Cell-seeded Constructs Research Articles

435 OPTIMIZATION OF A NATURAL COLLAGEN SCAFFOLD TO AID CELL–MATRIX PENETRATION FOR URETHRAL TISSUE ENGINEERING

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2010435 OPTIMIZATION OF A NATURAL COLLAGEN SCAFFOLD TO AID CELL–MATRIX PENETRATION FOR URETHRAL TISSUE ENGINEERING Lucy Liu, Shantaram Bharadwaj, Sang Lee, Anthony Atala, and Yuanyuan Zhang Lucy LiuLucy Liu More articles by this author , Shantaram BharadwajShantaram Bharadwaj More articles by this author , Sang LeeSang Lee More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Yuanyuan ZhangYuanyuan Zhang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.506AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Despite the advantages of natural collagen-based acellular matrices for urethral tissue engineering, two major obstacles currently limit their clinical application: (1) the high density of collagen on the mucosal side of the matrix hinders cell–matrix penetration when cells are seeded, and (2) retention of cellular compounds within the acellular matrix have the potential to cause chronic immunoreactions. The goal of this study was to fabricate a 3-dimensional (3-D) porous acellular scaffold derived from bladder submucosa (BSM) and recellularize the scaffold with human bladder urothelial and smooth muscle cells for use in urethral reconstruction applications of tissue engineering. METHODS Fresh porcine BSM was soaked with peracetic acid (PAA) at different concentrations (0,1, 3, 5 and 10%) and then treated with Triton X-100 for decellularization. Cellular/nuclear components and the porosity of the matrix were assessed by H&E, Masson's trichrome and 4,6-diamidino-2-phenylindole (DAPI) stains, DNA content analysis, and scanning electron microscopy. Biomechanics of the matrixes were analyzed with tensile testing. Bladder urothelial and smooth muscle cells were co-cultured on the BSM using dynamic culture conditions for one week. The cell-seeded BSM constructs were implanted in athymic mice for one month. Cell growth, cell-matrix infiltration and cell differentiation were evaluated in bladder cells seeded on decellularized BSM in vitro and in vivo. RESULTS DNA content analysis showed that the majority of nuclear material was removed from the BSM scaffold. Treatment with 5% PAA led to high porosity on the surface of the matrix with retention of less cellular material, while the treated matrix maintained about 75% of its original tensile strength. In dynamic culture, cells formed multiple, even layers on the surface of matrix. Bladder cells also penetrated deeper into the lamina propria of the treated matrix compared to untreated matrix. Immunocytochemical staining indicated that the grafted bladder cells expressed urothelial and smooth muscle specific markers both in vitro and in vivo. CONCLUSIONS This study demonstrates that decellularized/oxidized BSM possesses adequate porosity for cell infiltration into the matrix. Further, the use of decellularized/oxidized BSM combined with dynamic culture conditions significantly promoted bladder cell–matrix penetration in vitro and promoted cell growth in vivo. Scaffolds with such characteristics have potential applications in cell-based urethral tissue engineering. Winston-Salem, NC© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e172 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lucy Liu More articles by this author Shantaram Bharadwaj More articles by this author Sang Lee More articles by this author Anthony Atala More articles by this author Yuanyuan Zhang More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

In Vivo Assessment of a Tissue-Engineered Vascular Graft Combining a Biodegradable Elastomeric Scaffold and Muscle-Derived Stem Cells in a Rat Model

