Showing papers in "Tissue Engineering in 2001"

Multilineage cells from human adipose tissue: implications for cell-based therapies.

Abstract: Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

The Design of Scaffolds for Use in Tissue Engineering. Part I. Traditional Factors

Abstract: In tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate mammalian cells and guide their growth and tissue regeneration in three dimensions. However, existing three-dimensional scaffolds for tissue engineering proved less than ideal for actual applications, not only because they lack mechanical strength, but they also do not guarantee interconnected channels. In this paper, the authors analyze the factors necessary to enhance the design and manufacture of scaffolds for use in tissue engineering in terms of materials, structure, and mechanical properties and review the traditional scaffold fabrication methods. Advantages and limitations of these traditional methods are also discussed.

Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition.

Abstract: The aim of this study was to determine the influence of two key scaffold design parameters, void fraction (VF) and pore size, on the attachment, growth, and extracellular matrix deposition by sever...

Biodegradable polymer scaffolds with well-defined interconnected spherical pore network.

Abstract: Scaffolding plays pivotal role in tissue engineering. In this work, a novel processing technique has been developed to create three-dimensional biodegradable polymer scaffolds with well-controlled interconnected spherical pores. Paraffin spheres were fabricated with a dispersion method, and were bonded together through a heat treatment to form a three-dimensional assembly in a mold. Biodegradable polymers such as PLLA and PLGA were dissolved in a solvent and cast onto the paraffin sphere assembly. After dissolving the paraffin, a porous polymer scaffold was formed. The fabrication parameters were studied in relation to the pore shape, interpore connectivity, pore wall morphology, and mechanical properties of the polymer scaffolds. The compressive modulus of the scaffolds decreased with increasing porosity. Longer heat treatment time of the paraffin spheres resulted in larger openings between the pores of the scaffolds. Foams of smaller pore size (100-200 μm) resulted in significantly lower compressive mod...

Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion.

Abstract: Directed cell adhesion remains an important goal of implant and tissue engineering technology. In this study, surface energy and surface roughness were investigated to ascertain which of these properties show more overall influence on biomaterial–cell adhesion and colonization. Jet impingement was used to quantify cellular adhesion strength. Cellular proliferation and extracellular matrix secretion were used to characterize colonization of 3T3MC fibroblasts on: HS25 (a cobalt based implant alloy, ASTM F75), 316L stainless steel, Ti-6Al4V (a titanium implant alloy), commercially pure tantalum (Ta), polytetrafluoroethylene (PTFE), silicone rubber (SR), and high-density polyethylene (HDPE). The metals exhibited a nearly five-fold greater adhesion strength than the polymeric materials tested. Generally, surface energy was proportional to cellular adhesion strength. Only polymeric materials demonstrated significant increased adhesion strength associated with increased surface roughness. Cellular adhesion on me...

Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells.

Abstract: Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, β-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D3, adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers—leptin, lipoprotein lipase, and peroxisome proliferator activated receptor γ2—are reduced...

Mechanisms of Enhanced Osteoblast Adhesion on Nanophase Alumina Involve Vitronectin

Abstract: The role, including concentration, conformation, and bioactivity, of adsorbed vitronectin in enhancing osteoblast adhesion on nanophase alumina was investigated in the present study. Vitronectin adsorbed in a competitive environment in the highest concentration on nanophase alumina compared to conventional alumina. Enhanced adsorption of vitronectin on nanophase alumina was possibly due to decreased adsorption of apolipoprotein A-I and/or increased adsorption of calcium on nanophase alumina. In a novel manner, the present study utilized surface-enhanced Raman scattering (SERS) to determine the conformation of vitronectin adsorbed on nanophase alumina. These results provided the first evidence of increased unfolding of vitronectin adsorbed on nanophase alumina. Increased adsorption of calcium on nanophase alumina may affect the conformation of adsorbed vitronectin specifically to promote unfolding of the macromolecule to expose cell-adhesive epitopes recognized by specific cell-membrane receptors. Results ...

Differentiation of Osteoblasts and in Vitro Bone Formation from Murine Embryonic Stem Cells

Abstract: Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.

