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The contractile behavior of cells is relevant in understanding wound healing and scar formation. In tissue engineering, inhibition of the cell contractile response is critical for the regeneration of physiologically normal tissue rather than scar tissue. Previous studies have measured the contractile response of cells in a variety of conditions (e.g. on two-dimensional solid substrates, on free-floating tissue engineering scaffolds and on scaffolds under some constraint in a cell force monitor). Tissue engineering scaffolds behave mechanically like open-cell elastomeric foams: between strains of about 10 and 90%, cells progressively buckle struts in the scaffold. The contractile force required for an individual cell to buckle a strut within a scaffold has been estimated based on the strut dimensions (radius, r, and length, l) and the strut modulus, E(s). Since the buckling force varies, according to Euler's law, with r(4)/l(2), and the relative density of the scaffold varies as (r/l)(2), the cell contractile force associated with strut buckling is expected to vary with the square of the pore size for scaffolds of constant relative density. As the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease. Here we model the contractile response of fibroblasts by analyzing the response of a single tetrakaidecahedron to forces applied to individual struts (simulating cell contractile forces) using finite element analysis. We model tetrakaidecahedra of different strut lengths, corresponding to different scaffold pore sizes, and of varying numbers of loaded struts, corresponding to varying cell densities. We compare our numerical model with the results of free-floating contraction experiments of normal human dermal fibroblasts (NHDF) in collagen-GAG scaffolds of varying pore size and with varying cell densities., ((c) 2010 Elsevier Ltd. All rights reserved.)

This paper is the second in a series of papers describing the design and development of an osteochondral scaffold using collagen-glycosaminoglycan and calcium phosphate technologies engineered for the regenerative repair of articular cartilage defects. The previous paper described a technology (concurrent mapping) for systematic variation and control of the chemical composition of triple coprecipitated collagen, glycosaminoglycan, and calcium phosphate (CGCaP) nanocomposites without using titrants. This paper describes (1) fabricating porous, three-dimensional scaffolds from the CGCaP suspensions, (2) characterizing the microstructure and mechanical properties of such scaffolds, and (3) modifying the calcium phosphate mineral phase. The methods build on the previously demonstrated ability to vary the composition of a CGCaP suspension (calcium phosphate mass fraction between 0 and 80 wt %) and enable the production of scaffolds whose pore architecture (mean pore size: 50-1000 microm), CaP phase chemistry (brushite, octacalcium phosphate, apatite) and crosslinking density (therefore mechanical properties and degradation rate) can be independently controlled. The scaffolds described in this paper combine the desirable biochemical properties and pore architecture of porous collagen-glycosaminoglycan scaffolds with the strength and direct bone-bonding properties of calcium phosphate biomaterials in a manner that can be tailored to meet the demands of a range of applications in orthopedics and regenerative medicine., ((c) 2009 Wiley Periodicals, Inc.)

Collagen-glycosaminoglycan scaffolds for the regeneration of skin have previously been fabricated by freeze-drying a slurry containing a co-precipitate of collagen and glycosaminoglycan. The mechanical properties of the scaffold are low (e.g. the dry compressive Young's modulus is roughly 30kPa and the dry compressive strength is roughly 5kPa). There is interest in using these scaffolds for tendon and ligament regeneration where there is a need for improved mechanical properties. Previous attempts to increase the mechanical properties of the scaffold by increasing the solid volume fraction of the scaffolds were limited by the increasing viscosity of the slurry, making it more difficult to mix and giving inhomogeneous scaffolds. Our recent work on mineralized collagen-glycosaminoglycan scaffolds used a vacuum filtration technique to increase the volume fraction of solids in the slurry, thereby increasing the density and mechanical properties of the scaffolds. In this work, we used this technique to fabricate collagen-glycosaminoglycan scaffolds with dry densities between 0.0076 and 0.0311gcm(-3) and pore sizes between 250 and 350microm, values appropriate for soft tissue growth. The compressive Young's modulus and strength in the dry state increased from 32 to 127kPa and from 5 to 19kPa, respectively, with increasing density. The tensile Young's modulus in the dry state increased from 295 to 3.1MPa with increasing density. Finally, we showed that the attachment of cells onto the scaffold was directly proportional to the specific surface area of the scaffold, which defines the total internal surface area per volume of scaffold.

