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Optimal segmentation of microcomputed tomographic images of porous tissue-engineering scaffolds.

The morphometric properties of the porous tissue-engineering scaffolds play a dominant role in the initial cell attachment and subsequent tissue regeneration. These properties can be derived nondestructively with the use of quantitative analysis of high-resolution microcomputed tomography (microCT) imaging of scaffolds. Accurate segmentation of these acquired images into solid and porous subspaces is critical to the integrity of morphometric analysis. The absence of a single image-processing technique to provide such accurate separability immune to all the intricacies of the acquired data makes this seemingly simple task significantly error prone. Consequently, an optimal segmentation has to be selected by ranking the segmentations produced by a multiplicity of methods. This article proposes a robust, easy-to-implement, unambiguous, signal-processing-based, ground-truth-free, segmentation rating metric that correlates with visual acuity. With the use of this metric it is possible, for the first time, to threshold the data with a wide range of techniques and select automatically the technique that best delineates the acquired image. The proposed solution has been extensively tested on microCT images of scaffolds fabricated with biodegradable poly (propylene fumarate) (PPF) with the use of a solvent casting particulate leaching process. The approaches proposed and the results obtained may have profound implications for accurate image-based characterization of tissue-engineering scaffolds.

Biocompatible Materials↗

Porous polylactide/chitosan scaffolds for tissue engineering.

Novel porous scaffolds were fabricated using biodegradable polylactide/chitosan blends. A combinational technique involving solvent-extracting, liquid-solid separation, and freeze-drying paths were employed. The processing parameters were optimized in order to produce desired porous scaffolds and thus obtained scaffolds showed well distributed and interconnected porous structures with controllable porosities varying from around 50-85% and regulative pore sizes being distributed within a region between 2 and 190 microm. These scaffolds exhibited remarkably improved hydrophilicity based on the measurements for their swelling index. The results obtained from in vitro incubation of scaffolds in phosphate buffered saline solutions at 37 degrees C during various periods up to 10 weeks indicated that chitosan component inside the scaffolds, on the one hand, effectively buffered the acidic degradation products of polylactide/chitosan scaffolds and on the other hand, the degradation of the scaffolds was also conspicuously delayed. These porous scaffolds maintained well-defined compressive mechanical properties and by well-blending polylactide with chitosan component, improved toughness on the resultant porous scaffolds was also observed.

Biocompatible Materials↗

A three-phase, fully resorbable, polyester/calcium phosphate scaffold for bone tissue engineering: Evolution of scaffold design.

Bone tissue engineering strategies are fundamentally based upon porous scaffold materials that serve as a support for ingrowth of host cells and/or provide a substrate for exogenously delivered cells. Here we report the application of a surface calcium phosphate (CaP) mineral layer to a macroporous polymeric/CaP composite biomaterial, with a macroporous interconnectivity, and its subsequent in vivo evaluation in a rodent femoral defect. The application of the mineral layer eliminates the fibrous tissue encapsulation and foreign body giant cell response commonly seen at the interface of polymeric materials, yet retains the unique characteristics of the parent material as being macroporous, completely biodegradable and possessing a high degree of interconnectivity. This represents the third generation of this scaffold material, incorporating iterative changes to the scaffold design in response to both materials and biological design criteria to produce a material with enhanced in vitro and in vivo performance.

Absorbable Implants↗

Cell sheet engineering: recreating tissues without biodegradable scaffolds.

While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.

Animals↗

Mechanical characterization of collagen fibers and scaffolds for tissue engineering.

Engineered tissues must utilize scaffolding biomaterials that support desired cellular functions and possess or can develop appropriate mechanical characteristics. This study assessed properties of collagen as a scaffolding biomaterial for ligament replacements. Mechanical properties of extruded bovine achilles tendon collagen fibers were significantly affected by fiber diameter, with smaller fibers displaying higher tangent moduli and peak stresses. Mechanical properties of 125 micrometer-diameter extruded fibers (tangent modulus of 359.6+/-28.4MPa; peak stress of 36.0+/-5.4MPa) were similar to properties reported for human ligaments. Scaffolds of extruded fibers did not exhibit viscoelastic creep properties similar to natural ligaments. Collagen fibers from rat tail tendon (a well-studied comparison material) displayed characteristic strain-softening behavior, and scaffolds of rat tail fibers demonstrated a non-intuitive relationship between tangent modulus and specimen length. Composite scaffolds (extruded collagen fibers cast within a gel of Type I rat tail tendon collagen) were maintained with and without fibroblasts under standard culture conditions for 25 days; cell-incorporated scaffolds displayed significantly higher tangent moduli and peak stresses than those without cells. Because tissue-engineered products must possess appropriate mechanical as well as biological/chemical properties, data from this study should help enable the development of improved tissue analogues.

