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PubMed2005

Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications.

In the engineering of soft tissues, scaffolds with high elastance and strength coupled with controllable biodegradable properties are necessary. To fulfill such design criteria we have previously synthesized two kinds of biodegradable polyurethaneureas, namely poly(ester urethane)urea (PEUU) and poly(ether ester urethane)urea (PEEUU) from polycaprolactone, polycaprolactone-b-polyethylene glycol-b-polycaprolactone, 1,4-diisocyanatobutane and putrescine. PEUU and PEEUU were further fabricated into scaffolds by thermally induced phase separation using dimethyl sulfoxide (DMSO) as a solvent. The effect of polymer solution concentration, quenching temperature and polymer type on pore morphology and porosity was investigated. Scaffolds were obtained with open and interconnected pores having sizes ranging from several mum to more than 150 microm and porosities of 80-97%. By changing the polymer solution concentration or quenching temperature, scaffolds with random or oriented tubular pores could be obtained. The PEUU scaffolds were flexible with breaking strains of 214% and higher, and tensile strengths of approximately 1.0 MPa, whereas the PEEUU scaffolds generally had lower strengths and breaking strains. Scaffold degradation in aqueous buffer was related to the porosity and polymer hydrophilicity. Smooth muscle cells were filtration seeded in the scaffolds and it was shown that both scaffolds supported cell adhesion and growth, with smooth muscle cells growing more extensively in the PEEUU scaffold. These biodegradable and flexible scaffolds demonstrate potential for future application as cell scaffolds in cardiovascular tissue engineering or other soft tissue applications.

Absorbable Implants↗

PubMed2006

Porogen-based solid freeform fabrication of polycaprolactone-calcium phosphate scaffolds for tissue engineering.

Drop on demand printing (DDP) is a solid freeform fabrication (SFF) technique capable of generating microscale physical features required for tissue engineering scaffolds. Here, we report results toward the development of a reproducible manufacturing process for tissue engineering scaffolds based on injectable porogens fabricated by DDP. Thermoplastic porogens were designed using Pro/Engineer and fabricated with a commercially available DDP machine. Scaffolds composed of either pure polycaprolactone (PCL) or homogeneous composites of PCL and calcium phosphate (CaP, 10% or 20% w/w) were subsequently fabricated by injection molding of molten polymer-ceramic composites, followed by porogen dissolution with ethanol. Scaffold pore sizes, as small as 200 microm, were attainable using the indirect (porogen-based) method. Scaffold structure and porosity were analyzed by scanning electron microscopy (SEM) and microcomputed tomography, respectively. We characterized the compressive strength of 90:10 and 80:20 PCL-CaP composite materials (19.5+/-1.4 and 24.8+/-1.3 Mpa, respectively) according to ASTM standards, as well as pure PCL scaffolds (2.77+/-0.26 MPa) fabricated using our process. Human embryonic palatal mesenchymal (HEPM) cells attached and proliferated on all scaffolds, as evidenced by fluorescent nuclear staining with Hoechst 33258 and the Alamar Blue assay, with increased proliferation observed on 80:20 PCL-CaP scaffolds. SEM revealed multilayer assembly of HEPM cells on 80:20 PCL-CaP composite, but not pure PCL, scaffolds. In summary, we have developed an SFF-based injection molding process for the fabrication of PCL and PCL-CaP scaffolds that display in vitro cytocompatibility and suitable mechanical properties for hard tissue repair.

Calcium Phosphates↗

PubMed2004

Development of novel tissue engineering scaffolds via electrospinning.

Electrospinning has recently been developed as an efficient technique to develop polymeric nanofibres. Various synthetic and natural biodegradable polymers have been electrospun into fibres with diameters in the nanometre range (< 1 microm). The fibre diameter, structure and physical properties of the nanofibre matrices can be effectively tuned by controlling various parameters that affect the electrospinning process. The dimension and structure of electrospun polymeric nanofibre mats resembles mostly the collagen phase of natural extracellular matrix. This, combined with excellent physical properties such as high surface area, high porosity, interconnective pores of the nanofibre matrices and appropriate mechanical properties, well-controlled degradation rates and biocompatibility of the base polymer, make biodegradable polymeric nanofibre matrices ideal candidates for developing scaffolds for tissue engineering. This article reviews the recent advances in the development of synthetic biodegradable nanofibre-based matrices as scaffolds for tissue engineering.