Thermo-Responsive Culture Dishes Allow the Intact Harvest of Multilayered Keratinocyte Sheets without Dispase by Reducing Temperature

Abstract: To develop new technology for harvesting transplantable cultured epithelium without dispase treatment, human keratinocytes were plated on culture dishes grafted with a thermo-responsive polymer, po...

Tissue-Engineered Fabrication of an Osteochondral Composite Graft Using Rat Bone Marrow-Derived Mesenchymal Stem Cells

Abstract: This study tested the tissue engineering hypothesis that construction of an osteochondral composite graft could be accomplished using multipotent progenitor cells and phenotype-specific biomaterials. Rat bone marrow-derived mesenchymal stem cells (MSCs) were culture-expanded and separately stimulated with transforming growth factor-beta1 (TGF-beta1) for chondrogenic differentiation or with an osteogenic supplement (OS). MSCs exposed to TGF-beta1 were loaded into a sponge composed of a hyaluronan derivative (HYAF-11) for the construction of the cartilage component of the composite graft, and MSCs exposed to OS were loaded into a porous calcium phosphate ceramic component for bone formation. Cell-loaded HYAFF-11 sponge and ceramic were joined together with fibrin sealant, Tisseel, to form a composite osteochondral graft, which was then implanted into a subcutaneous pocket in syngeneic rats. Specimens were harvested at 3 and 6 weeks after implantation, examined with histology for morphologic features, and stained immunohistochemically for type I, II, and X collagen. The two-component composite graft remained as an integrated unit after in vivo implantation and histologic processing. Fibrocartilage was observed in the sponge, and bone was detected in the ceramic component. Observations with polarized light indicated continuity of collagen fibers between the ceramic and HYAFF-11 components in the 6-week specimens. Type I collagen was identified in the neo-tissue in both sponge and ceramic, and type II collagen in the fibrocartilage, especially the pericellular matrix of cells in the sponge. These data suggest that the construction of a tissue-engineered composite osteochondral graft is possible with MSCs and different biomaterials and bioactive factors that support either chondrogenic or osteogenic differentiation.

Evaluation of Nanostructured Composite Collagen–Chitosan Matrices for Tissue Engineering

Abstract: The development of suitable three-dimensional matrices for the maintenance of cellular viability and differentiation is critical for applications in tissue engineering and cell biology. The structure and composition of the extracellular matrix (ECM) has been shown to modulate cell behavior with respect to shape, movement, proliferation, and differentiation. Although collagen and chitosan have separately been proposed as in vitro ECM materials, the influence of chitosan-collagen composite matrices on cell morphology, differentiation, and function is not well studied. To this end, gel matrices of different proportions of collagen and chitosan were examined ultrastructurally and characterized for their ability to regulate cellular activity. A three-chamber system with circulating hydraulic fluids was used to evaluate the gel stability under fluid force. Results indicated that overall matrix integrity increased with the proportion of chitosan. Scanning electron microscopy indicated that the addition of chitos...

Two-Dimensional Manipulation of Cardiac Myocyte Sheets Utilizing Temperature-Responsive Culture Dishes Augments the Pulsatile Amplitude

Abstract: Although cardiac myocytes adherent to tissue culture polystyrene (TCPS) dishes retain the spontaneous beating, the pulsatile amplitude is highly limited compared to that in vivo. One of the main reasons for the limited pulsation may be the interface between the cells and the TCPS surfaces. Release of these cells from rigid TCPS surfaces may augment their pulsatile amplitude. With this perspective, we have developed a novel cell manipulation technique to detach cultured cardiac myocytes from rigid surfaces and to rescue higher pulsatile amplitude of the cells using temperature-responsive culture dishes and discuss the possibility of improving this heart tissue model. Primary cardiac myocytes were cultured on the slightly hydrophobic dish surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide). Cells adhered and proliferated, forming confluent cardiac myocyte sheets in a fashion similar to those on ungrafted TCPS dishes. Decrease in culture temperature resulted in surface change of the polymer from slight hydrophobic to highly hydrophilic due to extensive hydration of the grafted polymer on the dishes. This results in release of cardiac myocyte sheets from the dishes without enzymatic or EDTA treatment. When no support was used, the detached cardiac myocyte sheets shrank to one-tenth size, which ceased their pulsation. When chitin membranes were used to support the confluent sheets to prevent cell shrinkage, the detached cell sheets could be transferred and readily adhered onto another virgin TCPS dishes. These transferred cell sheets preserved the similar cell morphology and pulsation to those before the detachment. When polyethylene meshes were used to support cell sheet transfer, detached cardiac myocyte sheets partially attached to the mesh threads. Then, the constructs were inverted and placed in another culture dish to prevent direct association to dish surfaces. Moreover, the cardiac myocyte sheets were reorganized to heart tissue-like structures by the unisotropic contraction orientated by the mesh threads, and the pulsatile amplitude increased more than 10 times higher. This technique would bring about new insight in tissue engineering as well as cultured heart model.