Limited autologous vascular graft availability and poor patency rates of synthetic grafts for bypass or replacement of small-diameter arteries remain a concern in the surgical community. These limitations could potentially be improved by a tissue engineering approach. We report here our progress in the development and in vivo testing of a stem-cell-based tissue-engineered vascular graft for arterial applications. Poly(ester urethane)urea scaffolds (length = 10 mm; inner diameter = 1.2 mm) were created by thermally induced phase separation (TIPS). Compound scaffolds were generated by reinforcing TIPS scaffolds with an outer electrospun layer of the same biomaterial (ES-TIPS). Both TIPS and ES-TIPS scaffolds were bulk-seeded with 10 x 10(6) allogeneic, LacZ-transfected, muscle-derived stem cells (MDSCs), and then placed in spinner flask culture for 48 h. Constructs were implanted as interposition grafts in the abdominal aorta of rats for 8 weeks. Angiograms and histological assessment were performed at the time of explant. Cell-seeded constructs showed a higher patency rate than the unseeded controls: 65% (ES-TIPS) and 53% (TIPS) versus 10% (acellular TIPS). TIPS scaffolds had a 50% mechanical failure rate with aneurysmal formation, whereas no dilation was observed in the hybrid scaffolds. A smooth-muscle-like layer of cells was observed near the luminal surface of the constructs that stained positive for smooth muscle alpha-actin and calponin. LacZ+ cells were shown to be engrafted in the remodeled construct. A confluent layer of von Willebrand Factor-positive cells was observed in the lumen of MDSC-seeded constructs, whereas acellular controls showed platelet and fibrin deposition. This is the first evidence that MDSCs improve patency and contribute to the remodeling of a tissue-engineered vascular graft for arterial applications.

Online Monitoring of the Mechanical Behavior of Collagen Hydrogels: Influence of Corneal Fibroblasts on Elastic Modulus

Collagen hydrogels have been widely used to model biological systems and examine cell behavior in vitro. Of increasing interest is how cells affect the mechanical characteristics of their surrounding matrix and vice versa over long culture periods. In this study, the change in mechanical properties of collagen hydrogels embedded with human corneal fibroblasts was examined over a 6-week culture period using a novel online spherical indentation system. The elastic modulus of the hydrogels was found to increase during the first 2 weeks of culture and decrease after 4 weeks in culture. The effects of actin polymerization and matrix metalloproteinase inhibitors were also examined, which verified some mechanisms involved in the alteration of the mechanical properties such as cell tensile forces and other potential factors. This online monitoring technique demonstrates the ability to examine the mechanical properties of cell-seeded constructs in response to the culture environments--in particular, the response to the addition of drugs or chemical reagents--which will provide a useful tool in studying the mechano-feedback loop between cells and their surrounding matrix.

Tissue Equivalents Based on Cell-Seeded Biodegradable Microfluidic Constructs

One of the principal challenges in the field of tissue engineering and regenerative medicine is the formation of functional microvascular networks capable of sustaining tissue constructs. Complex tissues and vital organs require a means to support oxygen and nutrient transport during the development of constructs both prior to and after host integration, and current approaches have not demonstrated robust solutions to this challenge. Here, we present a technology platform encompassing the design, construction, cell seeding and functional evaluation of tissue equivalents for wound healing and other clinical applications. These tissue equivalents are comprised of biodegradable microfluidic scaffolds lined with microvascular cells and designed to replicate microenvironmental cues necessary to generate and sustain cell populations to replace dermal and/or epidermal tissues lost due to trauma or disease. Initial results demonstrate that these biodegradable microfluidic devices promote cell adherence and support basic cell functions. These systems represent a promising pathway towards highly integrated three-dimensional engineered tissue constructs for a wide range of clinical applications.

Dynamic Culture of Osteogenic Cells in Biomimetically Coated Poly(Caprolactone) Nanofibre Mesh Constructs

In our previous work, biomimetic calcium phosphate-coated poly(caprolactone) nanofibre meshes (BCP-NMs) were demonstrated to be more effective for supporting cell attachment and proliferation under static conditions, when compared with poly(caprolactone) nanofibre meshes (PCL-NMs). In many applications, in vitro cultivation of constructs using bioreactors that support efficient nutrition of cells has appeared as an important step toward the development of functional grafts. This work aimed at studying the effects of dynamic culture conditions and biomimetic coating on bone cells grown on the nanofibre meshes. BCP-NM and PCL-NM were seeded with osteoblast-like cells (MG63--human osteosarcoma-derived cell line). The cell-seeded constructs were cultured within a rotating bioreactor that simulated microgravity, at a fixed rotating speed, for different time periods, and then characterized. Cell morphology, viability, and phenotype were assessed. PCL-NM constructs presented a higher number of dead cells than BCP-NM constructs. Under dynamic conditions, the production of proteins associated with the extracellular matrix of bone was higher on BCP-NM constructs than in the PCL-NM ones, which indicates that coated samples may provide cells with a better environment for tissue growth. It is suggested that improved mass transfer in the bioreactor in combination with the appropriate substrate were decisive factors for this highly positive outcome for generating bone.

