Tissue engineering of bovine articular cartilage within porous poly(ether ester) copolymer scaffolds with different structures

The potential of porous poly(ether ester) scaffolds made from poly(ethylene glycol) terephthalate: poly(butylene terephthalate) (PEGT:PBT) block copolymers produced by various methods to enable cartilaginous tissue formation in vitro was studied. Scaffolds were fabricated by two different processes: paraffin templating (PT) and compression molding (CM). To determine whether PEGT:PBT scaffolds are able to support chondrogenesis, primary bovine chondrocytes were seeded within cylindrical scaffolds under dynamic seeding conditions. On day 3, constructs were transferred to six-well plates and evaluated for glycosaminoglycan (GAG) distribution (3, 10, and 24 days), type II collagen distribution, cellularity, and total collagen and GAG content (10 and 24 days). It was observed that better cell distribution during infiltration within PT scaffolds allowed greater chondrogenesis, and at later time points, than in CM scaffolds. The amount of GAG remained constant for all groups from 10 to 24 days, whereas collagen content increased significantly. These data suggest that PEGT:PBT scaffolds are suitable for cartilage tissue engineering, with the PT process enabling greater chondrogenesis than CM.     

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.     

Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique

In this study, we present and characterize a fiber deposition technique for producing three-dimensional poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT/PBT) block co-polymer scaffolds with a 100% interconnecting pore network for engineering of articular cartilage. The technique allowed us to "design-in" desired scaffold characteristics layer by layer by accurately controlling the deposition of molten co-polymer fibers from a pressure-driven syringe onto a computer controlled x-y-z table. By varying PEGT/PBT composition, porosity and pore geometry, 3D-deposited scaffolds were produced with a range of mechanical properties. The equilibrium modulus and dynamic stiffness ranged between 0.05-2.5 and 0.16-4.33 MPa, respectively, and were similar to native articular cartilage explants (0.27 and 4.10 MPa, respectively). 3D-deposited scaffolds seeded with bovine articular chondrocytes supported a homogeneous cell distribution and subsequent cartilage-like tissue formation following in vitro culture as well as subcutaneous implantation in nude mice. This was demonstrated by the presence of articular cartilage extra cellular matrix constituents (glycosaminoglycan and type II collagen) throughout the interconnected pore volume. Similar results were achieved with respect to the attachment of expanded human articular chondrocytes, resulting in a homogeneous distribution of viable cells after 5 days dynamic seeding. The processing methods and model scaffolds developed in this study provide a useful method to further investigate the effects of scaffold composition and pore architecture on articular cartilage tissue formation.     

Synthetic scaffold morphology controls human dermal connective tissue formation

Engineering tissues in bioreactors is often hampered by disproportionate tissue formation at the surface of scaffolds. This hinders nutrient flow and retards cell proliferation and tissue formation inside the scaffold. The objective of this study was to optimize scaffold morphology to prevent this from happening and to determine the optimal scaffold geometric values for connective tissue engineering. After comparing lyophilized crosslinked collagen, compression molded/salt leached PEGT/PBT copolymer and collagen-PEGT/PBT hybrid scaffolds, the PEGT/PBT scaffold was selected for optimization. Geometric parameters were determined using SEM, microcomputed tomography, and flow permeability measurements. Fibroblast were seeded and cultured under dynamic flow conditions for 2 weeks. Cell numbers were determined using CyQuant DNA assay, and tissue distribution was visualized in H&E- and Sirius Red-stained sections. Scaffolds 0.5 and 1.5 mm thick showed bridged connected tissue from top-to-bottom, whereas 4-mm-thick scaffolds only revealed tissue ingrowth until a maximum depth of 0.6-0.8 mm. Rapid prototyped scaffold were used to assess the maximal void space (pore size) that still could be filled with tissue. Tissue bridging between fibers was only found at fiber distances < or =401 +/- 60 microm, whereas filling of void spaces in 3D-deposited scaffolds only occurred at distances < or =273 +/- 55 microm. PEGT/PBT scaffolds having similar optimal porosities, but different average interconnected pore sizes of 142 +/- 50, 160 +/- 56 to 191 +/- 69 microm showed comparable seeding efficiencies at day 1, but after 2 weeks the total cell numbers were significantly higher in the scaffolds with intermediate and high interconnectivity. However, only scaffolds with an intermediate interconnectivity revealed homogenous tissue formation throughout the scaffold with complete filling of all pores. In conclusion, significant amount of connective tissue was formed within 14 days using a dynamic culture process that filled all void spaces of a PEGT/PBT scaffolds with the following geometric parameters: thickness 1.5-1.6 mm, pore size range 90-360 microm, and average interconnecting pore size of 160 +/- 56 microm.     