Toward Tissue Engineering of the Knee Meniscus

Abstract: This review details current efforts to tissue engineer the knee meniscus successfully. The meniscus is a fibrocartilaginous tissue found within the knee joint that is responsible for shock absorption, load transmission, and stability within the knee joint. If this tissue is damaged, either through tears or degenerative processes, then deterioration of the articular cartilage can occur. Unfortunately, there is a dearth in the amount of work done to tissue engineer the meniscus when compared to other musculoskeletal tissues, such as bone. This review gives a brief overview of meniscal anatomy, biochemical properties, biomechanical properties, and wound repair techniques. The discussion centers primarily on the different components of attempting to tissue engineer the meniscus, such as scaffold materials, growth factors, animal models, and culturing conditions. Our approach for tissue engineering the meniscus is also discussed.

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

Abstract: 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.

The growth of tissue engineering.

Abstract: This report draws upon data from a variety of sources to estimate the size, scope, and growth rate of the contemporary tissue engineering enterprise. At the beginning of 2001, tissue engineering research and development was being pursued by 3,300 scientists and support staff in more than 70 startup companies or business units with a combined annual expenditure of over $600 million. Spending by tissue engineering firms has been growing at a compound annual rate of 16%, and the aggregate investment since 1990 now exceeds $3.5 billion. At the beginning of 2001, the net capital value of the 16 publicly traded tissue engineering startups had reached $2.6 billion. Firms focusing on structural applications (skin, cartilage, bone, cardiac prosthesis, and the like) comprise the fastest growing segment. In contrast, efforts in biohybrid organs and other metabolic applications have contracted over the past few years. The number of companies involved in stem cells and regenerative medicine is rapidly increasing, and this area represents the most likely nidus of future growth for tissue engineering. A notable recent trend has been the emergence of a strong commercial activity in tissue engineering outside the United States, with at least 16 European or Australian companies (22% of total) now active.

Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes.

Abstract: The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.

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

Abstract: 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.

Liver Tissue Engineering: A Role for Co-culture Systems in Modifying Hepatocyte Function and Viability

Abstract: A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

Development and characterization of tissue-engineered aortic valves.

Abstract: Tissue-engineered aortic valves, known as recellularized heart valves, were developed by seeding human neonatal fibroblasts onto decellularized, porcine aortic valves. Recellularized heart valves w...

Micropatterned Schwann cell-seeded biodegradable polymer substrates significantly enhance neurite alignment and outgrowth

Abstract: Biomimetic strategies were employed to promote directional outgrowth of neurites in vitro by using a synergistic combination of physical, chemical, and cellular cues. Compression molded and solvent...

Functional Development of Engineered Skeletal Muscle from Adult and Neonatal Rats

Abstract: A myooid is a three-dimensional skeletal muscle construct cultured from mammalian myoblasts and fibroblasts. The purpose was to compare over several weeks in culture the morphology, excitability, and contractility of myooids developed from neonatal and adult rat cells. The hypotheses tested were as follows: (1) baseline forces of myooids correlate with the cross-sectional area (CSA) of the myooids composed of fibroblasts, and (2) peak isometric tetanic forces normalized by total CSA (specific P(o)) of neonatal and adult rat myooids are not different. Electrical field stimulation was used to measure the excitability and peak tetanic forces. The proportion of the CSA composed of fibroblasts was greater for neonatal (40%) than adult (17%) myooids. For all myooids the baseline passive force normalized by fibroblast CSA (mean = 5.5 kPa) correlated with the fibroblast CSA (r(2) = 0.74). A two-element cylindrical model was analyzed to determine the contributions of fibroblasts and myotubes to the baseline force. At each measurement period, the specific P(o) of the adult myooids was greater than that of the neonatal myooids. The specific P(o) of the adult myooids was approximately 1% of the control value for adult muscles and did not change with time in culture, while that of neonatal myooids increased.