Synthesis and microfabrication of biomaterials for soft-tissue engineering

Abstract Biomaterials synthesis and scaffold fabrication will play an increasingly important role in the design of systems for regenerative medicine and tissue engineering. These rapidly growing fields are converging as scaffold design must begin to incorporate multidisciplinary aspects in order to effectively organize cell-seeded constructs into functional tissue. This review article examines the use of synthetic biomaterials and fabrication strategies across length scales with the ultimate goal of guiding cell function and directing tissue formation. This discussion is parsed into three subsections: (1) biomaterials synthesis, including elastomers and gels; (2) synthetic micro- and nanostructures for engineering the cell–biomaterial interface; and (3) complex biomaterials systems design for controlling aspects of the cellular microenvironment.

Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone–starch scaffold and the in vitro development of vascularization

The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and organized into microcapillary-like structures. These vascular-like structures evolved from a cord-like configuration to a more complex branched morphology, had a lumen and stained in the perivascular region for type IV collagen. Genetic profiling of 84 osteogenesis-related genes was performed on co-culture vs. monoculture. Osteoblasts in co-culture showed a significant up-regulation of type I collagen and immunohistochemistry revealed that the scaffold was filled with a dense matrix stained for type I collagen. In direct contact with HDMEC hOBs secreted higher amounts of VEGF in relation to monoculture and the highest peak in the release profile correlated with the formation of microcapillary-like structures. The heterotypic communication between the two cell types was also assured by direct cell–cell contact as shown by the expression of the gap junction connexin 43. In summary, by making use of heterotypic cellular crosstalk this co-culture system is a strategy to form vascular-like structures in vitro on a 3D scaffold.

Parametric Finite Element Analysis of Physical Stimuli Resulting From Mechanical Stimulation of Tissue Engineered Cartilage

While mechanical stimulation of cells seeded within scaffolds is widely thought to be beneficial, the amount of benefit observed is highly variable between experimental systems. Although studies have investigated specific experimental loading protocols thought to be advantageous for cartilage growth, less is known about the physical stimuli (e.g., pressures, velocities, and local strains) cells experience during these experiments. This study used results of a literature survey, which looked for patterns in the efficacy of mechanical stimulation of chondrocyte seeded scaffolds, to inform the modeling of spatial patterns of physical stimuli present in mechanically stimulated constructs. The literature survey revealed a large variation in conditions used in mechanical loading studies, with a peak to peak strain of 10% (i.e., the maximum amount of deformation experienced by the scaffold) at 1 Hz on agarose scaffolds being the most frequently studied parameters and scaffold. This loading frequency was then used as the basis for simulation in the finite element analyses. 2D axisymmetric finite element models of 2x4 mm2 scaffolds with 360 modulus/permeability combinations were constructed using COMSOL MULTIPHYSICS software. A time dependent coupled pore pressure/effective stress analysis was used to model fluid/solid interactions in the scaffolds upon loading. Loading was simulated using an impermeable frictionless loader on the top boundary with fluid and solid displacement confined to the radial axis. As expected, all scaffold materials exhibited classic poro-elastic behavior having pressurized cores with low fluid flow and edges with high radial fluid velocities. Under the simulation parameters of this study, PEG scaffolds had the highest pressure and radial fluid velocity but also the lowest shear stress and radial strain. Chitosan and KLD-12 simulated scaffold materials had the lowest radial strains and fluid velocities, with collagen scaffolds having the lowest pressures. Parametric analysis showed maximum peak pressures within the scaffold to be more dependent on scaffold modulus than on permeability and velocities to depend on both scaffold properties similarly. The dependence of radial strain on permeability or modulus was more complex; maximum strains occurred at lower permeabilities and moduli, and the lowest strain occurred at the stiffest most permeable scaffold. Shear stresses within all scaffolds were negligible. These results give insight into the large variations in metabolic response seen in studies involving mechanical stimulation of cell-seeded constructs, where the same loading conditions produce very different results due to the differences in material properties.