The effect of PEGT/PBT scaffold architecture on oxygen gradients in tissue engineered cartilaginous constructs

Repair of articular cartilage defects using tissue engineered constructs composed of a scaffold and cultured autologous cells holds promise for future treatments. However, nutrient limitation (e.g. oxygen) has been suggested as a cause of the onset of chondrogenesis solely within the peripheral boundaries of larger constructs. In the present study, oxygen gradients were evaluated by microelectrode measurements in two porous polyethylene glycol terephthalate/polybutylene terephthalate (PEGT/PBT) scaffold architectures, a compression-molded and particle-leached sponge (CM) and a 3D-deposited fiber (3DF) scaffold. During the first 14 days in vitro, gradients intensified, after which a gradual decrease of the gradients was observed in vitro. In vivo, however, gradients changed instantly and became less pronounced. Although similar gradients were observed regardless of scaffold type, significantly more cells were present in the center of 3DF constructs after 2 weeks of in vivo culture. Our results stress the importance of a rationally designed scaffold for tissue-engineering applications. Organized structures, such as the 3DF PEGT/PBT polymer scaffolds, offer possibilities for regulation of nutrient supply and, therefore, hold promise for clinical approaches for cartilage repair.     

Design of biphasic polymeric 3-dimensional fiber deposited scaffolds for cartilage tissue engineering applications

This report describes a novel system to create rapid prototyped 3-dimensional (3D) fibrous scaffolds with a shell-core fiber architecture in which the core polymer supplies the mechanical properties and the shell polymer acts as a coating providing the desired physicochemical surface properties. Poly[(ethylene oxide) terephthalate-co-poly(butylene) terephthalate] (PEOT/PBT) 3D fiber deposited (3DF) scaffolds were fabricated and examined for articular cartilage tissue regeneration. The shell polymer contained a higher molecular weight of the initial poly(ethylene glycol) (PEG) segments used in the copolymerization and a higher weight percentage of the PEOT domains compared with the core polymer. The 3DF scaffolds entirely produced with the shell or with the core polymers were also considered. After 3 weeks of culture, scaffolds were homogeneously filled with cartilage tissue, as assessed by scanning electron microscopy. Although comparable amounts of entrapped chondrocytes and of extracellular matrix formation were found for all analyzed scaffolds, chondrocytes maintained their rounded shape and aggregated during the culture period on shell-core 3DF scaffolds, suggesting a proper cell differentiation into articular cartilage. This finding was also observed in the 3DF scaffolds fabricated with the shell composition only. In contrast, cells spread and attached on scaffolds made simply with the core polymer, implying a lower degree of differentiation into articular cartilaginous tissue. Furthermore, the shell-core scaffolds displayed an improved dynamic stiffness as a result of a "prestress" action of the shell polymer on the core one. In addition, the dynamic stiffness of the constructs increased compared with the stiffness of the bare scaffolds before culture. These findings suggest that shell-core 3DF PEOT/PBT scaffolds with desired mechanical and surface properties are a promising solution for improved cartilage tissue engineering.     

