Interconnected Scaffolds Research Articles

Osteoblast behaviour on in situ photopolymerizable three-dimensional scaffolds based on d,l-lactide and ε-caprolactone: influence of pore volume, pore size and pore shape

Bone marrow cells were cultured on in situ photopolymerizable scaffolds based on D,L-lactide and epsilon-caprolactone. The influence of pore volume, size and shape were evaluated. Bone formation was demonstrated by ALP activity, osteocalcin secretion and histological analysis. TEM at the polymer interface revealed osteoblasts which secreted an extracellular matrix containing matrix vesicles loaded with apatite. Cellular infiltration was possible for scaffolds with a porosity of 70 and gelatin particle size of 250-355 microm. Scaffolds with a porosity less than 70 had the tendency to form a polymer top layer. Although increasing the gelatin particle size to 355-500 microm, leads to infiltration even in scaffolds with a porosity of 60. No infiltration was possible in scaffolds with sodium chloride as porogen. On the contrary, sucrose and gelatin leads to better interconnected scaffolds at the same porosity. Hence, spherical gelatin particles are suitable to use as porogen in photopolymerizable scaffolds.

Characterization of porous injectable poly‐(propylene fumarate)‐based bone graft substitute

The use of bone grafts for orthopedic applications have increased steadily over the past decade. With improvements in surgical technique, combined with an increasing aged population requiring orthopedic treatment, the need for bone grafts substitutes have also increased. To be useful clinically, the bone graft substitute must be biocompatible, bioabsorbable, and have convenient handling properties. In addition, it must possess a microarchitecture that allows cellular ingrowth and remodeling while simultaneously providing mechanical strength. Poly(propylene fumarate) (PPF) has been investigated as an injectable, biodegradable scaffold for orthopedic applications. Various methods to create a porous, interconnected polymer scaffold are available. The foaming technique is a convenient method to accomplish this task. Reactions between bicarbonate salts and weak acids generate CO(2) gas, causing a bubbling reaction during the polymerization process. This technique allows the porosity of the scaffold to be modulated. Image analysis and mechanical testing of porous PPF fabricated using the foaming technique shows that a highly porous, interconnected scaffold can be produced. At approximately 50% porosity, the scaffold has excellent handling properties, contains pore sizes ranging from 50 to 500 mum with an elastic modulus ranging from 20 to 40 MPa. The foaming technique provides an additional method by which clinically useful polymers can be fabricated for use in various bone tissue engineering applications.

Design and preparation of μ‐bimodal porous scaffold for tissue engineering

AbstractThe aim of this study was to prepare poly‐ϵ‐caprolactone (PCL) foams, with a well‐defined micrometric and bimodal open‐pore dimension distribution, suitable as scaffolds for tissue engineering. The porous network pathway was designed without using toxic agents by combining gas foaming (GF) and selective polymer extraction techniques. PCL was melt‐mixed with thermoplastic gelatin (TG) in concentrations ranging from 40 to 60 wt %, to achieve a cocontinuous blend morphology. The blends were subsequently gas foamed by using N2‐CO2 mixtures, with N2 amount ranging from 0 to 80 vol %. Foaming temperature was changed from 38 to 110°C and different pressure drop rates were used. After foaming, TG was removed by soaking in H2O. The effect of blend compositions and GF process parameters on foam morphologies was investigated. Results showed that different combinations of TG weight ratios and GF parameters allowed the modulation of macroporosity fraction, microporosity dimension, and degree of interconnection. By optimizing the process parameters it was possible to tailor the morphologies of highly interconnected PCL scaffolds for tissue engineering. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Porous methacrylate tissue engineering scaffolds: using carbon dioxide to control porosity and interconnectivity

Porous scaffold structures are used in tissue engineering to provide structural guidance for regenerating tissues. The use of carbon dioxide (CO2) to create such scaffolds has received some attention in the past but many researchers believe that although CO2 processing of polymers can lead to porous scaffolds there is limited interconnectivity between the pores. In this study, highly porous (greater than 85%) and well interconnected scaffolds were obtained in which the size, distribution and number of pores could be controlled. This control was achieved by altering the rate of venting from polymer discs saturated with CO2 under modest temperature and pressure. The polymer used is a blend of poly (ethyl methacrylate) and tetrahydrofurfuryl methacrylate (PEMA/THFMA). This polymer system has shown promise for potential applications in cartilage repair.