Animals↗

PubMedDate not supplied

Assessment of tissue ingrowth rates in polyurethane scaffolds for tissue engineering.

The continuous development of new biomaterials for tissue engineering and the enhancement of tissue ingrowth into existing scaffolds, using growth factors, create the necessity for developing adequate tools to assess tissue ingrowth rates into porous biomaterials. Current histomorphometric techniques evaluating rates of tissue ingrowth tend either to measure the overall tissue content in an entire sample or to depend on the user to indicate a front of tissue ingrowth. Neither method is particularly suitable for the assessment of tissue ingrowth rates, as these methods either lack the sensitivity required or are problematic when there is a tissue ingrowth gradient rather than an obvious tissue ingrowth front. This study describes a histomorphometric method that requires little observer input, is sensitive, and renders detailed information for the assessment of tissue ingrowth rates into porous biomaterials. This is achieved by examining a number of computer-defined concentric zones, which are based on the distance of a pixel from the scaffold edge. Each zone is automatically analyzed for tissue content, eliminating the need for user definition of a tissue ingrowth front and thus reducing errors and observer dependence. Tissue ingrowth rates in two biodegradable polyurethane scaffolds (Estane and polycaprolactone-polyurethane [PCLPU]) specifically designed for tissue engineering of the knee meniscus were assessed. Samples were subcutaneously implanted in rats with follow-up until 6 months. Especially at the earlier follow-up points, PCLPU scaffolds showed significantly higher tissue ingrowth rates than Estane scaffolds, making the PCLPU scaffold a promising candidate for further studies investigating meniscus tissue engineering.

Animals↗

PubMedDate not supplied

In vitro reconstructed tissues on hyaluronan-based temporary scaffolding.

Tissue engineering offers the possibility to reconstruct tissue substitutes in order to replace lost or damaged tissues. The availability of appropriate biomaterial devices is essential to allow in vitro cultured cells to behave as in the original tissues in vivo. In our studies we utilized a seminatural biomaterial made up by the benzyl ester of hyaluronan to grow keratinocytes, fibroblasts and chondrocytes. Keratinocytes and fibroblasts were isolated from human foreskin. Cells were separately cultured on two different hyaluronan based biomaterial devices for the first 15 days and then co-cultured for an additional period of 2 weeks. Keratinocytes gave rise to a well-differentiated epithelial layer, while fibroblasts were able to synthesize all the main extracellular molecules inside the biomaterial spaces, forming dermal-like tissues. When these two tissues were co-cultured, a skin equivalent was formed with a dermal-epidermal junction. Chondrocytes were obtained from chick-embryo sterna and cultured for 21 days inside a non-woven scaffolding made up of a hyaluronan-based biomaterial. Cells were able to organize themselves into nodules embedded in a dense metachromatic substance in which type II collagen was present. Data from this study suggest that this novel class of hyaluronan derived biomaterials is suitable for different cell culture and in vitro tissue reconstruction.

Journal Article↗

PubMed2004

In vivo characterisation of a novel bioresorbable poly(lactide-co-glycolide) tubular foam scaffold for tissue engineering applications.

Polylactide-co-glycolide (PLGA) foams of tubular shape were assessed for their use as soft-tissue engineering scaffolds in vitro and in vivo. Porous membranes were fabricated by a thermally induced phase separation process of PLGA solutions in dimethylcarbonate. The parameters investigated were the PLGA concentration and the casting volume of solution. Membranes produced from 5 wt/v % polymer solutions and a 6 ml casting volume of polymer solution were selected for fabricating tubes of 3 mm diameter, 20 mm length and a nominal wall thickness of 1.5 mm. Scanning electron microscopy revealed that the structure of the tubular foams consisted of radially oriented and highly interconnected pores with a large size distribution (50-300 microm). Selected tubes were implanted subcutaneously into adult male Lewis rats. Although the lumen of the tubes collapsed within one week of implantation, histological examination of the implanted scaffolds revealed that the foam tubes were well tolerated. Cellular infiltration into the foams, consisting mainly of fibrovascular tissue, was evident after two weeks and complete within eight weeks of implantation. The polymer was still evident in the scaffolds after eight weeks of implantation. The results from this study demonstrate that the PLGA tubular foams may be useful as soft-tissue engineering scaffolds with modification holding promise for the regeneration of tissues requiring a tubular shape scaffold such as intestine.