Evaluation of two biodegradable polymeric systems as substrates for bone tissue engineering

The aim of this study was to evaluate two biodegradable polymeric systems as scaffolds for bone tissue engineering. Rat bone marrow cells were seeded and cultured for 1 week on two biodegradable porous polymeric systems, one composed of poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) and the other composed of cornstarch blended with poly(epsilon-caprolactone) (SPCL). Porous hydroxyapatite granules were used as controls. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay and by scanning electron microscopy examination; their osteogenic differentiation was screened by the expression of alkaline phosphatase. In addition, the in vivo osteogenic potential of the engineered constructs was evaluated through ectopic implantation in a nude mouse model. Results revealed that cells were able to proliferate, differentiate, and form extracellular matrix on all materials tested. Moreover, all constructs induced abundant formation of bone and bone marrow after 4 weeks of implantation. The extent of osteogenesis (approximately 30% of void volume) was similar in all types of implants. However, the amount of bone marrow and the degree of bone contact were higher on HA scaffolds, indicating that the polymers still need to be modulated for higher osteoconductive capacity. Nevertheless, the findings suggest that both PEGT/PBT and SPCL systems are excellent candidates to be used as scaffolds for a cell therapy approach in the treatment of bone defects.     

Evaluation of chondrogenesis within PEGT: PBT scaffolds with high PEG content

Porous poly(ethylene glycol) terephthalate:poly (butylene terephthalate) (PEGT:PBT) scaffolds with high PEG molecular weight (1000 g/mole) and PEGT content (60%) were fabricated using two different processes-paraffin templating and compression molding-for cartilage engineering applications. This polymer composition has previously been shown to enable chondrocyte adhesion and maintain differentiated phenotype in 2D monolayer culture. The influence of 3D polymer scaffold processing on the formation of cartilaginous tissue was studied by seeding primary immature bovine chondrocytes within cylindrical scaffolds in mixed flask reactors for 3 days, followed by cultivation in culture plates for a total of 10 or 24 days. Tissue-polymer constructs were evaluated morphologically by SEM and histology, and quantitatively for cellularity, total collagen, and glycosaminoglycan content, all of which remained statistically equivalent for each time point tested, irrespective of fabrication method. These data demonstrate that the polymers engineered for this study were able to support chondrogenesis independent of scaffold fabrication process, with the influence of pore architecture lessened by the highly hydrated scaffold microenvironments induced by high PEG content.     

Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition

We investigated whether the post-expansion redifferentiation and cartilage tissue formation capacity of adult human nasal chondrocytes can be regulated by controlled modifications of scaffold composition and architecture. As a model system, we used poly(ethylene glycol)-terephthalate-poly(butylene)-terephthalate block copolymer scaffolds from two compositions (low or high PEG content, resulting in different wettability) and two architectures (generated by compression molding or three-dimensional (3D) fiber deposition) with similar porosity and mechanical properties, but different interconnecting pore architectures. Scaffolds were seeded with expanded human chondrocytes and the resulting constructs assessed immunohistochemically, biochemically and at the mRNA expression level following up to 4 weeks of static culture. For a given 3D architecture, the more hydrophilic scaffold enhanced cell redifferentiation and cartilaginous tissue formation after 4 weeks culture, as assessed by higher mRNA expression of collagen type II, increased deposition of glycosaminoglycan (GAG) and predominance of type II over type I collagen immunostain. The fiber-deposited scaffolds, with a more accessible pore volume and larger interconnecting pores, supported increased GAG deposition, but only if a more hydrophilic composition was used. By applying controlled and selective modifications of chemico-physical scaffold parameters, we demonstrate that both scaffold composition and architecture are instructive for expanded human chondrocytes in the generation of 3D cartilaginous tissues. The observed effects of composition and architecture were likely to have been mediated, respectively, by differential serum protein adsorption and efficiency of nutrient/waste exchange.     

Dynamic mechanical properties of 3D fiber-deposited PEOT/PBT scaffolds: an experimental and numerical analysis