Animals↗

Bone formation on two-dimensional poly(DL-lactide-co-glycolide) (PLGA) films and three-dimensional PLGA tissue engineering scaffolds in vitro.

For some bone tissue engineering strategies, direct contact of newly synthesized bone with a scaffold is important for structural continuity and stability at the scaffold/bone interface. Thus, as the polymer degrades, the support function of the scaffold could be adopted by the developing bone structure. This study was designed to determine whether poly(DL-lactide-co-glycolide) with a comonomer ratio of 75:25 supports bone apposition in vitro. Osteogenic cells derived from rat bone marrow cells were cultured for 2 weeks on polymeric two-dimensional films and three-dimensional tissue engineering scaffolds. Bacteriological grade polystyrene and tissue culture polystyrene dishes served as negative and positive controls for interfacial bone deposition, respectively. The surfaces of the prepared substrates were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle, scanning electron microscopy, and atomic force microscopy. After cell culture, the elaborated matrix was examined using scanning electron microscopy, transmission electron microscopy, and energy dispersive spectroscopy. The results show that poly(DL-lactide-co-glycolide) supports appositional bone growth on both two-dimensional films and three-dimensional scaffolds, including the formation of a mineralized cement line matrix. Furthermore, surface topographical features are not required for the adherence of the cement line matrix to the polymer.

Animals↗

Different substitute biomaterials as potential scaffolds in tissue engineering.

PURPOSE: To find the optimal scaffold for tissue-engineered bone, one approach is to test existing biomaterials on their suitability as scaffolds. In this study, the suitability of different alloplastic and xenogenic biomaterials as scaffolds for ex vivo osteoblast cultivation was investigated. MATERIALS AND METHODS: Normal human osteoblast cells were cultured on the surface of bovine collagenous materials, bovine hydroxyapatite, porcine gelatin, synthetic polymer, and collagen-containing bovine hydroxyapatite, and the investigation of proliferation was performed after 24, 72, and 120 hours. Measurement of the differentiation marker alkaline phosphatase and osteocalcin was made after 20 days of incubation. RESULTS: The obtained data showed significantly higher proliferation and differentiation rates in cells cultivated on collagen-rich biomaterials in comparison to noncollagenous or collagen-poor biomaterials (P < .05). DISCUSSION: In tissue engineering the scaffold should be biocompatible and serve as a proper matrix for the cells to produce the new structural environment of extracellular matrix ex vivo. Collagen supports initial cell attachment and cell proliferation, allowing immature osteogenic cells to differentiate into mature osteoblasts, but collagen may not be the only dominating factor for cell-matrix interaction during ex vivo bone formation. CONCLUSION: These data suggest that a 3-dimensional collagen matrix can provide a more favorable environment for the attachment, proliferation, and differentiation of in vitro osteoblastlike cells, at least until the initial stage of differentiation, than noncollagenous biomaterials.

Absorbable Implants↗

Tissue engineering of blood vessels: characterization of smooth-muscle cells for culturing on collagen-and-elastin-based scaffolds.

Tissue engineering offers the opportunity to develop vascular scaffolds that mimic the morphology of natural arteries. We have developed a porous three-dimensional scaffold consisting of fibres of collagen and elastin interspersed together. Scaffolds were obtained by freeze-drying a suspension of insoluble type I collagen and insoluble elastin. In order to improve the stability of the obtained matrices, they were cross-linked by two different methods. A water-soluble carbodi-imide, alone or in combination with a diamine, was used for this purpose: zero- or non-zero-length cross-links were obtained. The occurrence of cross-linking was verified by monitoring the thermal behaviour and the free-amino-group contents of the scaffolds before and after cross-linking. Smooth-muscle cells (SMCs) were cultured for different periods of time and their ability to grow and proliferate was investigated. SMCs were isolated from human umbilical and saphenous veins, and the purity of the cultures obtained was verified by staining with a specific monoclonal antibody (mAb). Cultured cells were also identified by mAbs against muscle actin and vimentin. After 14 days, a confluent layer of SMCs was obtained on non-cross-linked scaffolds. As for the cross-linked samples, no differences in cell attachment and proliferation were observed between scaffolds cross-linked using the two different methods. Cells cultured on the scaffolds were identified with an anti-(alpha-smooth-muscle actin) mAb. The orientation of SMCs resembled that of the fibres of collagen and elastin. In this way, it may be possible to develop tubular porous scaffolds resembling the morphological characteristics of native blood vessels.

