Scaffold Permeability Research Articles

Permeability Evaluation of Lay-down Patterns and Pore Size of Pcl Scaffolds

The success of tissue engineering is strongly dependent on the ability to produce biomimetic scaffolds that mimic the biomechanical properties of the native host tissues. Additive Manufacturing techniques, namely Biomanufacturing are increasingly being recognized as the ideal methods to produce 3D porous structures. These systems produce scaffolds with an effective control over pore size/shape and spatial distribution. The BioExtruder system is one of the existing additive biomanufacturing systems for tissue engineering applications. The working principle is based on the extrusion of thin filaments of low melting point biomaterials in a layer-by-layer fashion. There are several process and geometric parameters controlled by the BioExtruder system with a direct influence on the morphological and mechanical properties of the extruded scaffolds. This research work is focused on the geometric parameters, namely the pore architecture (lay-down pattern of the filaments) and the pore size regarding the scaffold's permeability.

Hydraulic permeability of multilayered collagen gel scaffolds under plastic compression-induced unidirectional fluid flow

Under conditions of free fluid flow, highly hydrated fibrillar collagen gels expel fluid and undergo gravity driven consolidation (self-compression; SC). This process can be accelerated by the application of a compressive stress (plastic compression; PC) in order to generate dense collagen scaffolds for tissue engineering. To define the microstructural evolution of collagen gels under PC, this study applied a two-layer micromechanical model that was previously developed to measure hydraulic permeability (k) under SC. Radially confined PC resulted in unidirectional fluid flow through the gel and the formation of a dense lamella at the fluid expulsion boundary which was confirmed by confocal microscopy of collagen immunoreactivity. Gel mass loss due to PC and subsequent SC were measured and applied to Darcy’s law to calculate the thickness of the lamella and hydrated layer, as well as their relative permeabilities. Increasing PC level resulted in a significant increase in mass loss fraction and lamellar thickness, while the thickness of the hydrated layer dramatically decreased. Permeability of lamella also decreased from 1.8×10−15 to 1.0×10−15m2 in response to an increase in PC level. Ongoing SC, following PC, resulted in a uniform decrease in mass loss and k with increasing PC level and as a function SC time. Experimental k data were in close agreement with those estimated by the Happel model. Calculation of average k values for various two-layer microstructures indicated that they each approached 10−15–10−14m2 at equilibrium. In summary, the two-layer micromechanical model can be used to define the microstructure and permeability of multi-layered biomimetic scaffolds generated by PC.

The fish scale‐derived Biocornea as a scaffold for human corneal cells

AbstractPurpose Collagen matrices are a promising alternative for corneal donor transplants. Existing collagen matrices are synthesized or derived from animal corneas. We investigated whether fish scale‐derived collagen type I matrix could serve as a scaffold for in vitro corneal regeneration.Methods Primary human keratocytes were cultured for 2 weeks onto uncoated and collagen type IV coated scaffolds. Additionally a cell line of human corneal epithelial cells (HCEC) were co‐cultured with the scaffold. Cell morphology and tissue organization were assessed using fluorescence staining. Furthermore, epithelial morphogenesis, cell proliferation, cell infiltration and the effect of different protease treatments on scaffold permeability were analyzed as well.Results Keratocytes and HCECs cultured onto the micro‐patterned side covered the whole surface of the scaffold as well as keratocytes cultured onto the smooth side of the scaffold. No difference in cell attachment was observed between the uncoated and coated scaffolds. Cross sections showed no cellular infiltration into the scaffold. Dispase treatment separated the lamellae at the edges of the scaffold, but this did not induce cell penetration.Conclusion The fish scale‐derived scaffold is biocompatible with human corneal epithelial and stromal cells and could therefore be a promising non‐expensive basis for corneal regeneration. Additional stromal‐scaffold interaction and cell infiltration studies will be the focus of our research. Commercial interest