Differential Cell Viability of Chondrocytes and Progenitor Cells in Tissue-engineered Constructs Following Implantation into Osteochondral Defects

Animal studies in cartilage tissue engineering usually include the transfer of cultured cells into chondral or osteochondral defects. Immediately at implantation, the cells are exposed to a dramatically changed environment. The aim of this study was to determine the viability of two cell types currently considered for cellular therapies of cartilage defects-chondrocytes and progenitor cells-shortly after exposure to an osteochondral defect in rabbit knees. To that end, autogenic chondrocytes and periosteal cells were labeled with CM-DiI fluorochrome, seeded or cultured in PEGT/PBT scaffolds for periods up to 2 weeks, transferred into osteochondral defects, harvested 5 days postimplantation, and analyzed for cell viability. In order to further elucidate factors effecting cell viability within our model system, we investigated the effect of serum, 2) extracellular matrix surrounding implanted cells, 3) scaffold interconnectivity, and 4) hyaluronan, as a known cell protectant. Controls included scaffolds with devitalized cells and scaffolds analyzed at implantation. We found that the viability of periosteum cells (14%), but not of chondrocytes (65-95%), was significantly decreased after implantation. The addition of hyaluronan increased periostium cell viability to 44% (p < 0.05). Surprisingly, cell viability in less interconnected compression-molded scaffolds was higher compared to that of fully interconnected scaffolds produced by rapid prototyping. All other factors tested did not affect viability significantly. Our data suggest chondrocytes as a suitable cell source for cartilage repair in line with clinical data on several chondrocyte-based therapies. Although we did not test progenitor cells other the periosteum cells, tissue-engineering approaches using such cell types should take cell viability aspects into consideration.

Systematic selection of solvents for the fabrication of 3D combined macro- and microporous polymeric scaffolds for soft tissue engineering

In this study, we investigate the fabrication of 3D porous poly(lactic-co-glycolic acid) (PLGA) scaffolds using the thermally-induced phase separation technique. The current study focuses on the selection of alternative solvents for this process using a number of criteria, including predicted solubility, toxicity, removability and processability. Solvents were removed via either vacuum freeze-drying or leaching, depending on their physical properties. The residual solvent was tested using gas chromatography-mass spectrometry. A large range of porous, highly interconnected scaffold architectures with tunable pore size and alignment was obtained, including combined macro- and microporous structures and an entirely novel 'porous-fibre' structure. The morphological features of the most promising poly(lactic-co-glycolic acid) scaffolds were analysed via scanning electron microscopy and X-ray micro-computed tomography in both two and three dimensions. The Young's moduli of the scaffolds under conditions of temperature, pH and ionic strength similar to those found in the body were tested and were found to be highly dependent on the architectures.

Macroporous interconnected dextran scaffolds of controlled porosity for tissue-engineering applications

Dextran hydrogels have been studied as drug delivery vehicles but not as scaffolds for tissue-engineering likely because previously synthesized dextran hydrogels had pores too small for cell penetration. Our goal was to create macroporous, interconnected dextran scaffolds. To this end, we took advantage of the liquid–liquid immiscibility of poly(ethylene glycol) and methacrylated dextran during radical crosslinking of the methacrylated moieties. By controlling the degree of methacrylate substitution on dextran, dextran molar mass and PEG concentration, macroporous hydrogels were created. The presence of PEG in solution had a significant effect on the final morphology of the dextran hydrogel leading to the formation of different types of structures, from microporous gel to macroporous gel-wall to a macroporous interconnected-beaded structure. A series of formulation diagrams were prepared which allowed us to determine which conditions led to the formation of macroporous interconnected-beaded scaffolds. Dextran macroporous interconnected-beaded gels had a high water content, between 89% and 94%, a homogeneous morphology, determined by scanning electron microscopy, with interconnected macroporous pores, as determined by protein diffusivity where the effective diffusion coefficients of BSA were calculated to be 3.1×10 −7 cm 2/s for Dex-MA 40 kDa DS 5 and 1×10 −7 cm 2/s for Dex-MA 6 kDa DS10, which are similar to that of BSA in water, 5.9×10 −7 cm 2/s. Mercury intrusion porosimetry showed that the macroporous interconnected-beaded scaffolds had a bimodal distribution of macropores, with a median diameter of 41 μm, interconnected by throats, which had a median diameter of 11 μm. Together, these data suggest that the dextran scaffolds will be advantageous in applications that require an interconnected macroporous geometry, such as those of tissue engineering where cell penetration and nutrient diffusion are necessary for tissue regeneration.

