Carl G. Simon

The Effect of 3D Hydrogel Scaffold Modulus on Osteoblast Differentiation and Mineralization Revealed by Combinatorial Screening

Cells are known to sense and respond to the physical properties of their environment and those of tissue scaffolds. Optimizing these cell-material interactions is critical in tissue engineering. In this work, a simple and inexpensive combinatorial platform was developed to rapidly screen three-dimensional (3D) tissue scaffolds and was applied to screen the effect of scaffold properties for tissue engineering of bone. Differentiation of osteoblasts was examined in poly(ethylene glycol) hydrogel gradients spanning a 30-fold range in compressive modulus ( approximately 10 kPa to approximately 300 kPa). Results demonstrate that material properties (gel stiffness) of scaffolds can be leveraged to induce cell differentiation in 3D culture as an alternative to biochemical cues such as soluble supplements, immobilized biomolecules and vectors, which are often expensive, labile and potentially carcinogenic. Gel moduli of approximately 225 kPa and higher enhanced osteogenesis. Furthermore, it is proposed that material-induced cell differentiation can be modulated to engineer seamless tissue interfaces between mineralized bone tissue and softer tissues such as ligaments and tendons. This work presents a combinatorial method to screen biological response to 3D hydrogel scaffolds that more closely mimics the 3D environment experienced by cells in vivo.

Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation

The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (beta-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins generally lack bioactive peptides for enhancing cell functions. Thus, a synthetic spider silk, spidroin, containing two RGD cell adhesive sequences (RGD-spidroin) was engineered. RGD-spidroin was blended with different ratios of fibroin and spun coat into films on glass coverslips. beta-Sheet formation, contact angle, surface topography and RGD surface presentation were characterized and correlated with cell behavior. We found that the amount of beta-sheet formation was directly related to the RGD-spidroin content of the blends after annealing, with the pure RGD-spidroin demonstrating the highest amount of beta-sheet content. The increased beta-sheet content improved film stability under culture conditions. A new visualization technique demonstrated that the RGD presentation on the film surface was affected by both the RGD-spidroin content and annealing conditions. It was determined that 10mass% RGD-spidroin was necessary to improve film stability and to achieve osteoblast attachment and differentiation.

Freeform fabricated scaffolds with roughened struts that enhance both stem cell proliferation and differentiation by controlling cell shape.

We demonstrate that freeform fabricated (FFF) scaffolds with a roughened surface topography can support hBMSC proliferation, while also inducing osteogenic differentiation, for maximized generation of calcified, bone-like tissue. Previously, hBMSCs rapidly proliferated, without osteogenic differentiation, during culture in FFF scaffolds. In contrast, hBMSCs underwent osteogenic differentiation, with slow proliferation, during culture in nanofiber scaffolds. Analysis of cell morphology showed that the topography presented by the nanofiber scaffolds drove hBMSC differentiation by guiding them into a morphology that induced osteogenic differentiation. Herein, we hypothesized that using the high-surface area architecture of FFF scaffolds to present a surface roughness that drives hBMSCs into a morphology that induces osteogenic differentiation would yield a maximum amount differentiated hBMSCs and bone-like tissue. Thus, a solvent etching method was developed that imparted a 5-fold increase in roughness to the surface of the struts of poly(ε-caprolactone) (PCL) FFF scaffolds. The etched scaffolds induced osteogenic differentiation of the hBMSCs while un-etched scaffolds did not. The etched scaffolds also supported the same high levels of hBMSC proliferation that un-etched scaffolds supported. Finally, hBMSCs on un-etched scaffolds had a large spread area, while hBMSCs on etched scaffolds has a smaller area and were more rounded, indicating that the surface roughness from the etched scaffolds dictated the morphology of the hBMSCs. The results demonstrate that FFF scaffolds with surface roughness can support hBMSC proliferation, while also inducing osteogenic differentiation, to maximize generation of calcified tissue. This work validates a rational approach to scaffold fabrication where the structure of the scaffold was designed to optimize stem cell function by controlling cell morphology.