Absorbable Implants↗

PubMed2006

Manufacturing and morphology structure of polylactide-type microtubules orientation-structured scaffolds.

Tissue engineering using scaffold not only should have biodegradability and a certain 3D structure, but also its morphology structure should be mimetic to that of the repaired natural tissue. So to manufacture the scaffold with a biomimetic structure as the natural tissues is important. In this research, highly porous poly(L-lactic acid) (PLLA) and poly(L-lactic-co-glycolic acid) (PLGA) scaffolds with microtubules orientation structure were designed and fabricated by using dioxane as solvent and an improved thermal-induced phase separation (TIPS) technique. All the factors which will affect solvent crystallization and microtubules orientation structure of the scaffold, such as the type of the solvent and polymer, concentration of the polymer solution, and temperature-gradient of the system have been studied carefully. So the porosity, diameter, tubular morphology and orientation of the microtubules could be controlled by adjusting the concentration of the polymer solution and temperature-gradient of the system. The scaffold with diameter of microtubules from 40 to 240microm and high porosity up to 96% could be obtained by adjusting temperature-gradient during the TIPS process. By increasing concentration of the polymer solution the regularity of the microtubular scaffold has been improved and the thickness of wall of the microtubules has been increased as well. In vitro cell culture results show that after the scaffolds have been improved by the ammonia plasma treatment and then collagen anchorage method, the human transparent cartilage cells H144, could be seeded deeply into the microtubules orientation-structured scaffolds and grew well there.

Biocompatible Materials↗

PubMedDate not supplied

Feasibility of polysaccharide hybrid materials for scaffolds in cartilage tissue engineering: evaluation of chondrocyte adhesion to polyion complex fibers prepared from alginate and chitosan.

The ideal cell-carrier material for cartilage regeneration should be one that closely mimics the natural environment in a living articular cartilage matrix. In the current study, we considered that alginate-based chitosan hybrid biomaterials could provide excellent supports for chondrocyte adhesion. To test this hypothesis, we investigated the adhesion behavior of rabbit chondrocytes onto an alginate polymer versus the adhesion of the chondrocytes onto some alginate-based chitosan hybrid polymer fibers in vitro. We demonstrated that the alginate-based chitosan hybrid polymer fibers showed much improved adhesion capacity with chondrocytes in comparison with alginate polymer fiber. Additionally, morphologic studies revealed maintenance of the characteristic round morphology of the chondrocyte and the dense fiber of the type II collagen produced by the chondrocytes in the hybrid polymer. On the basis of these results, we conclude that an alginate-based chitosan hybrid polymer fiber has considerable potential as a desirable biomaterial for cartilage tissue scaffolds.

Alginates↗

PubMed2002

Novel peptide-based biomaterial scaffolds for tissue engineering.

Biomaterial scaffolds are components of cell-laden artificial tissues and transplantable biosensors. Some of the most promising new synthetic biomaterial scaffolds are composed of self-assembling peptides that can be modified to contain biologically active motifs. Peptide-based biomaterials can be fabricated to form two- and three-dimensional structures. Recent studies show that biomaterial promotion of multi-dimensional cell-cell interactions and cell density are crucial for proper cellular differentiation and for subsequent tissue formation. Other refinements in tissue engineering include the use of stem cells, cell pre-selection and growth factor pre-treatment of cells that are used for seeding scaffolds. These cell-culture technologies, combined with improved processes for defining the dimensions of peptide-based scaffolds, might lead to further improvements in tissue engineering. Novel peptide-based biomaterial scaffolds seeded with cells show promise for tissue repair and for other medical applications.

Animals↗

PubMed2000

Smooth muscle cell adhesion to tissue engineering scaffolds.

Synthetic polyesters of lactic and glycolic acid, and the extracellular matrix molecule collagen are among the most widely-utilized scaffolding materials in tissue engineering. However, the mechanism of cell adhesion to these tissue engineering scaffolds has not been extensively studied. In this paper, the mechanism of adhesion of smooth muscle cells to these materials was investigated. Vitronectin was found to be the predominant matrix protein adsorbed from serum-containing medium onto polyglycolic acid, poly(lactic co-glycolic) acid, and collagen two-dimensional films and three-dimensional scaffolds. Fibronectin adsorbed to both materials as well, although to a much lower density. Smooth muscle cell adhesion was mediated through specific integrin receptors interacting with these adsorbed proteins, as evidenced by both immunostaining and blocking studies. The receptors involved in adhesion included the alpha(v)beta5 to vitronectin, the alpha5beta1 to fibronectin and the alpha2beta1 to collagen I. Identification of the specific receptors used to adhere to these polymers clarifies why smooth muscle tissue development differs on these scaffolds, and may allow one to design tissue formation by controlling the surface chemistry of tissue engineering scaffolds.