Mineralized collagen-glycosminoglycan scaffolds have previously been fabricated by freeze-drying a slurry containing a co-precipitate of calcium phosphate, collagen and glycosaminoglycan. The mechanical properties of the scaffold are low (e.g. the dry Young's modulus for a 50 wt.% mineralized scaffold is roughly 780 kPa). Our previous attempt to increase the mechanical properties of the scaffold by increasing the mineralization (from 50 to 75 wt.%) was unsuccessful due to defects in the more mineralized scaffold. In this paper, we describe a new technique to improve the mechanical properties by increasing the relative density of the scaffolds. The volume fraction of solids in the slurry was increased by vacuum-filtration. The slurry was then freeze-dried in the conventional manner to produce scaffolds with relative densities between 0.045 and 0.187 and pore sizes of about 100-350 microm, values appropriate for bone growth. The uniaxial compressive stress-strain curves of the scaffolds indicated that the Young's modulus in the dry state increased from 780 to 6500 kPa and that the crushing strength increased from 39 to 275 kPa with increasing relative density. In the hydrated state, the Young's modulus increased from 6.44 to 34.8 kPa and the crushing strength increased from 0.55 to 2.12 kPa; the properties were further increased by cross-linking. The modulus and strength were well described by models for cellular solids.

Mineralized collagen-glycosaminoglycan scaffolds designed for bone regeneration have been synthesized via triple co-precipitation in the absence of a titrant phase. Here, we characterize the microstructural and mechanical properties of these newly developed scaffolds with 50 and 75 wt.% mineral content. The 50 wt.% scaffold had an equiaxed pore structure with isotropic mechanical properties and a Ca-P-rich mineral phase comprised of brushite; the 75 wt.% scaffold had a bilayer structure with a pore size varying in the through-thickness direction and a mineral phase comprised of 67% brushite and 33 wt.% monetite. The compressive stress-strain response of the scaffolds was characteristic of low-density open-cell foams with distinct linear elastic, collapse plateau and densification regimes. The elastic modulus and strength of individual struts within the scaffolds were measured using an atomic force microscopy cantilevered beam-bending technique and compared with the composite response under indentation and unconfined compression. Cellular solids models, using the measured strut properties, overestimated the overall mechanical properties for the scaffolds; the discrepancy arises from defects such as disconnected pore walls within the scaffold. As the scaffold stiffness and strength decreased with increasing overall mineral content and were less than that of natural, mineralized collagen scaffolds, these microstructural/mechanical relations will be used to further improve scaffold design for bone regeneration applications.

Cell-mediated contraction plays a critical role in many physiological and pathological processes, notably organized contraction during wound healing. Implantation of an appropriately formulated (i.e., mean pore size, chemical composition, degradation rate) three-dimensional scaffold into an in vivo wound site effectively blocks the majority of organized wound contraction and results in induced regeneration rather than scar formation. Improved understanding of cell contraction within three-dimensional constructs therefore represents an important area of study in tissue engineering. Studies of cell contraction within three-dimensional constructs typically calculate an average contractile force from the gross deformation of a macroscopic substrate by a large cell population. In this study, cellular solids theory has been applied to conventional column buckling relationships to quantify the magnitude of individual cell contraction events within a three-dimensional, collagen-glycosaminoglycan scaffold. This new technique can be used for studying cell mechanics with a wide variety of porous scaffolds that resemble low-density, open-cell foams. It extends previous methods for analyzing cell buckling of two-dimensional substrates to three-dimensional constructs. From data available in the literature, the mean contractile force (Fc) generated by individual dermal fibroblasts within the collagen-glycosaminoglycan scaffold was calculated to range between 11 and 41 nN (Fc=26+/-13 nN, mean+/-SD), with an upper bound of cell contractility estimated at 450 nN.