Mechanical properties of three-dimensional (3D) scaffolds can be appropriately modulated through novel fabrication techniques like 3D fiber deposition (3DF), by varying scaffold's pore size and shape. Dynamic stiffness, in particular, can be considered as an important property to optimize the scaffold structure for its ultimate in vivo application to regenerate a natural tissue. Experimental data from dynamic mechanical analysis (DMA) reveal a dependence of the dynamic stiffness of the scaffold on the intrinsic mechanical and physicochemical properties of the material used, and on the overall porosity and architecture of the construct. The aim of this study was to assess the relationship between the aforementioned parameters, through a mathematical model, which was derived from the experimental mechanical data. As an example of how mechanical properties can be tailored to match the natural tissue to be replaced, articular bovine cartilage and porcine knee meniscus cartilage dynamic stiffness were measured and related to the modeled 3DF scaffolds dynamic stiffness. The dynamic stiffness of 3DF scaffolds from poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) copolymers was measured with DMA. With increasing porosity, the dynamic stiffness was found to decrease in an exponential manner. The influence of the scaffold architecture (or pore shape) and of the molecular network properties of the copolymers was expressed as a scaffold characteristic coefficient alpha, which modulates the porosity effect. This model was validated through an FEA numerical simulation performed on the structures that were experimentally tested. The relative deviation between the experimental and the finite element model was less than 15% for all of the constructs with a dynamic stiffness higher than 1 MPa. Therefore, we conclude that the mathematical model introduced can be used to predict the dynamic stiffness of a porous PEOT/PBT scaffold, and to choose the biomechanically optimal structure for tissue engineering applications.     

Anatomical 3D fiber-deposited scaffolds for tissue engineering: designing a neotrachea

The advantage of using anatomically shaped scaffolds as compared to modeled designs was investigated and assessed in terms of cartilage formation in an artificial tracheal construct. Scaffolds were rapid prototyped with a technique named three-dimensional fiber deposition (3DF). Anatomical scaffolds were fabricated from a patient-derived computerized tomography dataset, and compared to cylindrical and toroidal tubular scaffolds. Lewis rat tracheal chondrocytes were seeded on 3DF scaffolds and cultured for 21 days. The 3-(4,5-dimethylthiazol-2yl)-2,5-dyphenyltetrazolium bromide (MTT) and sulfated glycosaminoglycan (GAG) assays were performed to measure the relative number of cells and the extracellular matrix (ECM) formed. After 3 weeks of culture, the anatomical scaffolds revealed a significant increase in ECM synthesis and a higher degree of differentiation as shown by the GAG/MTT ratio and by scanning electron microscopy analysis. Interestingly, a lower scaffold's pore volume and porosity resulted in more tissue formation and a better cell differentiation, as evidenced by GAG and GAG/MTT values. Scaffolds were compliant and did not show any signs of luminal obstruction in vitro. These results may promote anatomical scaffolds as functional matrices for tissue regeneration not only to help regain the original shape, but also for their improved capacity to support larger tissue formation.     

Three-dimensional fiber-deposited PEOT/PBT copolymer scaffolds for tissue engineering: influence of porosity, molecular network mesh size, and swelling in aqueous media on dynamic mechanical properties

Among novel scaffold fabrication techniques, 3D fiber deposition (3DF) has recently emerged as a means to fabricate well-defined and custom-made scaffolds for tissue regeneration, with 100% interconnected pores. The mechanical behavior of these constructs is dependent not only on different three-dimensional architectural and geometric features, but also on the intrinsic chemical properties of the material used. These affect the mechanics of the solid material and eventually of 3D porous constructs derived from them. For instance, poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymers are known to have mechanical properties, depending on the PEOT/PBT weight ratio in block form and on the molecular weight of the initial poly(ethylene glycol) (PEG) blocks. These differences are enhanced even more by their different swelling properties in aqueous media. Therefore, this article examines the influence of copolymer compositions in terms of their swelling on dynamic mechanical properties of solid material and porous 3DF scaffolds. The molecular weight of the starting PEG blocks used in the copolymer synthesis varied from 300 to 1000 g/mol. The PEOT/PBT weight ratio in the blocks used varied from 55/45 to 80/20. This corresponded to an increase of the swelling ratio Q from 1.06 to 2.46, and of the mesh size xi from approximately 9 Angstrom to approximately 47 Angstrom. With increased swelling, dynamic mechanical analysis (DMA) revealed a decrease in elastic response and an increase of viscoelasticity. Thus, by coupling structural and chemical characteristics, the viscoelastic properties of PEOT/PBT 3DF scaffolds may be fine tuned to achieve mechanical requirements for a variety of engineered tissues. Ultimately, the combination of 3DF and DMA may be useful to validate the hypothesis that mimicking the biomechanical behavior of a specific tissue for its optimal replacement is an important issue for at least some tissue-engineering applications.     