A new strategy to produce sustained growth of central nervous system axons: continuous mechanical tension.

Abstract: Although a primary strategy to repair spinal cord and other nerve injuries is to bridge the damage with axons, producing axons of sufficient length and number has posed a significant challenge. Here, we explored the ability of integrated central nervous system (CNS) axons to grow long distances in response to continuous mechanical tension. Neurons were plated on adjacent membranes and allowed to integrate, including the growth of axons across a 50-microm border between the two membranes. Using a microstepper motor system, we then progressively separated the two membranes further apart from each other at the rate of 3.5 microm every 5 min. In the expanding gap, we found thick bundles comprised of thousands of axons that responded to this tensile elongation by growing a remarkable 1 cm in length by 10 days of stretch. This is the first evidence that the center portion of synapsed CNS axons can exhibit sustained "stretch-induced growth." This may represent an important growth mechanism for the elongation of established white matter tracts during development. We also found by doubling the stretch rate to 7 microm/5 min that the axon bundles could not maintain growth and disconnected in the center of the gap by 3 days of stretch, demonstrating a tolerance limit for the rate of axonal growth. We propose that this newfound stretch-induced growth ability of integrated CNS axons may be exploited to produce transplant materials to bridge extensive nerve damage.

Validation of a Simple Histological-Histochemical Cartilage Scoring System

Abstract: In this study, we assessed the validity of a subjective histological-histochemical scoring system as compared to an automated histomorphometry program for analyzing cartilage repair tissue. In the first part of the study, we assessed the ability of the human eye to estimate the percent cartilage in a histological section. Twenty-nine rabbit periosteal explants that had been cultured in agarose transforming growth factor-beta (TGF-beta) were selected so that the percentage of cartilage in the specimens was distributed equally from 0% to 100%. Color photomicrographs were evaluated by 5 expert observers who gave a visual estimate of the percent cartilage. There was a strong correlation between the estimated and actual percent cartilage (R(2) = 0.92, p < 0.0001) and among the observers (I.C.C. = 0.89). On average, the estimated percent cartilage was within ten percent of the actual percent measured. In the second part, we compared the data derived using a simple cartilage score with those obtained by automated image analysis. The histological slides from 159 explants cultured under various experimental conditions (14 treatment groups) in two different experiments were analyzed. The cartilage content was estimated visually and a score from 0 to 3 was assigned. A previously validated, computerized image analysis system was used to measure the actual percent cartilage. Statistical analyses revealed a good linear regression (R(2) = 0.84, p = 0.0001), and even better polynomial correlation between the actual measurement and the score (R(2) = 0.88, p = 0.0001). These data demonstrate the validity of a simple histological-histochemical subjective scoring system. A computerized automated program such as the one employed in this study is preferable due to its many advantages. However, a subjective scoring system may be appropriate to use when the funding and expertise required for a computerized image analysis program are not available.

Cultured Chondrocytes Produce Injectable Tissue-Engineered Cartilage in Hydrogel Polymer