Tissue engineering scaffolds are used extensively as three-dimensional analogs of the extracellular matrix (ECM). However, less attention has been paid to characterizing the scaffold microstructure and mechanical properties than to the processing and bioactivity of scaffolds. Collagen-glycosaminoglycan (CG) scaffolds have long been utilized as ECM analogs for the regeneration of skin and are currently being considered for the regeneration of nerve and conjunctiva. Recently a series of CG scaffolds with a uniform pore microstructure has been developed with a range of sizes of equiaxed pores. Experimental characterization and theoretical modeling techniques have previously been used to describe the pore microstructure, specific surface area, cell attachment and permeability of these variants. The results of tensile and compressive tests on these CG scaffolds and of bending tests on the individual struts that define the scaffold network are reported here. The CG scaffold variants exhibited stress-strain behavior characteristic of low-density, open-cell foams with distinct linear elastic, collapse plateau and densification regimes. Scaffolds with equiaxed pores were found to be mechanically isotropic. The independent effects of hydration level, pore size, crosslink density and relative density on the mechanical properties was determined. Independent control over scaffold stiffness and pore size was obtained. Good agreement was observed between experimental results of scaffold mechanical characterization and low-density, open-cell foam model predictions for uniform scaffolds. The characterized scaffold variants provide a standardized framework with defined extracellular environments (microstructure, mechanics) for in vitro studies of the mechanical interactions between cells and scaffolds as well as in vivo tissue engineering studies.

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.

The objective of the study was to directly measure the force of contraction of adult articular chondrocytes and to examine their contractile behavior in collagen-glycosaminoglycan analogs of extracellular matrix by live cell imaging in vitro. The contractile forces generated by passages 2 and 3 adult canine articular chondrocytes were measured using a cell force monitor. The contractile behavior of the cells was also directly imaged as they were cultured in collagen-glycosaminoglycan scaffolds. Passage 2 cells seeded in a collagen-glycosaminoglycan scaffold were capable of generating a force of 0.3 nN/cell. Chondrocytes subcultured through a third passage generated a force twice that level, paralleling the increase in the alpha-smooth muscle actin (SMA) content of the cells as demonstrated by Western blot analysis. Treatment of passage 3 cells with staurosporine reduced the force of contraction by approximately one-half, reflecting the effects of this agent in reducing the SMA content of the cells and disrupting the microfilaments. These values compare with 1 nN previously reported for lapine dermal fibroblasts of passage 5-7, using the same apparatus. Direct live cell imaging documented the contractile behavior of the articular chondrocytes in the collagen-glycosaminoglycan matrix in the time frame in which the force was directly measured in the cell force monitor. This imaging demonstrated how the cells acted individually and in unison to buckle the collagen struts making up the matrix. Adult articular chondrocytes are capable of generating a SMA-enabled force of contraction sufficient to model extracellular matrix molecules.

Abstract: Tissue engineering scaffolds are used extensively as three-dimensional analogs of the extracellular matrix (ECM). Collagen-glycosaminoglycan (CG) scaffolds have long been utilized as ECM analogs for the regeneration of skin and are currently being considered for the regeneration of nerve, conjunctiva, and a host or orthopedic tissues. Recently a series of CG scaffolds with a uniform pore microstructure has been developed with a range of sizes of equiaxed pores. Experimental characterization and theoretical modeling techniques have been used to describe the pore microstructure, specific surface area, tensile and compressive mechanical properties, cell attachment, and permeability of these variants. Here we describe the fabrication, and characterization, and modeling of a series of CG and mineralized CG scaffolds. We then discuss their use in vivo to induce tissue regeneration following injury and in vitro as standardized 3D materials to study the influence of microstructural and mechanical features on cell behaviors such as motility and contraction. [Copyright &y& Elsevier]