In vitro chondrocyte behavior on porous biodegradable poly (e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.     

新型可降解聚合物聚乙二醇对苯二甲酸酯/聚对苯二甲酸丁二醇酯的细胞相容性研究

目的：研究新型可降解聚合物聚乙二醇对苯二甲酸酯／聚对苯二甲酸丁二醇酯(PEGT／PBT)的人脐静脉内皮细胞(HUVEC)相容性，对其在组织工程血管中的应用进行探讨。方法：对PEGT／PBT进行细胞毒性评价。观察并检测HUVEC在PEGB／PBT、Ⅰ型胶原改性(Col-)的PEGT／PBT、纤维连接蛋白改性(Fn-)的PEGT／PBT上的粘附和增殖，对细胞在粘附过程中的粘着斑蛋白进行免疫荧光染色观察。结果：PEGT／PBT细胞毒性不大于1级，能支持脐静脉内皮细胞的粘附和增殖。纤维连接蛋白和Ⅰ型胶原处理可促进HLIVEC在PEGT／PBT膜上的增殖，而且纤维连接蛋白可增加HUVEC在PEGT／PBT上20min、2h的粘附率，并促进细胞形成局部粘附结构。结论：新型可降解聚合物PEGT／PBT具有良好的血管细胞相容性，对其在组织工程血管中的应用值得进一步开发研究。     

Tissue engineering of fish skin: behavior of fish cells on poly(ethylene glycol terephthalate)/poly(butylene terephthalate) copolymers in relation to the composition of the polymer substrate as an initial step in constructing a robotic/living tissue hybrid

This study presents the development of a biosynthetic fish skin to be used on aquatic robots that can emulate fish. Smoothness of the external surface is desired in improving high propulsive efficiency and maneuvering agility of autonomous underwater vehicles such as the RoboTuna (Triantafyllou, M., and Triantafyllou, G. Sci. Am. 272, 64, 1995). An initial step was to determine the seeding density and select a polymer for the scaffolds. The attachment and proliferation of chinook salmon embryo (CHSE-214) and brown bullhead (BB) cells were studied on different compositions of a poly(ethylene glycol terephthalate) (PEGT) and poly(butylene terephthalate) (PBT) copolymer (Polyactive). Polymer films were used, cast of three different compositions of PEGT/PBT (weight ratios of 55/45, 60/40, and 70/30) and two different molecular masses of PEGT (300 and 1000 Da). When a 55 wt% and a 300-Da molecular mass form of PEGT was used, maximum attachment and proliferation of CHSE-214 and BB cells were achieved. Histological studies and immunostaining indicate the presence of collagen and cytokeratins in the extracellular matrix formed after 14 days of culture. Porous scaffolds of PEGT/PBT copolymers were also used for three-dimensional tissue engineering of fish skin, using BB cells. Overall, our results indicate that fish cells can attach, proliferate, and express fish skin components on dense and porous Polyactive scaffolds.     

Photo-patterning of porous hydrogels for tissue engineering

Since pore size and geometry strongly impact cell behavior and in vivo reaction, the ability to create scaffolds with a wide range of pore geometries that can be tailored to suit a particular cell type addresses a key need in tissue engineering. In this contribution, we describe a novel and simple technique to design porous, degradable poly(2-hydroxyethyl methacrylate) hydrogel scaffolds with well-defined architectures using a unique photolithography process and optimized polymer chemistry. A sphere-template was used to produce a highly uniform, monodisperse porous structure. To create a patterned and porous hydrogel scaffold, a photomask and initiating light were employed. Open, vertical channels ranging in size from 360+/-25 to 730+/-70 microm were patterned into approximately 700 microm thick hydrogels with pore diameters of 62+/-8 or 147+/-15 microm. Collagen type I was immobilized onto the scaffolds to facilitate cell adhesion. To assess the potential of these novel scaffolds for tissue engineering, a skeletal myoblast cell line (C2C12) was seeded onto scaffolds with 147 microm pores and 730 microm diameter channels, and analyzed by histology and digital volumetric imaging. Cell elongation, cell spreading and fibrillar formation were observed on these novel scaffolds. In summary, 3D architectures can be patterned into porous hydrogels in one step to create a wide range of tissue engineering scaffolds that may be tailored for specific applications.     