Adsorption↗

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↗

PubMed2004

Global gene expression of cells attached to a tissue engineering scaffold.

A goal of tissue engineering is to produce a scaffold material that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Little is known about how cells respond on a molecular level to tissue engineering scaffold materials. In this work we used oligonucleotide microarrays to interrogate gene expression profiles associated with cell-biomaterial interactions. We seeded collagen-glycosaminoglycan meshes, a widely used tissue engineering scaffold material, with human IMR-90 fibroblasts and compared transcript levels with control cells grown on tissue culture polystyrene. Genes involved in cell signaling, extracellular matrix remodeling, inflammation, angiogenesis and hypoxia were all activated in cells on the collagen-GAG mesh. Understanding the impact of a scaffold on attached cells will facilitate the design of improved tissue engineering materials.

CD55 Antigens↗

PubMed2006

Surface adsorption of DNA to tissue engineering scaffolds for efficient gene delivery.

Gene delivery from tissue engineering scaffolds has potential to promote localized transgene expression that can induce the formation of functional tissues. Substrate-mediated delivery, an alternative delivery strategy to sustained release, is based on immobilization of DNA complexes to the polymer surface for subsequent delivery to cells cultured on the substrate. We investigate polyethylenimine (PEI)/DNA complex immobilization and subsequent cellular transfection on tissue engineering scaffolds fabricated from poly(lactide-co-glycolide) (PLG). The properties of the substrate and the complex affect both immobilization and cellular transfection. PLG promotes binding of PEI/DNA complexes, with percent bound independent of the N/P ratio or the DNA dosage. The levels of transgene expression are similar to or greater than control studies based on bolus DNA delivery, with orders of magnitude less DNA. Immobilization also homogeneously distributes the DNA throughout the scaffold, resulting in large numbers of transfected cells (>60%) at low surface quantities (<50 ng). Importantly, this approach can be employed to transfect cells throughout a three-dimensional scaffold. Tissue engineering scaffolds that are prefabricated into various shapes from a range of materials could potentially employ this strategy for numerous applications.

Adsorption↗

PubMed2006

Microsphere-integrated collagen scaffolds for tissue engineering: effect of microsphere formulation and scaffold properties on protein release kinetics.

A promising approach to control the time and space distribution of signalling molecules inside tissue engineering scaffolds consists in entrapping biodegradable microspheres releasing the protein locally for long time frames. However, a rational design of microsphere-integrated scaffolds requires the knowledge of protein release profiles directly within the polymeric template. In this work, PLGA microspheres encapsulating rhodamine-labelled bovine serum albumin (BSA-Rhod) as a model protein were produced in different formulation conditions and tested for their release features in solution and in collagen and collagen/hyaluronic acid (HA) scaffolds. BSA-Rhod release profiles from single microspheres in solution and within the scaffold were assessed by using a confocal laser scanning microscopy (CLSM)-assisted method. Results suggest that the same diffusion-erosion process controls BSA-Rhod release from microspheres in solution and collagen. Nonetheless, two main factors contribute protein release within the scaffold, that is water activity in the release environment and transport properties of the protein in the gel. While microsphere formulation mainly controls the induction time necessary to activate protein release, polymer scaffold composition governs the release rate. Thus, the fine regulation of a tissue engineering construct may be obtained by an appropriate combination of microspheres and scaffolds, providing a spatial and temporal control over signalling molecule delivery.

Animals↗

PubMed2003

The fundamentals of tissue engineering: scaffolds and bioreactors.

Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate, and (iii) bioreactors that provide the conditions necessary for the cells to regenerate functional cartilaginous tissues. Here we explore the paradigm of tissue-engineered cartilage repair that is based on the generation of immature but functional constructs in vitro, and the remodelling and maturation of these constructs in vivo.

Adult↗

PubMed2006

Porogen-induced surface modification of nano-fibrous poly(L-lactic acid) scaffolds for tissue engineering.