Evaluation of a biodegradable scaffold‐ and stem cell‐based tissue‐engineered vascular graft in rat and pig models

Bi‐layered, synthetic, biodegradable, elastomeric tubular scaffolds comprised of a highly porous medial layer and an external, reinforcing fibrous layer were bulk‐seeded with allogeneic (rat studies) or autologous (pig studies), LacZ+ muscle‐derived stem cells (MDSCs) using a previously described rotational vacuum seeding device, then placed in spinner flask culture for 24 hours. Cellular integration was assessed via nuclear and cytoskeletal staining. Constructs were implanted as interposition grafts in the abdominal aorta of rats for 8 weeks or carotid arteries of pigs for 30 days. Angiogram, histology, immunostaining and SEM were performed at time of explant. In the rat studies, cell‐seeded constructs showed a higher patency rate than the unseeded controls (63% vs. 0%). A smooth muscle‐like layer of cells was observed near the luminal surface of the constructs. LacZ+ cells were shown to be engrafted in the remodeled construct. A confluent layer of vWF‐positive cells was observed in the lumen of seeded constructs, whereas controls showed thrombosis. In the pig studies to date, all TEVGs were patent, though intimal hyperplasia was observed. We have demonstrated the successful integration of allogeneic stem cell‐based TEVGs in a rat model and the feasibility of a completely autologous approach in a pig model. Support from NIH BRP #R01 HL069368.

Invited Commentary

In recent years, stem cell therapy has received considerable attention as a novel therapeutic option for refractory congestive heart failure. Although the mechanisms through which these cells elicit functional improvement are yet to be fully determined, recent studies suggest that paracrine effects, rather than direct trans-differentiation of donor cells into cardiomyocytes, may predominate in augmenting postinfarction cardiac function. Progress in cell therapy, however, is hampered by suboptimal engraftment of donor cells into the myocardium, related to the extensive cell death that ensues from the hostile milieu into which the donor cells are introduced. This problem has stimulated further investigation of the use of bioengineered scaffolds as cell delivery vehicles, with the hope that these constructs may enhance cell retention and functional recovery. In this study, Hamdi and colleagues [1Hamdi H. Furuta A. Bellamy V. et al.Cell delivery: intramyocardial injections or epicardial deposition? A head-to-head comparison.Ann Thorac Surg. 2009; 87: 1196-1204Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar] evaluated the effect of differing cell delivery techniques on postinfarct heart function. Using skeletal myoblasts as donor cells in a rat infarct model, the authors compared the conventional intramyocardial injection technique with epicardial placement of two cell-seeded constructs, one made of a double-layered myoblast cell sheet and the other constructed from a cell-seeded Gelfoam (Pharmacia & Upjohn/Pfizer, New York, NY) patch. The authors concluded that the two epicardially delivered tissue scaffolds led to significant improvements in left ventricular ejection fraction (LVEF) compared with the controls injected with culture medium, whereas intramyocardial injection prevented further deterioration of ventricular function but did not improve LVEF. Histologic analysis also showed greater angiogenesis in the two epicardially treated groups. The major quandary posed by these findings is that the effects of the varying techniques of cell transfer were unrelated to the detectable persistence of the myoblasts at the studied time point. Analysis of cell engraftment revealed minimal donor cell retention, with residual human cells detected in only 2 of 7 hearts in each of the two groups treated with the epicardial scaffold. The authors attribute the between-group differences in LVEF to donor cell effects at earlier, unevaluated time points (ie, earlier than 1 month). Their analysis of cytokines 1 month after cell delivery showed no differences between groups that might explain differences in functional recovery, but this, like the absence of persistent donor cells, may merely be a reflection of the time point chosen. Somewhat surprisingly, end-systolic and end-diastolic volumes did not differ between groups. Thus, although the cell sheets and Gelfoam patch improved LVEF, they did not do so by reducing infarct expansion compared with intramyocardial injection. Ultimately, the lack of a demonstrable mechanism to explain the differences observed between groups somewhat limits the strength of the conclusions that can be drawn about the relative merits of these different cell delivery techniques. Regardless, this is a topical study that compares the conventional intramyocardial delivery method to two potentially attractive methods of epicardial delivery. The results are provocative, and should spur further investigation to determine whether there are intrinsic benefits in using biomaterials in the setting of a recent or remote myocardial infarction. If there are, what are the mechanisms through which these constructs exert synergistic effects with the seeded cells? The optimal cell type or mixture of cell types that is best suited to a particular scaffold material has not been determined, nor have the physical, electrical, or surface characteristics that render one cell type more appropriate for seeding than another. Those studies may eventually lead to the development of off-the-shelf bioengineered cell-seeded constructs that may sidestep the issues of cell harvesting, expansion, and delivery that complicate current applications of cell therapy. Cell Delivery: Intramyocardial Injections or Epicardial Deposition? A Head-to-Head ComparisonThe Annals of Thoracic SurgeryVol. 87Issue 4PreviewMultiple needle-based injections of cells in the myocardium are associated with a low engraftment rate, which may limit the benefits of the procedure. This study used skeletal myoblasts to perform a head-to-head comparison of conventional injections with epicardial deposition of scaffold-embedded cells. Full-Text PDF