The use of PEGT/PBT as a dermal scaffold for skin tissue engineering

Human skin equivalents (HSEs) were engineered using biodegradable-segmented copolymer PEGT/PBT as a dermal scaffold. As control groups, fibroblast-populated de-epidermized dermis, collagen, fibrin and hybrid PEGT/PBT-collagen matrices were used. Two different approaches were used to generate full-thickness HSE. In the 1-step approach, keratinocytes were seeded onto the fibroblast-populated scaffolds and cultured at the air-liquid (A/L) interface. In the 2-step approach, fully differentiated epidermal sheets were transferred onto fibroblast-populated scaffolds and cultured at the A/L. In a 1-step procedure, keratinocytes migrated into the porous PEGT/PBT scaffold. This was prevented by incorporating fibroblast-populated collagen into the pores of the PEGT/PBT matrix or using the 2-step procedure. Under all experimental conditions, fully differentiated stratified epidermis and basement membrane was formed. Differences in K6, K16, K17, collagen type VII, laminin 5 and nidogen staining were observed. In HSE generated with PEGT/PBT, the expression of these keratins was higher, and the deposition of collagen type VII, laminin 5 and nidogen at the epidermal/matrix junction was retarded compared to control HSEs. Our results illustrate that the copolymer PEGT/PBT is a suitable scaffold for the 2-step procedure, whereas the incorporation of fibroblast-populated collagen or fibrin into the pores of the scaffold is required for the 1-step procedure.     

Porous PEOT/PBT scaffolds for bone tissue engineering: preparation,characterization, and in vitro bone marrow cell culturing

The preparation, characterization, and in vitro bone marrow cell culturing on porous PEOT/PBT copolymer scaffolds are described. These scaffolds are meant for use in bone tissue engineering. Previous research has shown that PEOT/PBT copolymers showed in vivo degradation, calcification, and bone bonding. Despite this, several of these copolymers do not support bone marrow cell growth in vitro. Surface modification, such as gas-plasma treatment, is needed to improve the in vitro cell attachment. Porous structures were prepared using a freeze-drying and a salt-leaching technique, the latter one resulting in highly porous interconnected structures of large pore size. Gas-plasma treatment with CO(2) generated a surface throughout the entire structure that enabled bone marrow cells to attach. The amount of DNA was determined as a measure for the amount of cells present on the scaffolds. No significant effect of pore size on the amount of DNA present was seen for scaffolds with pore sizes between 250-1000 microm. Light microscopy data showed cells in the center of the scaffolds, more cells were observed in the scaffolds of 425-500 microm and 500-710 microm pore size compared to the ones with 250-425 microm and 710-1000 microm pores.     

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

Biocompatibility and degradation of poly(ether-ester) microspheres: in vitro and in vivo evaluation

Microspheres of a hydrophobic and a hydrophilic poly(ether–ester) copolymer were evaluated for their in vitro and in vivo biocompatibility and degradation. The microspheres prior to and after sterilization were tested for in vitro cytotoxicity. The in vivo biocompatibility of the poly(ethylene glycol) terephthalate and poly(butylene terephthalate) (PEGT/PBT) microspheres was evaluated subcutaneously and intramuscularly for 24 weeks in rabbits. The in vivo degradation of the microspheres was studied microscopically and compared to the in vitro degradation. The in vitro and in vivo studies showed the biocompatibility of the microspheres of both the hydrophobic and the hydrophilic PEGT/PBT copolymer. Extracts of these microspheres showed no cytotoxic reactivity in the in vitro cytotoxicity test. Sterilization of the microspheres by gamma irradiation did not affect the cytotoxicity. PEGT/PBT microspheres injected subcutaneously and intramuscularly in rabbits showed a mild tissue response in vivo, in terms of the inflammatory response, the foreign body reaction and the granulation tissue response. Although an in vitro degradation experiment showed a decrease in molecular weight due to hydrolysis, the in vivo degradation of the microspheres was slower than previously published.