Abstract: The purpose of this study was to determine if chondrocytes cultured through several subcultures at very low plating density would produce new cartilage matrix after being reimplanted in vivo with or without a hydrogel polymer scaffold. Chondrocytes were initially plated in low-density monolayer culture, grown to confluence, and passaged four times. After each passage cells were suspended in purified porcine fibrinogen and injected into the subcutaneous space of nude mice while simultaneously polymerizing with thrombin to reach a final concentration of 40 million cells/cc. Controls were made by injecting fresh, uncultured cells with fibrin polymer and by injecting the cultured cells in saline (without polymer). All samples were harvested at 6 weeks. When injected in polymer, both fresh cells and cells that had undergone only one passage in culture produced cartilaginous nodules. Cultured cells did not produce cartilage, regardless of length of time spent in culture, when injected without polymer. Cartilage was also not recovered from samples with cells kept in culture for longer than one passage, even when provided with a polymer matrix. All samples harvested were subjected to histological analysis and assayed for total DNA, glycosaminoglycan (GAG), and type II collagen. There was histological evidence of cartilage in the groups that used fresh cells and cultured cells suspended in fibrin polymer that only underwent one passage. No other group contained areas that would be consistent with cartilage histologically. All experimental samples had a higher percent of DNA than native swine cartilage, and there was no statistical difference between the DNA content of the groups containing cultured or fresh cells in fibrin polymer. Whereas the GAG content of native cartilage was 8.39% of dry weight and fresh cells in fibrin polymer was 12.85%, cultured cells in fibrin polymer never exceded the 2.48% noted from first passage cells. In conclusion, this study demonstrates that porcine chondrocytes that have been cultured in monolayer for one passage will produce cartilage in vivo when suspended in fibrin polymer.

A novel use of alginate hydrogel as Schwann cell matrix.

Abstract: The use of bioresorbable conduits supplemented with Schwann cells (SCs) is a promising tissue engineering technique to replace nerve grafting. Alginate hydrogel (AH), as a SC tissue engineering matrix, has many advantages over previously used matrices but has not been evaluated for this purpose. In this study, the viability and proliferation of SCs together with SC function in AH was evaluated in vitro. AlamarBlue cell assay was used to monitor the viability of SCs in AH and compared to SC viability in collagen gel, fibrin glue, hyaluronic acid, Matrigel, and standard culture plate over 5 days in culture. The results showed that the viability and growth of SCs in different matrices over the culture period did not significantly differ to culture plate culture. SC function when suspended in AH was monitored using chick embryo dorsal root ganglia (CDRG) growth assay. Growth of CDRG in AH with or without SCs was compared to CDRG growth without AH matrix. After 3 days in culture, the mean length of neurite sprouting was measured. The results showed that there was neurite growth in AH but was reduced to 43% of control. The neurite growth in AH was, however, enhanced by 170% when SCs were suspended in the gel. In conclusion, AH supported SC viability and function in vitro and may be useful in peripheral nerve tissue engineering in reconstructive procedures.

Three-dimensional cell-scaffold constructs promote efficient gene transfection: implications for cell-based gene therapy.

Abstract: To date, introduction of gene-modified cells in vivo is still a critical limitation for cell-based gene therapy. In this study, based on tissue engineering techniques, we developed a three-dimensional (3-D) transfection system to be cell-based gene delivery vehicle. Human trophoblast-like ED(27) and fibroblastic NIH3T3 cells were used as model cell lines. Cells were seeded onto PET fibrous matrices and plated on polyethylene terephathalate (PET) films as 2-D transfection control. The cell-matrices and cell-films were transfected with pCMV-betagal and pEGFP (green fluorescent protein) reporter gene vectors using LipofectAmine reagent. Gene expression on 3-D versus 2-D growth surface were investigated. The effects of seeding method, seeding density, porosity of the PET matrix, and culturing time of the cell-matrix complex on cDNA transfection and expression in the 3-D cell-matrix complex were also investigated. The beta-gal assay and GFP detection showed that 3-D transfection promoted a higher gene expression level and longer expression time as compared to 2-D transfection. There existed an optimal initial cell seeding density for gene transfection of 3-D cell-matrix complex. Cells seeded on PET matrices with a lower porosity ( approximately 87%) had higher gene expression activities than cells in the matrices with a higher porosity ( approximately 90%). Also, Higher gene expression levels of beta-gal were obtained for the more uniformly seeded matrices that were seeded with a depth-filtration method. The results from this study demonstrate the potential utility of cells seeded onto 3-D fibrous matrices as cell-based gene delivery vehicle for in vitro study of gene expression or in vivo gene therapy.

Porous, resorbable, fiber-reinforced scaffolds tailored for articular cartilage repair.