Cell Delivery: Intramyocardial Injections or Epicardial Deposition? A Head-to-Head Comparison

Multiple needle-based injections of cells in the myocardium are associated with a low engraftment rate, which may limit the benefits of the procedure. This study used skeletal myoblasts to perform a head-to-head comparison of conventional injections with epicardial deposition of scaffold-embedded cells. Four weeks after ligation-induced myocardial infarction, 40 rats were randomly allocated to receive intramyocardial injections of 5 million human skeletal myoblasts or control medium or to have the infarcted area covered with either a bilayer myoblast cell sheet prepared from a fibrin-coated culture plate or a myoblast-seeded collagen sponge (Gelfoam; Pharmacia & Upjohn, Kalamazoo, MI). End points, assessed after 1 month, included left ventricular function blindly measured by echocardiography, quantification of cell engraftment by quantitative real-time polymerase chain reaction and immunostaining, histologic assessment of fibrosis and angiogenesis, and tissue levels of host-specific angiogenic and antifibrotic cytokines. Compared with control medium- or myoblast-injected hearts, those receiving the two cell constructs demonstrated the highest recoveries of left ventricular function (p = 0.004 versus controls). Both myoblast cell sheets and myoblast-seeded Gelfoam sponges also resulted in significantly greater angiogenesis compared with controls. The Gelfoam group was associated with the best outcome with regard to the number of engrafted donor cells (p = 0.03 versus myoblasts) and the reduction of fibrosis (p = 0.02 and p = 0.04 versus the control and myoblast groups, respectively). Compared with injections, delivery of myoblasts in a construct overlaying the infarcted area is associated with better graft functionality, possibly because of maintenance of improved cell patterning. The cell-seeded Gelfoam construct was found to feature a user-friendly, reproducible, and atraumatic technique.