Permeability analysis of scaffolds for bone tissue engineering

Porous artificial bone substitutes, especially bone scaffolds coupled with osteobiologics, have been developed as an alternative to the traditional bone grafts. The bone scaffold should have a set of properties to provide mechanical support and simultaneously promote tissue regeneration. Among these properties, scaffold permeability is a determinant factor as it plays a major role in the ability for cells to penetrate the porous media and for nutrients to diffuse. Thus, the aim of this work is to characterize the permeability of the scaffold microstructure, using both computational and experimental methods. Computationally, permeability was estimated by homogenization methods applied to the problem of a fluid flow through a porous media. These homogenized permeability properties are compared with those obtained experimentally. For this purpose a simple experimental setup was used to test scaffolds built using Solid Free Form techniques. The obtained results show a linear correlation between the computational and the experimental permeability. Also, this study showed that permeability encompasses the influence of both porosity and pore size on mass transport, thus indicating its importance as a design parameter. This work indicates that the mathematical approach used to determine permeability may be useful as a scaffold design tool.

The influence of collagen–glycosaminoglycan scaffold relative density and microstructural anisotropy on tenocyte bioactivity and transcriptomic stability

Biomaterials for orthopedic tissue engineering must balance mechanical and bioactivity concerns. This work describes the fabrication of a homologous series of anisotropic collagen–GAG (CG) scaffolds with aligned tracks of ellipsoidal pores but increasing relative densities (ρ∗/ρs), and we report the role scaffold relative density plays in directing tenocyte bioactivity. Scaffold permeability and mechanical properties, both in tension and compression, were significantly influenced by relative density in a manner predicted by cellular solids models. Equine tenocytes showed greater levels of attachment, metabolic activity, soluble collagen synthesis, and alignment as well as less cell-mediated scaffold contraction in anisotropic CG scaffolds of increasing relative density. Notably, the lowest density scaffolds experienced significant cell-mediated contraction with associated decreases in tenocyte number as well as loss of microstructural integrity, aligned contact guidance cues, and preferential tenocyte orientation over a 14 day culture period. Gene expression analyses suggested tenocyte de-differentiation in the lowest density scaffold while indicating that the highest density scaffold supported significant increases in COMP (4-fold), tenascin-C (3-fold), and scleraxis (15-fold) expression as well as significant decreases in MMP-1 (9-fold) and MMP-13 (13-fold) expression on day 14. These results suggest that anisotropic scaffold relative density can help to modulate the maintenance of a more tendon-like microenvironment and aid long-term tenocyte transcriptomic stability. Overall, this work demonstrates that relative density is a critical scaffold parameter, not only for insuring mechanical competence, but also for directing cell transcriptomic stability and behavior.

Prediction of permeability of regular scaffolds for skeletal tissue engineering: A combined computational and experimental study

Scaffold permeability is a key parameter combining geometrical features such as pore shape, size and interconnectivity, porosity and specific surface area. It can influence the success of bone tissue engineering scaffolds, by affecting oxygen and nutrient transport, cell seeding efficiency, in vitro three-dimensional (3D) cell culture and, ultimately, the amount of bone formation. An accurate and efficient prediction of scaffold permeability would be highly useful as part of a scaffold design process. The aim of this study was (i) to determine the accuracy of computational fluid dynamics (CFD) models for prediction of the permeability coefficient of three different regular Ti6Al4V scaffolds (each having a different porosity) by comparison with experimentally measured values and (ii) to verify the validity of the semi-empirical Kozeny equation to calculate the permeability analytically. To do so, five CFD geometrical models per scaffold porosity were built, based on different geometrical inputs: either based on the scaffold’s computer-aided design (CAD) or derived from 3D microfocus X-ray computed tomography (micro-CT) data of the additive manufactured (AM) scaffolds. For the latter the influence of the spatial image resolution and the image analysis algorithm used to determine the scaffold’s architectural features on the predicted permeability was analysed. CFD models based on high-resolution micro-CT images could predict the permeability coefficients of the studied scaffolds: depending on scaffold porosity and image analysis algorithm, relative differences between measured and predicted permeability values were between 2% and 27%. Finally, the analytical Kozeny equation was found to be valid. A linear correlation between the ratio Φ3/Ss2 and the permeability coefficient k was found for the predicted (by means of CFD) as well as measured values (relative difference of 16.4% between respective Kozeny coefficients), thus resulting in accurate and efficient calculation of the permeability of regular AM scaffolds.

Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture

Quantifying the effects of mechanical loading on the metabolic response of chondrocytes is difficult due to complicated structure of cartilage ECM and the coupled nature of the mechanical stimuli presented to the cells. In this study we describe the effects of fluid flow, particularly hydrostatic pressure and wall shear stress, on the Ca(2+) signaling response of bovine articular chondrocytes in 3D culture. Using well-established alginate hydrogel system to maintain spherical chondrocyte morphology, we altered solid volume fraction to change scaffold mechanics. Fluid velocities in the bulk of the scaffolds were directly measured via an optical technique and scaffold permeability and aggregate modulus was characterized to quantify the mechanical stimuli presented to cells. Ca(2+) signaling response to direct perfusion of chondrocyte-seeded scaffolds increased monotonically with flow rate and was found more directly dependent on fluid velocity rather than shear stress or hydrostatic pressure. Chondrocytes in alginate scaffolds responded to fluid flow at velocities and shear stresses 2-3 orders of magnitude lower than seen in previous monolayer studies. Our data suggest that flow-induced Ca(2+) signaling response of chondrocytes in alginate culture may be due to mechanical signaling pathways, which is influenced by the 3D nature of cell shape.

The realistic prediction of oxygen transport in a tissue-engineered scaffold by introducing time-varying effective diffusion coefficients

An adequate oxygen supply is one of the most important factors needed in order to regenerate or engineer thick tissues or complex organs. To devise a method for maximizing the amount of oxygen available to cells, it is necessary to understand and to realistically predict oxygen transport within an engineered tissue. In this study, we focused on the fact that oxygen transport through a tissue-engineered scaffold may vary with time as cells proliferate. To confirm this viewpoint, effective oxygen diffusion coefficients ( D e , s ) of scaffolds were deduced from experimental measurements and simulations of oxygen-concentration profiles were performed using these D e , s values in a two-dimensional (2-D) perfusion model. The results of this study indicate that higher porosity, hydraulic permeability and interconnectivity of scaffolds with no cells are responsible for the prominent diffusion capability quantified using D e , s . On the other hand, the D e , s of scaffolds with cells has a negative linear relationship with cell density. Cell proliferation with time leads to a significant decrease in oxygen concentration in the 2-D perfusion model. This result demonstrates the gradual restriction of oxygen transport in a porous scaffold during cell culture. Therefore, the realistic prediction of oxygen transport using a time-varying D e , s will provide an appropriate basis for designing optimal transport networks within a thick scaffold.

Evaluation of Scaffolds based on α-Tricalcium Phosphate Cements for Tissue Engineering Applications

Growth of cells in 3-D porous scaffolds has gained importance in the field of tissue engineering. The scaffolds guide cellular growth, synthesize extracellular matrix and other biological molecules, and make the formation of tissues and functional organs easier. The aim of this study is to use α-tricalcium phosphate cement in order to obtain new types of scaffolds with the aid of paraffin spheres as pore generators. The porosity of the scaffolds produced with paraffin spheres was analyzed and compared to the literature, and the study of scaffold permeability using the Forchheimer equation allowed the analysis of pore interconnectivity. In vitro tests showed the behavior of scaffolds in solutions of simulated body fluid, and viability and cell proliferation were also evaluated. The results show the potential use of the materials developed for scaffolds for use in tissue engineering applications.

Mechanical and Biochemical Assessments of Three-Dimensional Poly(1,8-Octanediol-co-Citrate) Scaffold Pore Shape and Permeability Effects onIn VitroChondrogenesis Using Primary Chondrocytes

