-free layers and release of the protein was restricted to zones within the scaffold. Cell response to rhBMP-2 was tuneable by changing the dose of the rhBMP-2 released by varying the ratio of protein-loaded and protein-free microparticles within scaffolds. Zonal activity of rhBMP-2 on C2C12 cells was demonstrated. The scaffolds may find utility in applications where gradients of growth factors within 3D templates are required or where zonation of tissue growth is required.

Animals↗

PubMed2005

Nano- and micro-fiber combined scaffolds: a new architecture for bone tissue engineering.

One possible interesting way of designing a scaffold for bone tissue engineering is to base it on trying to mimic the biophysical structure of natural extracellular matrix (ECM). This work was developed in order to produce scaffolds for supporting bone cells. Nano and micro fiber combined scaffolds were originally produced from starch based biomaterials by means of a fiber bonding and a electrospinning, two step methodology. The cell culture studies with SaOs-2 human osteoblast-like cell line and rat bone marrow stromal cells demonstrated that presence of nanofibers influenced cell shape and cytoskeletal organization of the cells on the nano/micro combined scaffolds. Moreover, cell viability and Alkaline Phosphatase (ALP) activity for both cell types was found to be higher in nano/micro combined scaffolds than in control scaffolds based on fiber meshes without nanofibers. Consequently, the developed structures are believed have a great potential on the 3D organization and guidance of cells that is provided for engineering of 3-dimensional bone tissues.

Alkaline Phosphatase↗

PubMed2005

Microtensile testing of collagen fibril for cardiovascular tissue engineering.

As a candidate scaffold material for cardiac tissue engineering, Type I collagen material shows different mechanical properties due to the differences in the structural features such as orientation of fibril alignment and the environmental factors such as ultraviolet (UV) irradiation. In this article one-dimensional tensile stress tests were performed with the use of a nanoscale tensile tester on four groups of Type I collagen samples that were differentiated in terms of fibril orientation and/or UV irradiation. The collagen surface structure was studied with the use of an atomic force microscope (AFM). Effects of fibril orientation and UV irradiation on their mechanical properties were investigated. The mechanical properties and deformation mechanisms are discussed in conjunction with collagen structure, fibril orientation, and dispersion of the generated crosslinking by UV irradiation.

Biomechanical Phenomena↗

PubMed1999

De novo reconstitution of a functional mammalian urinary bladder by tissue engineering.

Human organ replacement is limited by a donor shortage, problems with tissue compatibility, and rejection. Creation of an organ with autologous tissue would be advantageous. In this study, transplantable urinary bladder neo-organs were reproducibly created in vitro from urothelial and smooth muscle cells grown in culture from canine native bladder biopsies and seeded onto preformed bladder-shaped polymers. The native bladders were subsequently excised from canine donors and replaced with the tissue-engineered neo-organs. In functional evaluations for up to 11 months, the bladder neo-organs demonstrated a normal capacity to retain urine, normal elastic properties, and histologic architecture. This study demonstrates, for the first time, that successful reconstitution of an autonomous hollow organ is possible using tissue-engineering methods.

Animals↗

PubMed2001

Evaluation of ultra-thin poly(epsilon-caprolactone) films for tissue-engineered skin.

Various natural and synthetic polymeric materials have been used as scaffold matrices for tissue-engineered skin. However, the commercially available skin replacement products pose problems of poor mechanical properties and immunological rejection. We have thus developed a film of 5 microm thickness, via biaxial stretching of poly(epsilon-caprolactone) (PCL), as a potential matrix for living skin replacements. The aim of this study was to evaluate the feasibility of using biaxially stretched PCL films as matrices for culturing human dermal fibroblasts. For this purpose, we cultured human dermal fibroblasts for 7 days on the films. Glass cover slips and polyurethane (PU) sheets were used as controls. The data from phase contrast light, confocal laser, and scanning electron microscopy suggested that biaxially stretched PCL films support the attachment and proliferation of human dermal fibroblasts. Thymidine-labeling results showed quantitatively that cell proliferation on the PCL films was superior to that on the PU samples. These results indicated that biaxially stretched PCL films supported the growth of human dermal fibroblasts and might have potential to be applied in tissue engineering a dermal equivalent or skin graft.

Bioprosthesis↗

PubMed2003

Role of mechanics in vascular tissue engineering.

There is a clinical need for the tissue engineering of a small diameter blood vessel substitute. Ideally such a vascular substitute should possess the functional attributes of the native vessel which it will replace. This means not only having the appropriate mechanical properties, but also being non-thrombogenic and exhibiting vasoactivity. In each of these there is in some way a role of mechanics. For thrombogenicity, an "endothelial-like" lining is required, one which is responsive to its mechanical environment in a manner similar to that of the normal vascular endothelium. If vasoactivity is exhibited, then this lining must also serve as a signal transduction interface, communicating with the underlying smooth muscle cells which themselves must be of a contractile phenotype if they are to carry out the biomechanical function resulting in contraction and dilation. Having appropriate mechanical properties means not only exhibiting sufficient strength, but also a viscoelasticity which allows for a compliance that matches that of the vascular system into which the vascular substitute is to be placed. Finally, once implanted the biological responses which will occur at least in part are regulated by biomechanical factors. Thus, the proper incorporation of biomechanics into the design of a vascular substitute is critical to achieving success.