Interface integration of layered collagen scaffolds with defined matrix stiffness: implications for sheet-based tissue engineering

Successful application of sheet-based engineering for complex tissue reconstruction requires optimal integration of construct components. An important regulator of cellular responses (such as migration and collagen deposition) mediating interface integration is matrix stiffness. In this study we developed a sheet-based 3D model of interface integration that allows control of interface matrix stiffness. Fluid was removed from acellular or fibroblast-seeded bilayer collagen hydrogel constructs, using plastic compression to increase collagen density and matrix stiffness. Cell-seeded constructs were either compressed at day 0 and cultured for 7 days (compressed culture, high stiffness) or left uncompressed during culture and compressed on day 7 (compliant-compressed culture, low stiffness). Constructs were fitted onto a mechanical testing system to measure interface adhesive strength. Analysis of stresses by finite element modelling predicted a sharp rise of stress and rapid failure at the interface. While cell-seeded constructs showed a six-fold increase in interface adhesive strength compared to acellular control constructs (p < 0.05), there was no significant difference between low- and high-stiffness cultures after 1 week. Cell migration across the interface was greater in low- compared to high-stiffness constructs at 24 h (p < 0.05); however, no significant difference was observed after 1 week. Visualization of interfaces showed fusion of the two layers in low- but not in high-stiffness constructs after 1 week of culture. The ability to regulate cellular behaviour at an interface by controlling matrix stiffness could provide an important tool for modelling the integration of sheet-based bioengineered tissues in bioreactor culture or post-implantation.

Effects of TGF-β1 and hydrostatic pressure on meniscus cell-seeded scaffolds

The combinatorial effects of TGF-β1 and hydrostatic pressure (HP) were investigated on meniscus cell-seeded PLLA constructs using a two-phase sequential study. The objective was to identify potentially synergistic effects of these stimuli toward enhancing the biomechanical and compositional characteristics of the engineered constructs. In Phase I, the effects of TGF-β1 were examined on the ability of meniscus cells to produce ECM. In Phase II, meniscus cell-seeded PLLA constructs were cultured for 4 wks with a combination of TGF-β1 and HP (10 MPa, 0 Hz or 10 MPa, 0.1 Hz). TGF-β1 was found to increase collagen and GAG deposition in the scaffolds 15-fold and 8-fold, respectively, in Phase I. In Phase II, the combination of TGF-β1 and 10 MPa, 0 Hz HP resulted in 4-fold higher collagen deposition (additive increase), 3-fold higher GAG deposition and enhanced compressive properties (additive and synergistic increases), when compared to the unpressurized no growth factor culture control. Though significant correlations were observed between the compressive properties (moduli and viscosity), and the GAG and collagen content of the constructs, the correlations were stronger with collagen. This study provides robust evidence that growth factors and HP can be used successfully in combination to enhance the functional properties of in vitro engineered knee meniscus constructs.

Improving Linear Stiffness of the Cell-Seeded Collagen Sponge Constructs by Varying the Components of the Mechanical Stimulus

In vitro mechanical stimulation has been reported to induce cell alignment and increase cellular proliferation and collagen synthesis. Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases construct stiffness and repair biomechanics after surgery. However, these studies used a single mechanical stimulation profile, the latter composed of multiple components whose individual and combined effects on construct properties remain unknown. Thus, the purpose of this study was to understand the relative importance of a subset of these components on construct stiffness. To try to optimize the resulting mechanical stimulus, we used an iterative process to vary peak strain, cycle number, and cycle repetition while controlling cycle frequency (1 Hz), rise and fall times (25% and 17% of the period, respectively), hours of stimulation/day (8 h/day), and total time of stimulation (12 days). Two levels of peak strain (1.2 % and 2.4%), cycle number (100 and 3000 cycles/day), and cycle repetition (1 and 20) were first examined. Higher levels of peak strain and cycle number were then examined to optimize the stimulus using response surface methodology. Our results indicate that constructs stimulated with 2.4% strain, 3000 cycles/day, and one cycle repetition produced the stiffest constructs. Given the significant positive correlations we have previously found between construct stiffness and repair biomechanics at 12 weeks post-surgery, these in vitro enhancements offer the prospect of further improving repair biomechanics.