Biocompatible Materials↗

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds.

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.

Coated Materials, Biocompatible↗

Solvent-assisted room-temperature compression molding approach to fabricate porous scaffolds for tissue engineering.

This study investigated the room-temperature compression molding/particle leaching approach to fabricate three-dimensional porous scaffolds for tissue engineering. Scaffolds with anatomical shapes (ear, joint, tube, cylinder) were made from biodegradable poly(D,L-lactide) and poly[(D,L-lactide)-co-glycolide]. The utility of this room-temperature compression approach comes from the effect of solvent assistance, but the tendency for post-molding scaffold shrinkage is a problem unique to this method and is thus examined with emphasis in this paper. Scaffold shrinkage was found to be tolerable under normal fabrication conditions with high salt contents, which is just what the preparation of highly porous scaffolds requires. Furthermore, the resultant porosities after salt leaching were measured as well as the initial scaffold shrinkages after solvent evaporation, and the relation between them was revealed by theoretical analysis and confirmed by comparison with experimental measurements. The pores were interconnected, and porosity can exceed 90%. The effects of porosity on the mechanical properties of porous scaffolds were also investigated. This convenient fabrication approach is a prospective method for the tailoring of porous scaffolds for a variety of possible applications in tissue engineering and tissue reconstruction.

Biocompatible Materials↗

In vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering.

In vitro degradation behaviors of three-dimensional tissue engineering porous scaffolds made from amorphous poly(D,L-lactide-co-glycolide) with three different formulations have been systematically investigated up to 26 weeks in phosphate buffer saline solution at 37 degrees C. The following properties of the scaffolds were measured as a function of degradation time: dimensions, weight, compressive strength and modulus, polymer molecular weight and its distribution, and pore morphology. Of special interest was the determination of mechanical properties in wet environment. The pH of the PBS media was also detected. According to the characteristic changes of the various properties of porous scaffolds, the degradation process is suggested to be roughly divided into three stages tentatively named as quasi-stable stage, decrease-of-strength stage, loss-of-weight and disruption-of-scaffold stage.

Biocompatible Materials↗

Mechanical and structural properties of a novel hybrid heart valve scaffold for tissue engineering.

Hybrid heart valve scaffolds were fabricated from decellularized porcine aortic heart valve matrices and enhanced with bioresorbable polymers using different protocols: (i) dip coating of lyophilized decellularized matrices, and (ii) impregnation of wet decellularized matrices. The following polymers were evaluated: poly(4-hydroxybutyrate) and poly(3-hydroxybutyrate-co4-hydroxybutyrate). Tensile tests were conducted to assess the biomechanical behavior of valve leaflet strips. Suture retention strength was evaluated for the adjacent conduit. A pulse duplicator system was used for functional testing of the valves under physiological systemic load conditions. The properties of the hybrid structures were compared with native, decellularized, and glutaraldehyde-fixed specimens. Mechanisms of the polymer impregnation process were studied with IR spectroscopy, fluorescent microscopic imaging, and SEM. Altogether this study demonstrates the feasibility and improved biomechanical function of a novel hybrid heart valve scaffold for an application in tissue engineering.

Animals↗

Chitosan sponges as tissue engineering scaffolds for bone formation.

Rat calvarial osteoblasts were grown in porous chitosan sponges fabricated by freeze drying. The prepared chitosan sponges had a porous structure with a 100-200 microm pore diameter, which allowed cell proliferation. Cell density, alkaline phosphatase activity and calcium deposition were monitored for up to 56 d culture. Cell numbers were 4 x 10(6) (day 1), 11 x 10(6) (day 28) and 12 x 10(6) (day 56) per g sponge. Calcium depositions were 9 (day 1), 40 (day 28) and 48 (day 56) microg per sponge. Histological results corroborated that bone formation within the sponges had occurred. These results show that chitosan sponges can be used as effective scaffolding materials for tissue engineered bone formation in vitro.