Biomechanical Phenomena↗

PubMedDate not supplied

Automatic algorithm for generating complex polyhedral scaffold structures for tissue engineering.

In this article, an approach for tissue-engineering (TE) scaffold fabrication by way of integrating computer-based medical imaging, computer graphics, data manipulation techniques, computer-aided design (CAD), and rapid prototyping (RP) technologies is introduced. The aim is to provide a generic solution for the production of scaffolds that can potentially meet the diverse requirements of TE applications. In the work presented, a novel parametric library of open polyhedral unit cells is developed to assist the user in designing the microarchitecture of the scaffold according to the requirements of its final TE application. Once an open polyhedral unit cell design is selected and sized, a specially developed algorithm is employed to assemble the microarchitecture of the scaffold while adhering to the external geometry of the patient's anatomy generated from medical imaging data. RP fabrication techniques are then employed to build the scaffolds according to the CAD-generated designs. The combined application of such technologies promises unprecedented scaffold qualities with spatially and anatomically accurate three-dimensional forms as well as highly consistent and reproducible microarchitectures. The integrated system also has great potential in providing new cost-effective and rapid solutions to customized made-to-order TE scaffold production.

Algorithms↗

PubMed2005

Esophageal epithelial cell interaction with synthetic and natural scaffolds for tissue engineering.

As an initial step towards a tissue-engineered esophagus, rat esophageal epithelial cells (REEC) were isolated and characterized for epithelial identity, adhesion protein preference, and in vitro interaction with natural and synthetic scaffolds. The scaffolds consisted of AlloDerm (LifeCell Corporation, Branchburg, NJ), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic) acid (75:25) (PLGA75), poly(lactic-co-glycolic) acid (50:50) (PLGA50), and polycaprolactone/poly(L-lactic acid) (50:50) (PCL/PLLA). Various factors-including calcium concentration, scaffold composition, and pore size--were evaluated for their influence on epithelial growth and differentiation. By day 18, keratinocytes seeded on AlloDerm cultured under high Ca(++) (1.5mm) conditions showed a proliferating basal cell layer, epithelial stratification (5--6 layers) and a thick keratin layer. The synthetic scaffolds (PLGA, PLLA, PCL/PLLA) also showed complete surface coverage, regions of proliferating basal cells, and evidence of stratification (2--3 layers) and keratinization. The highly porous nature of the synthetic scaffolds, however, limited the formation of a continuous epithelial layer and resulted in a lack of overall spatially-defined differentiation. In conclusion, rat esophageal epithelial cells were successfully isolated and characterized, with cells seeded on AlloDerm showing superior epithelial organization and stratification compared to synthetic scaffolds. Modification of the synthetic scaffold's surface properties and pore size may be necessary to mimic epithelial behavior on natural scaffolds.

Animals↗

PubMed2002

Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds.

Macroporous scaffolds composed of biodegradable polymers have found extensive use as three-dimensional substrates either for in vitro cell seeding followed by transplantation, or as conductive substrates for direct implantation in vivo. Methods abound for creation of macroporous scaffolds for tissue engineering, and common methods typically employ a solid porogen within a three-dimensional polymer matrix to create a well-defined pore size, pore structure, and total scaffold porosity. This study describes an approach to impart improved pore interconnectivity to polymer scaffolds for tissue engineering by partially fusing the solid porogen together prior to creation of a continuous polymer matrix. Three dimensional, porous scaffolds of the copolymer 85:15 poly(lactide-co-glycolide) were fabricated via either a solvent casting/particulate leaching process, or a gas foaming/particulate leaching process. Prior to creation of a continuous polymer matrix the NaCl crystals, which serve as the solid porogen, are partially fused via treatment in 95% humidity. Scanning electron micrographs clearly display fused salt crystals and an enhancement in pore interconnectivity in the salt fused scaffolds prepared via both solvent casting and gas foaming, and the extent of pore interconnectivity is enhanced with longer treatment times. Fusion of salt crystal for 24 h increased the radius of curvature of salt crystals, and led to a twofold increase in the compressive modulus of solvent cast scaffolds (total porosity of 97 +/- 1%). Fusion of NaCl crystals prior to gas foaming resulted in a decrease in scaffold compressive modulus from 277 +/- 60k Pa to 187 +/- 30k Pa (total porosity of 94 +/- 1%). The resulting highly interconnected scaffolds have implications for facilitated cell migration, abundant cell-cell interaction, and potentially improved neural and vascular growth within tissue engineering scaffolds.

Biocompatible Materials↗