Tissue Engineering for Total Meniscal Substitution: Animal Study in Sheep Model

The aim of the study was to investigate the use of a novel hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Two different surgical implantation techniques in a sheep model were evaluated. Twenty-four skeletally mature sheep were treated with total medial meniscus replacements, while two meniscectomies served as empty controls. The animals were divided into two groups: cell-free scaffold and scaffold seeded with autologous chondrocytes. Two different surgical techniques were compared: in 12 animals, the implant was sutured to the capsule and to the meniscal ligament; in the other 12 animals, also a transtibial fixation of the horns was used. The animals were euthanized after 4 months. The specimens were assessed by gross inspection and histology. All implants showed excellent capsular ingrowth at the periphery. Macroscopically, no difference was observed between cell-seeded and cell-free groups. Better implant appearance and integrity was observed in the group without transosseous horns fixation. Using the latter implantation technique, lower joint degeneration was observed in the cell-seeded group with respect to cell-free implants. The histological analysis indicated cellular infiltration and vascularization throughout the implanted constructs. Cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. The current study supports the potential of a novel HYAFF/polycaprolactone scaffold for total meniscal substitution. Seeding of the scaffolds with autologous chondrocytes provides some benefit in the extent of fibrocartilaginous tissue repair.

Growth and differentiation of alveolar bone cells in tissue‐engineered constructs and monolayer cultures

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.

Tensile properties of engineered cartilage formed from chondrocyte- and MSC-laden hydrogels

The objective of this study was to determine the capacity of chondrocyte- and mesenchymal stem cell (MSC)-laden hydrogel constructs to achieve native tissue tensile properties when cultured in a chemically defined medium supplemented with transforming growth factor-beta3 (TGF-beta3). Cell-laden agarose hydrogel constructs (seeded with bovine chondrocytes or MSCs) were formed as prismatic strips and cultured in a chemically defined serum-free medium in the presence or absence of TGF-beta3. The effects of seeding density (10 vs 30 million cells/mL) and cell type (chondrocyte vs MSC) were evaluated over a 56-day period. Biochemical content, collagenous matrix deposition and localization, and tensile properties (ramp modulus, ultimate strain, and toughness) were assessed biweekly. Results show that the tensile properties of cell-seeded agarose constructs increase with time in culture. However, tensile properties (modulus, ultimate strain, and toughness) achieved on day 56 were not dependent on either the initial seeding density or the cell type employed. When cultured in medium supplemented with TGF-beta3, tensile modulus increased and plateaued at a level of 300-400 kPa for each cell type and starting cell concentration. Ultimate strain and toughness also increased relative to starting values. Collagen deposition increased in constructs seeded with both cell types and at both seeding densities, with exposure to TGF-beta3 resulting in a clear shift toward type II collagen deposition as determined by immunohistochemical staining. These findings demonstrate that the tensile properties, an important and often overlooked metric of cartilage development, increase with time in culture in engineered hydrogel-based cartilage constructs. Under the free-swelling conditions employed in the present study, tensile moduli and toughness did not match that of the native tissue, though significant time-dependent increases were observed with the inclusion of TGF-beta3. Of note, MSC-seeded constructs achieved tensile properties that were comparable to chondrocyte-seeded constructs, confirming the utility of this alternative cell source in cartilage tissue engineering. Further work, including both modulation of the chemical and mechanical culture environment, is required to optimize the deposition of collagen and its remodeling to achieve tensile properties in engineered constructs matching the native tissue.

Matrix stiffness and serum concentration effects matrix remodelling and ECM regulatory genes of human bone marrow stem cells