Poly(1,8-octanediol-co-citrate) (POC) is a biocompatible, biodegradable elastomer with potential application for soft tissue applications such as cartilage. For chondrogenesis, permeability is a scaffold design target that may influence cartilage regeneration. Scaffold permeability is determined by many factors such as pore shape, pore size, pore interconnectivity, porosity, and so on. Our focus in this study was to examine the effects of pore shape and permeability of two different POC scaffold designs on matrix production, mRNA gene expression, and differentiation of chondrocytes in vitro and the consequent mechanical property changes of the scaffold/tissue constructs. Since type I collagen gel was used as a cell carrier in the POC scaffolds, we also examined the effects of collagen gel concentration on chondrogenesis. We found that lower collagen I gel concentration provides a favorable microenvironment for chondrocytes promoting better chondrogenic performance of chondrocytes. With regard to scaffold design, low permeability with a spherical pore shape better enhanced the chondrogenic performance of chondrocytes in terms of matrix production, and mRNA gene expressions in vitro compared to the highly permeable scaffold with a cubical pore shape.

Reduced hydraulic permeability of three-dimensional collagen scaffolds attenuates gel contraction and promotes the growth and differentiation of mesenchymal stem cells

Optimal scaffold characteristics are essential for the therapeutic application of engineered tissues. Hydraulic permeability (k) affects many properties of collagen gels, such as mechanical properties, cell-scaffold interactions within three dimensions (3D), oxygen flow and nutrient diffusion. However, the cellular response to 3D gel scaffolds of defined k values has not been investigated. In this study, unconfined plastic compression under increasing load was used to produce collagen gels with increasing solid volume fractions. The Happel model was used to calculate the resulting permeability values in order to study the interaction of k with gel mechanical properties and mesenchymal stem cell (MSC)-induced gel contraction, metabolism and differentiation in both non-osteogenic (basal medium) and osteogenic medium for up to 3 weeks. Collagen gels of fibrillar densities ranging from 0.3 to >4.1 wt.% gave corresponding k values that ranged from 1.00 to 0.03 microm(2). Mechanical testing under compression showed that the collagen scaffold modulus increased with collagen fibrillar density and a decrease in k value. MSC-induced gel contraction decreased as a direct function of decreasing k value. Relative to osteogenic conditions, non-osteogenic MSC cultures exhibited a more than 2-fold increase in gel contraction. MSC metabolic activity increased similarly under both osteogenic and non-osteogenic culture conditions for all levels of plastic compression. Under osteogenic conditions MSC differentiation and mineralization, as indicated by alkaline phosphatase activity and von Kossa staining, respectively, increased in response to an elevation in collagen fibrillar density and decreased gel permeability. In this study, gel scaffolds with higher collagen fibrillar densities and corresponding lower k values provided a greater potential for MSC differentiation and appear most promising for bone grafting purposes. Thus, cell-scaffold interactions can be optimized by defining the 3D properties of collagen scaffolds through k adjustment.

Mechanical, permeability, and degradation properties of 3D designed poly(1,8 octanediol‐co‐citrate) scaffolds for soft tissue engineering

Poly(1,8-octanediol-co-citric acid) (POC) is a synthetic biodegradable elastomer that can be processed into three-dimensional (3D) scaffolds for tissue engineering. We investigated the effect of designed porosity on the mechanical properties, permeability, and degradation profiles of the POC scaffolds. For mechanical properties, scaffold compressive data were fitted to a one-dimensional (1D) nonlinear elastic model, and solid tensile data were fitted to a Neohookean incompressible nonlinear elastic model. Chondrocytes were seeded on scaffolds to assess the biocompatibility of POC. Increased porosity was associated with increased degradation rate, increased permeability, and decreased mechanical stiffness, which also became less nonlinear. Scaffold characterization in this article will provide design guidance for POC scaffolds to meet the mechanical and biological parameters needed for engineering soft tissues such as cartilage.