Animals↗

Characterization of protein release from photocrosslinkable hyaluronic acid-polyethylene glycol hydrogel tissue engineering scaffolds.

The goal of this work was to utilize the naturally derived bioactive polymer hyaluronic acid (HA) to create a combination tissue engineering scaffold and protein delivery device. HA is a non-immunogenic, non-adhesive glycosaminoglycan that plays significant roles in several cellular processes, including angiogenesis and the regulation of inflammation. In previous work, we created photopolymerizable glycidyl methacrylate-hyaluronic acid (GMHA) hydrogels that had controlled degradation rates, were cytocompatible, and were able to be modified with peptide moieties. In the present studies, we characterized the release of a model protein, bovine serum albumin (BSA), from GMHA and GMHA-polyethylene glycol (PEG) hydrogels. Although BSA could be released rapidly (> 60% within 6 h) from 1% GMHA hydrogels, we found that increasing either the GMHA or the PEG concentrations could lengthen the duration of protein delivery. Preliminary size exclusion chromatography studies indicated that the released BSA was almost entirely in its native monomeric form. Lastly, protein release was extended to several weeks by suspending BSA-poly(lactic-co-glycolic acid) microspheres within the hydrogel bulk. These initial studies indicate that the naturally derived biopolymer HA can be employed to design novel photopolymerizable composites that are suitable for delivering stable proteins from scaffolding in tissue engineering applications.

Coated Materials, Biocompatible↗

Osteoblast response to PLGA tissue engineering scaffolds with PEO modified surface chemistries and demonstration of patterned cell response.

Because tissues are characterized by a well-defined three-dimensional arrangement of cells, tissue engineering scaffolds that facilitate the organization and differentiation of new tissue will have improved performance in comparison to scaffolds that only provide surfaces for cell attachment and growth. We hypothesize that instructions for cells can be incorporated into tissue engineering scaffolds by patterning the scaffold's architecture and surface chemistry. Our goals for the presented work were to collect data about cell response to three-dimensional, porous scaffolds with uniformly modified surfaces chemistries, and to demonstrate patterning of cell response by patterning surface chemistry. Our system was osteoblast response to poly(l-lactide-co-glycolide) scaffolds modified with poly(ethylene oxide) (PEO). Scaffolds were fabricated using the Three-Dimensional Printing (3DP) process which has control over scaffolds properties to a resolution of approximately 100 microm in all three dimensions. At higher PEO concentrations, adhesion, growth rates, and migration of rat osteoblasts were reduced; alkaline phosphate activity was increased, and cells were less spread and had microvilli. Patterned regions of low and high cell adhesion were demonstrated on scaffolds fabricated with 1 mm thick stripes of PEO and non-PEO regions.

Animals↗

Novel porous aortic elastin and collagen scaffolds for tissue engineering.

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.

Animals↗

Controlled release from coated polymer microparticles embedded in tissue-engineered scaffolds.

The controlled release of proteins in tissue-engineered implants is being examined with the potential application to improve vascularization and hasten tissue growth. Bovine serum albumin (BSA), was encapsulated within poly(D,L-lactic-co-glycolic acid) [PLGA] microparticles. The microparticles were coated with poly(vinyl alcohol) and incorporated into PLGA tissue-engineered scaffolds during fabrication. The release of BSA from PLGA microparticles, coated PLGA microparticles, and microparticles embedded in a porous PLGA scaffold was measured. We have developed a novel approach that will permit incorporation of coated polymeric microparticles during PLGA scaffold fabrication. Growth factors or drugs could be incorporated into the microparticles resulting in a long-term, controlled release.

Animals↗

Growth factor release from tissue engineering scaffolds.

Synthetic scaffold materials are used in tissue engineering for a variety of applications, including physical supports for the creation of functional tissues, protective gels to aid in wound healing and to encapsulate cells for localized hormone-delivery therapies. In order to encourage successful tissue growth, these scaffold materials must incorporate vital growth factors that are released to control their development. A major challenge lies in the requirement for these growth factor delivery mechanisms to mimic the in-vivo release profiles of factors produced during natural tissue morphogenesis or repair. This review highlights some of the major strategies for creating scaffold constructs reported thus far, along with the approaches taken to incorporate growth factors within the materials and the benefits of combining tissue engineering and drug delivery expertise.

Bioartificial Organs↗