In addition to suitable pore architecture of a tissue-engineering scaffold, surface modification after scaffolding fabrication is often needed to enhance the interactions between cells and the synthetic material. In this study, a novel one-step process was developed to fabricate surface-modified nano-fibrous poly(L-lactic acid) (NF-PLLA) scaffolds. First, gelatin spheres with smooth surface were prepared by non-surfactant emulsification, solvent extraction, and freeze-drying. A three-dimensional NF-PLLA scaffold was then fabricated by using gelatin spheres as porogen. Gelatin molecules were entrapped onto the scaffold surface during the fabrication process. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy analysis demonstrated the existence of gelatin molecules on the surface of the polymer scaffold. The amount of gelatin on the scaffold surface was controlled by the composition of the solvent mixture of gelatin solution. The compressive modulus of scaffold prepared with gelatin spheres was more than three times higher than that prepared with irregular gelatin particles of the same size range. The surface modification significantly improved initial cell adhesion and proliferation over a 2-week culture. SEM images indicated that cells spread on the gelatin-entrapped scaffolds in contrast to spherical or spindle morphology on the control 1 day after cell seeding. Furthermore, more matrix secretion was observed on the surface-modified scaffolds than on the control after 2 weeks of in vitro cultivation. In conclusion, this approach provides a simple one-step process to fabricate surface-modified collagen-like NF-PLLA scaffolds, which have improved cell adhesion and proliferation.

3T3 Cells↗

PubMed2004

Production and characterization of chitosan fibers and 3-D fiber mesh scaffolds for tissue engineering applications.

This study reports on the production of chitosan fibers and 3-D fiber meshes for the use as tissue engineering scaffolds. Both structures were produced by means of a wet spinning technique. Maximum strain at break and tensile strength of the developed fibers were found to be 8.5% and 204.9 MPa, respectively. After 14 d of immersion in simulated body fluid (SBF), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and inductively coupled plasma emission (ICP) spectroscopy analyses showed that a bioactive Ca-P layer was formed on the surface of the fibers, meaning that they exhibit a bioactive behavior. The samples showed around 120% max. swelling in physiological conditions. The pore sizes of 3-D chitosan fiber mesh scaffolds were observed to be in the range of 100-500 microm by SEM. The equilibrium-swelling ratio of the developed scaffolds was found to be around 170% (w/w) in NaCl solution at 37 degrees C. Besides that, the limit swelling strain was less than 30%, as obtained by mechanical spectroscopy measurements in the same conditions. The viscoelastic properties of the scaffolds were also evaluated by both creep and dynamic mechanical tests. By means of using short-term MEM extraction test, both types of structures (fibers and scaffolds) were found to be non-cytotoxic to fibroblasts. Furthermore, osteoblasts directly cultured over chitosan fiber mesh scaffolds presented good morphology and no inhibition of cell proliferation could be observed.Osteoblast-like cells proliferating over chitosan based fibers after 7 d of culture.

Animals↗

PubMed2004

Novel photopolymerizable biodegradable triblock polymers for tissue engineering scaffolds: synthesis and characterization.

For use in micro-patterned scaffolds in tissue engineering, novel diacrylated triblock macromers (PLA-b-PCL-b-PLA, PGA-b-PCL-b-PGA and PCL-b-PEO-b-PCL) were synthesized and characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). All diacrylated polymers were designed as triblock copolymers and involved biodegradable blocks of relatively non-polar epsilon-caprolactone (CL) and polar monomers such as glycolide (GA), lactide (LA) or ethylene oxide (EO). All triblock polymers were prepared in molecular weights of a few kilo daltons via the anionic ring-opening polymerization (ROP) of the corresponding lactide, glycolide or caprolactone using stannous octoate [Sn(Oct)(2)] as catalyst. The polymers had low polydispersity indices, ranging from 1.23 to 1.56. Biodegradable polymeric networks were prepared with conversions of 72-84% via photopolymerization of the triblock diacrylated polymers with 2,2-dimethoxy-2-phenylacetophenone (DMPA) as photoinitiator. PLA-b-PCL-b-PLA copolymers crumbled easily and were not suitable for micro-patterning. PGA-b-PCL-b-PGA copolymers had higher water contact angles than PCL-b-PEO-b-PCL and were also cytocompatible with Fibroblasts 3T3.

3T3 Cells↗