Differential effects of designed scaffold permeability on chondrogenesis by chondrocytes and bone marrow stromal cells

It has been widely postulated that scaffold permeability has a significant influence on chondrogenesis. However, since permeability has not been rigorously controlled in previous studies, there is no definitive conclusion as to how permeability affects cartilage regeneration by primary chondrocytes or progenitor cells. Here we explored the in vitro effects of scaffold permeability on matrix production and cellular differentiation of chondrocytes and bone marrow stromal cells (BMSCs) using precisely designed poly(ɛ-caprolactone) (PCL) scaffolds in which the high permeability design was 5.25 times more permeable than the lowest permeability design. We found that scaffold permeability affects the chondrogenic performance of chondrocytes and bone marrow stromal cells in opposite ways. Decreased scaffold permeability results in an increase in cartilaginous matrix production by chondrocytes, promoting increases in aggrecan content and collagen 2: collagen 1 gene expression ratios. On the other hand, increased scaffold permeability is more favorable for differentiation of BMSCs down a chondrogenic lineage in this model. The ability to direct cell differentiation and chondrogenesis through a physical design parameter, such as permeability, establishes the capability to incorporate chondrogenic potential into a material design.

Permeability of porous gelcast scaffolds for bone tissue engineering

The permeability of metallic and ceramic open-cell foams prepared by the gelcasting technique was assessed by fitting of Forchheimer’s equation to experimental pressure drop curves. The ceramic composition was based on pure hydroxyapatite, while the metallic composition was based on titanium metal. Experimental Darcian (k 1) and non-Darcian (k 2) permeability constants displayed values in the range 0.40–3.24 × 10−9 m2 and 3.11–175.8 × 10−6 m respectively. Tortuosity was evaluated by gas diffusion experiments and ranged from 1.67 to 3.60, with porosity between 72 and 81% and average hydraulic pore size between 325 and 473 μm. Such features were compared to data reported in the literature for cancellous bones and synthetic scaffolds for bone graft. A detailed discussion concerning the limitations of Darcy’s law for fitting laboratory data and for predicting fluid flow through scaffolds in real biomedical applications is also performed. Pore size was obtained by image analysis and was also derived from permeation-absorption-diffusion experiments. In both cases, values were within the range expected for porous scaffolds applications.

The pore size of polycaprolactone scaffolds has limited influence on bone regeneration in an in vivo model

Bone tissue engineering scaffolds should be designed to optimize mass transport, cell migration, and mechanical integrity to facilitate and enhance new bone growth. Although many scaffold parameters could be modified to fulfill these requirements, pore size is an important scaffold characteristic that can be rigorously controlled with indirect solid freeform fabrication. We explored the effect of pore size on bone regeneration and scaffold mechanical properties using polycaprolactone (PCL) scaffolds designed with interconnected, cylindrical orthogonal pores. Three scaffold designs with unique microarchitectures were fabricated, having pore sizes of 350, 550, or 800 microm. Bone morphogenetic protein-7 transduced human gingival fibroblasts were suspended in fibrin gel, seeded into scaffolds, and implanted subcutaneously in immuno-compromised mice for 4 or 8 weeks. We found that (1) modulus and peak stress of the scaffold/bone constructs depended on pore size and porosity at 4 weeks but not at 8 weeks, (2) bone growth inside pores depended on pore size at 4 weeks but not at 8 weeks, and (3) the length of implantation time had a limited effect on scaffold/bone construct properties. In conclusion, pore sizes between 350 and 800 microm play a limited role in bone regeneration in this tissue engineering model. Therefore, it may be advantageous to explore the effects of other scaffold structural properties, such as pore shape, pore interconnectivity, or scaffold permeability, on bone regeneration when designing PCL scaffolds for bone tissue engineering.

Permeability evaluation of 45S5 Bioglass ®-based scaffolds for bone tissue engineering

Permeability is a key parameter for microstructural design of scaffolds, since it is related to their capability for waste removal and nutrients/oxygen supply. In this framework, Darcy's experiments were carried out in order to determine the relationship between the pressure drop gradient and the fluid flow velocity in Bioglass ®-based scaffolds to obtain the scaffold's permeability. Using deionised water as working fluid, the measured average permeability value on scaffolds of 90–95% porosity was 1.96×10 −9 m 2. This value lies in the published range of permeability values for trabecular bone.