A facile preparation of highly interconnected macroporous PLGA scaffolds by liquid–liquid phase separation II

A regular and well-interconnected macroporous (from 50 to 200μm) poly(d,l-lactic acid-co-glycolic acid) (PLGA) scaffold was fabricated by means of the thermally induced phase separation (TIPS) method. Poly(l-lactic acid) (PLLA) was blended with PLGA to increase the viscosity of polymer solution; a block copolymer of poly(ethylene glycol) (PEG) with PLGA was added as a surfactant to decrease the interfacial tension between the polymer-rich and polymer-lean phases. The effect of TIPS parameters including the concentration of diblock copolymer and PLGA/PLLA ratio was also studied. The cloud-point curve shifted to higher temperatures with both increasing the PLLA composition in the PLGA/PLLA blend and the PEG contents in the additives (PEG itself and PEG–PLGA diblocks). This shifting to higher temperature increases the quenching depth during phase separation. Addition of a PEG–PLGA diblock copolymer (0.5wt% in solution) to the PLGA/PLLA (1/1) blend polymer in a dioxane/water solution stabilized the morphology development during TIPS with respect to interconnection and macropores, and avoided segregation or sedimentation in the late stage.

Architecture control of three-dimensional polymeric scaffolds for soft tissue engineering. I. Establishment and validation of numerical models.

One of the most important functions of artificial three-dimensional (3D) polymeric scaffolds is to serve as a physical support to provide tissues with an appropriate architecture for in vitro cell culture as well as in vivo tissue regeneration. The production of three-dimensional (3D) polymeric scaffolds with tailored macroporous architecture is thus a crucial step in promoting controlled vascularization and tissue growth within host environments. In this study, 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds were manufactured by a thermally induced phase-separation (TIPS) technique. By controlling the quenching strategy, 3D interconnected PLGA scaffolds with tunable pore size and alignment were obtained and characterized with the use of scanning electron microscopy (SEM). A series of numerical heat-transfer models were established in an effort to describe the cooling process within the PLGA freezing regime. Among them, a two-dimensional (2D) solidification model has proved to be the most successful in describing the quenching of the polymer solution and has the potential to be used to infer the various 3D macroporous architectures created from different quenching conditions.

A novel fused interconnected scaffold for bone tissue engineering

A novel fused interconnected scaffold for bone tissue engineering

A facile preparation of highly interconnected macroporous poly( d, l-lactic acid- co-glycolic acid) (PLGA) scaffolds by liquid–liquid phase separation of a PLGA–dioxane–water ternary system

Regular and highly interconnected macroporous scaffolds ranging in size from 50 to 150 μm were fabricated from poly( d, l-lactic acid- co-glycolic acid) (PLGA)–dioxane–water ternary systems via thermally induced phase separation (TIPS) without any surfactant or other additives. The effect on scaffold morphology of processing parameters including quenching temperature, polymer concentration, solvent composition and molecular weight, was investigated as a function of quenching time. The cloud-point temperature of the polymer solution was found to depend on polymer concentration, solvent composition, and polymer molecular weight. The water content in the solvent mixture had the greatest effect on the cloud-point temperature. The optimal quenching temperature for preparing macroporous inter-connected scaffolds from a 9 wt% PLGA solution (dioxane–water=87/13, by wt) was less than −7 °C. In low viscosity PLGA solutions, sedimentation of the polymer rich phase occurred due to the segregation of the separated phases under gravity. This led to the formation of scaffolds with irregular and closed pores.

Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds.

Macroporous scaffolds composed of biodegradable polymers have found extensive use as three-dimensional substrates either for in vitro cell seeding followed by transplantation, or as conductive substrates for direct implantation in vivo. Methods abound for creation of macroporous scaffolds for tissue engineering, and common methods typically employ a solid porogen within a three-dimensional polymer matrix to create a well-defined pore size, pore structure, and total scaffold porosity. This study describes an approach to impart improved pore interconnectivity to polymer scaffolds for tissue engineering by partially fusing the solid porogen together prior to creation of a continuous polymer matrix. Three dimensional, porous scaffolds of the copolymer 85:15 poly(lactide-co-glycolide) were fabricated via either a solvent casting/particulate leaching process, or a gas foaming/particulate leaching process. Prior to creation of a continuous polymer matrix the NaCl crystals, which serve as the solid porogen, are partially fused via treatment in 95% humidity. Scanning electron micrographs clearly display fused salt crystals and an enhancement in pore interconnectivity in the salt fused scaffolds prepared via both solvent casting and gas foaming, and the extent of pore interconnectivity is enhanced with longer treatment times. Fusion of salt crystal for 24 h increased the radius of curvature of salt crystals, and led to a twofold increase in the compressive modulus of solvent cast scaffolds (total porosity of 97 +/- 1%). Fusion of NaCl crystals prior to gas foaming resulted in a decrease in scaffold compressive modulus from 277 +/- 60k Pa to 187 +/- 30k Pa (total porosity of 94 +/- 1%). The resulting highly interconnected scaffolds have implications for facilitated cell migration, abundant cell-cell interaction, and potentially improved neural and vascular growth within tissue engineering scaffolds.

Drug-releasing scaffolds fabricated from drug-loaded microspheres.

Biodegradable scaffolds serve a central role in many strategies for engineering tissue replacements or in guiding tissue regeneration. Typically, these scaffolds function to create and maintain a space and to provide a support for cell adhesion. However, these scaffolds also can serve as vehicles for the delivery of bioactive factors (e.g., protein or DNA) in order to manipulate cellular processes within the scaffold microenvironment. This study presents a novel approach to fabricate tissue-engineering scaffolds capable of sustained drug delivery whereby drug-loaded microspheres are fabricated into structures with controlled porosity. A double-emulsion process was used to fabricate microspheres with encapsulated DNA that retained its integrity and was released from the microspheres within 24 h. These DNA-loaded microspheres subsequently were formed into a nonporous disk or an interconnected open-pore scaffold (>94% porosity) via a gas-foaming process. The disks and scaffolds exhibited sustained plasmid release for at least 21 days and had minimal burst during the initial phase of release. This approach of assembling drug-loaded microspheres into porous and nonporous structures may find great utility in the fabrication of synthetic matrices that direct tissue formation.

A novel fabrication method of macroporous biodegradable polymer scaffolds using gas foaming salt as a porogen additive.

Highly open porous biodegradable poly(L-lactic acid) ¿PLLA scaffolds for tissue regeneration were fabricated by using ammonium bicarbonate as an efficient gas foaming agent as well as a particulate porogen salt. A binary mixture of PLLA-solvent gel containing dispersed ammonium bicarbonate salt particles, which became a paste state, was cast in a mold and subsequently immersed in a hot water solution to permit the evolution of ammonia and carbon dioxide within the solidifying polymer matrix. This resulted in the expansion of pores within the polymer matrix to a great extent, leading to well interconnected macroporous scaffolds having mean pore diameters of around 300-400 microm, ideal for high-density cell seeding. Rat hepatocytes seeded into the scaffolds exhibited about 95% seeding efficiency and up to 40% viability at 1 day after the seeding. The novelty of this new method is that the PLLA paste containing ammonium bicarbonate salt particles can be easily handled and molded into any shape, allowing for fabricating a wide range of temporal tissue scaffolds requiring a specific shape and geometry.