Abstract: Porous 75:25 poly(D,L-lactide-co-glycolide) scaffolds reinforced with polyglycolide fibers were prepared with mechanical properties tailored for use in articular cartilage repair. Compression testing was performed to investigate the influence of physiological testing conditions, manufacturing method, anisotropic properties due to predominant fiber orientation, amounts of fiber reinforcement (0 to 20 wt, %), and viscoelasticity via a range of strain rates. Using the same testing modality, the mechanical properties of the scaffolds were compared with pig and goat articular cartilage. Results showed that mechanical properties of the scaffolds under physiological conditions (aqueous, 37 degrees C) were much lower than when tested under ambient conditions. The manufacturing method and anisotropy of the scaffolds significantly influenced the mechanical properties. The compressive modulus and yield strength proportionally increased with increasing fiber reinforcement up to 20%. From 0.01 to 10 mm/mm/min strain rate, the compressive modulus increased in a logarithmic fashion, and the yield strength increased in a semi-log fashion. The compressive modulus of the non-reinforced scaffolds was most similar to the pig and goat articular cartilage when compared using similar testing conditions and modality, but the improvement in yield strength using the stiffer scaffolds with fiber reinforcement could provide needed structural support for in vivo loads.

The use of fibrin beads for tissue engineering and subsequential transplantation.

Abstract: New biological technologies such as tissue engineering procedures require the transplantation of functionally active cells within supportive carrier matrices. This paper describes a sequential culture procedure for different types of cells. The technique includes the initial preparation of a mixed alginate-fibrin vehicle that guaranteed an initial cell proliferation and differentiation to establish a stable matrix structure, and the subsequent removal of the alginate component prior to transplantation to circumvent the problem of missing bioresorbability. The resulting biodegradable carrier is mechanically stable and promotes further tissue maturation. Chondrocytes, periosteal-derived cells, as well as nucleus pulposus cells were entrapped in fibrin-alginate beads and in fibrin beads. The results indicate a promising technical approach to create stable transplants for reconstructive surgery of cartilage and bone.

Expression of Liver-Specific Functions by Rat Hepatocytes Seeded in Treated Poly(Lactic-co-Glycolic) Acid Biodegradable Foams

Abstract: Techniques of liver replacement would benefit patients awaiting donor livers and may be a substitute for transplantation in patients whose livers can regenerate. Poly(lactic-co-glycolic acid) (PLGA) copolymers are biodegradable and have been shown to be useful as scaffolds for seeding and culturing various types of cells. In this study, foam disks were prepared from PLGA (lactic-to-glycolic mole ratio of 85:15) by lyophilization of benzene (5% w/v) solutions. These disks were then used as scaffolds for rat hepatocyte culture. Foams were coated with either a type I collagen gel (0.1% w/v), coated with gelatin (5% w/v), or treated with oxygen plasma (25 W, 90 s) to modify their surface chemistry and wettability. The disks were then seeded with rat hepatocytes (106/mL) and cultured for a period of 2 weeks. All surface treatments resulted in increased hydrophilicity, the greatest being obtained by collagen treatment (contact angle < 10°), and a minimal decrease in void fraction (5%). DNA content after a 2-wee...

Frequency- and duration-dependent effects of cyclic pressure on select bone cell functions.

Abstract: The present study demonstrated unique correlations between characteristic parameters of mechanical loading and osteoblast functions. Specifically, osteoblast proliferation was dependent on the frequency and on the duration of the applied cyclic pressure stimulus: decreased cell proliferation was only observed when these cells were exposed to cyclic pressure at 1.0-Hz (but not at 0.25-Hz) frequency for 1 h (but not for 20 min) daily for 5 days. In contrast, endothelial cells were not responsive to cyclic pressure, whereas fibroblast proliferation increased under similar test conditions. Most important, cyclic pressure affected various osteoblast genes differently: exposure of osteoblasts to cyclic pressure (at 1.0-Hz frequency for 1 h daily) resulted in enhanced transcription and translation of alkaline phosphatase after 5 days; the same mechanical stimulus, however, did not affect osteopontin mRNA expression during the same time periods. These findings provide cellular and molecular level information, which is not only important in elucidating the correlation between mechanical loading and bone homeostasis, but can be useful in development of new technology in skeletal tissue engineering.