The effects of mechanical stimulation of cell-seeded collagen constructs on cell orientation, intracellular signalling and molecular responses have been widely reported. In this study we investigated in vitro the contractile responses of human bone marrow stem cells (HBMSCs) to increasing collagen gel substrate stiffness and their effect on extracellular matrix (ECM) regulatory genes. Human dermal fibroblasts (HDFs) were used as controls. Cells were cultured in 10% and 20% FCS and embedded in collagen constructs at a density of 1 million cells/ml collagen. Matrix stiffness was achieved by subjecting the constructs to three different strain regimes (0%, 5% and 10%), using a computer-driven tensional culture force monitor (t-CFM) capable of uniaxial loading. The contraction forces generated by the cells were quantified over 24 h. Molecular outputs were quantified using RT-PCR. HBMSCs significantly increased force generation to increasing serum concentration (i.e 10% to 20%). 10% FCS concentration significantly reduced contraction as pre-strain stiffness was increased in HBMSCs and HDFs (0% > 5% > 10%). However, at 20% FCS HBMSCs generated similar peak force contraction at 24 h to 5% and 10% pre-strain (0% = 5% = 10%). The ECM regulatory gene for MMP2 showed upregulation at 5% pre-strain, but a 50% downregulation when pre-strain was increased to 10%. MMP9 was upregulated at 5% pre-strain and further upregulated at 10% pre-strain. In designing tissue-engineering solutions, predictable responses of cells, embedded within bio-artificial matrices, to external mechanical forces are critical. To take into account the increasing stiffness of the matrix as increasing ECM is deposited, it would be necessary to take mechanical stimulation into account to determine predictable cellular responses.

Establishing Pediatric Cardiovascular Services in the Developing World

There is increasing realization that the lack of facilities for sustainable pediatric cardiac services in the developing world results in a massive number of preventable deaths and suffering. It is estimated that 15 million children die or are crippled annually by potentially treatable or preventable cardiac diseases. Ignored for a long time, this issue is starting to be a cause of major concern to individuals, governments, and, most importantly, cardiovascular specialists who can appreciate the gravity of the problem and that the current situation is unacceptable. What then can be done to alleviate the problem, by whom, and how? In this issue of Circulation , Larrazabal et al describe the pioneering efforts of Castenada and his colleagues in Guatemala.1 This should act as a model and a source of inspiration in this field. This editorial is an attempt to outline some of the issues that relate to the problem, such as estimates of its size, and explores potential, long-lasting solutions. Article p 1882 The pediatric population constitutes a larger proportion of the community in developing countries, with ≈40% of individuals <18 years old in some countries. Children in developing countries have a significantly higher incidence and prevalence of serious cardiac diseases.2 This is contributed to by a variety of factors that include a lack of early correction of congenital cardiac abnormalities that results in accumulation of a large number of children with uncorrected anomalies who survive the neonatal period. In addition, many of these children develop more rapid deterioration in their clinical condition as a result of accelerated forms of secondary changes in the heart and other organs, such as pulmonary hypertension. Some of these changes could be the result of genetic and/or environmental factors such as pollution or infection. Furthermore, endemic “neglected” cardiac diseases such as rheumatic …

Expression Patterns of Ets2 Protein Correlate with Bone-Specific Proteins in Cell-Seeded Three-Dimensional Bone Constructs

The transcription factor Ets2 and its transcriptional targets osteopontin (OPN) and osteocalcin (OC) are expressed in tissue-engineered bone constructs in vitro. Up to now little is known about the role of Ets2 in tissue-engineering applications. This study was intended to investigate the hypothesis that protein expression of Ets2 is correlated with the expression of bone-specific proteinsin tissue-engineeredbone constructs. Cell-seeded three-dimensional bone constructs manufactured with osteoblastic cells and poly(lactic-co-glycolic acid) polymer fleeces over a period of 21 days were analyzed by SDS-PAGE and Western blotting. The protein expression of OPN, OC, osteonectin and collagen type I was analyzed. Cellularity, alkaline phosphatase-specific activity and histology confirmed the osteoblastic phenotype of the constructs. Correlations between Ets2 expression and OPN and Ets2 and collagen type I expression could be detected during the phase of late osteoblastic differentiation between days 9 and 21. The correlation between OC and collagen type I was significant in this late stage of osteoblastic differentiation. These results suggest that there is a strong interplay of Ets2 with bone-specific proteins in cell-seeded three-dimensional bone constructs. This study is a crucial step to elucidate the complex interplay of bone-related proteins in the application of bone tissue engineering.