Smooth muscle cell adhesion in surface‐modified three‐dimensional copolymer scaffolds prepared from co‐continuous blends

In this article, tissue-engineering scaffolds were fabricated from P(epsilon-CL-co-D,L-LA)-PEG-P(epsilon-CL-co-D,L-LA) copolymers using co-continuous blends with polystyrene as the porogen phase. By means of static annealing and following extraction of the porogen phase, pore sizes (channel widths) in the range of 15-350 microm were obtained. Smooth muscle cells were seeded in three-dimensional fibronectin-modified scaffolds of two different pore sizes. Considerably enhanced cell seeding efficiency was found for scaffolds with larger pore sizes, indicating the importance of this parameter to promote effective cell intrusion into bulk materials. Compressive moduli ranged from 2.3 +/- 0.3 to 67 +/- 15 MPa and decreased with increasing pore size. The reverse trend was found for scaffold permeability (kappa), which ranged from 8.5 x 10(-16) to 6.7 x 10(-11) m(2). This was comparable with permeabilities previously reported for scaffolds with higher pore sizes and void volumes, but more irregular pore morphologies. Taken together, the results obtained in this study motivate further investigation for possible future applications in tissue engineering.

Influence of pore size on tensile strength, permeability and porosity of hyaluronan-collagen scaffolds

Recent investigations have shown the importance of scaffold pore size on the realisation of tissue engineered cartilage which promotes cell adhesion, proliferation and differentiation. The objective of this study was to investigate the influence of pore size on the mechanical properties, the permeability and the porosity of hyaluronan-collagen scaffolds. Hyaluronan-collagen scaffolds with three different mean pore sizes (302.5, 402.5 and 525 microm) have been produced according to a standardised protocol. The maximum stress at rupture, the Young's Moduli, permeability and porosity of the scaffolds were investigated. The permeability was determined both empirically and mathematically. Increased pore sizes indicated a larger stress at rupture as well as increased Young's Moduli. Porosity and permeability were raised by increasing pore sizes. The mathematically calculated permeability showed the same trend. The results indicate a higher mechanical stability for scaffolds with larger pores. The experimental and mathematical experiments both show increased permeability and fluid mobility for larger pores in scaffolds. Morphological changes resulting from the alteration of pore size led to non-correlation between the calculated and the experimental permeability.

Scaffold permeability as a means to determine fiber diameter and pore size of electrospun fibrinogen

The purpose of this study was to construct a flowmeter that could accurately measure the hydraulic permeability of electrospun fibrinogen scaffolds, providing insight into the transport properties of electrospun scaffolds while making the measurement of their topographical features (fiber diameter and pore size) more accurate. Three different concentrations of fibrinogen were used (100, 120, and 150 mg/mL) to create scaffolds with three different fiber diameters and pore sizes. The fiber diameters and pore sizes of the electrospun scaffolds were first analyzed with scanning electron microscopy and image analysis software. The permeability of each scaffold was measured with the flowmeter and used to calculate permeability-based fiber diameters and pore sizes, which were compared to values obtained through image analysis. Permeability measurement revealed scaffold permeability to increase with fibrinogen concentration, much like average fiber diameter and pore size. Comparison between the two measurement methods demonstrated the efficacy of the flowmeter as a way to measure scaffold features.

The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering

The permeability of scaffolds and other three-dimensional constructs used for tissue engineering applications is important as it controls the diffusion of nutrients in and waste out of the scaffold as well as influencing the pressure fields within the construct. The objective of this study was to characterize the permeability/fluid mobility of collagen-GAG scaffolds as a function of pore size and compressive strain using both experimental and mathematical modeling techniques. Scaffolds containing four distinct mean pore sizes (151, 121, 110, 96 microns) were fabricated using a freeze-drying process. An experimental device was constructed to measure the permeability of the scaffold variants at different levels of compressive strain (0, 14, 29 and 40% while a low-density open-cell foam cellular solids model utilizing a tetrakaidecahedral unit cell was used to accurately model the permeability of each scaffold variant at all level of applied strain. The results of both the experimental and the mathematical analysis revealed that scaffold permeability increases with increasing pore size and decreases with increasing compressive strain. The excellent comparison between experimentally measured and predicted scaffold permeability suggests that cellular solids modelling techniques can be utilized to predict scaffold permeability under a variety of physiological loading conditions as well as to predict the permeability of future scaffolds with a wide variety of pore microstructures.