The support of bone marrow stromal cell differentiation by airbrushed nanofiber scaffolds

Nanofiber scaffolds are effective for tissue engineering since they emulate the fibrous nanostructure of native extracellular matrix (ECM). Although electrospinning has been the most common approach for fabricating nanofiber scaffolds, airbrushing approaches have also been advanced for making nanofibers. For airbrushing, compressed gas is used to blow polymer solution through a small nozzle which shears the polymer solution into fibers. Our goals were 1) to assess the versatility of airbrushing, 2) to compare the properties of airbrushed and electrospun nanofiber scaffolds and 3) to test the ability of airbrushed nanofibers to support stem cell differentiation. The results demonstrated that airbrushing could produce nanofibers from a wide range of polymers and onto a wide range of targets. Airbrushing was safer, 10-fold faster, 100-fold less expensive to set-up and able to deposit nanofibers onto a broader range of targets than electrospinning. Airbrushing yielded nanofibers that formed loosely packed bundles of aligned nanofibers, while electrospinning produced un-aligned, single nanofibers that were tightly packed and highly entangled. Airbrushed nanofiber mats had larger pores, higher porosity and lower modulus than electrospun mats, results that were likely caused by the differences in morphology (nanofiber packing and entanglement). Airbrushed nanofiber scaffolds fabricated from 4 different polymers were each able to support osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs). Finally, the differences in airbrushed versus electrospun nanofiber morphology caused differences in hBMSC shape where cells had a smaller spread area and a smaller volume on airbrushed nanofiber scaffolds. These results highlight the advantages and disadvantages of airbrushing versus electrospinning nanofiber scaffolds and demonstrate that airbrushed nanofiber scaffolds can support stem cell differentiation.

Fabrication of combinatorial polymer scaffold libraries

We have designed a novel combinatorial research platform to help accelerate tissue engineering research. Combinatorial methods combine many samples into a single specimen to enable accelerated experimentation and discovery. The platform for fabricating combinatorial polymer scaffold libraries can be used to rapidly identify scaffold formulations that maximize tissue formation. Many approaches for screening cell-biomaterial interactions utilize a two-dimensional format such as a film or surface to present test substrates to cells. However, cells in vivo exist in a three-dimensional milieu of extracellular matrix and cells in vitro behave more naturally when cultured in a three-dimensional environment than when cultured on a two-dimensional surface. Thus, we have designed a method for fabricating combinatorial biomaterial libraries where the materials are presented to cells in the form of three-dimensional, porous, salt-leached, polymer scaffolds. Many scaffold variations and compositions can be screened in a single experiment so that optimal scaffold formulations for tissue formation can be rapidly identified. In summary, we have developed a platform technology for fabricating combinatorial polymer scaffold libraries that can be used to screen cell response to materials in a three-dimensional, scaffold format.

X-ray microcomputed tomography for the measurement of cell adhesionand proliferation in polymer scaffolds

We have explored the use of X-ray microcomputed tomography (microCT) for assessing cell adhesion and proliferation in polymer scaffolds. Common methods for examining cells in scaffolds include fluorescence microscopy and soluble assays for cell components such as enzymes, protein or DNA. Fluorescence microscopy is generally qualitative and cannot visualize the scaffold interior. Soluble assays quantitatively measure cell number but do not yield information on cell spatial distribution. Herein, the ability of microCT to detect cells in scaffolds was compared with fluorescence microscopy and a soluble DNA assay. Comparisons were performed using polymer scaffolds that were seeded with cells at different densities and cultured for different times. The results showed that fluorescence microscopy had better resolution than muicroCT and that the soluble DNA assay was approximately 5x more sensitive than microCT under the conditions tested. However, microCT was able to image through opaque scaffolds to yield quantitative 3D imaging and analysis via a single, non-invasive modality. Quantitative microCT analysis of cell penetration into scaffolds was demonstrated. Further, quantitative microCT volume analysis required that the cell density in the scaffolds be greater than 1 million cells per mL indicating that microCT is best suited for quantifying cells at relatively high density during culture in scaffolds. In sum, the results demonstrate the benefits and limitations of using microCT for 3D imaging and analysis of cell adhesion and proliferation in polymer scaffolds.

Cell interactions with biomaterials gradients and arrays.

Gradients and arrays have become very useful to the fields of tissue engineering and biomaterials. Both gradients and arrays make efficient platforms for screening cell response to biomaterials. Graded biomaterials also have functional applications and make useful substrates for fundamental studies of cell phenomena such as migration. This article will review the use of gradients and arrays in tissue engineering and biomaterials research, with a focus on cellular and biologic responses.

In vitro Cytotoxicity of Amorphous Calcium Phosphate Composites

Calcium phosphate-based biomaterials are being increasingly used as bone substitutes in dentistry and in reconstructive and orthopedic applications because of their good biocompatibility, osteoconductivity and/or bone-bonding properties. In this study, the in vitro cytotoxicity of the amorphous calcium phosphate (ACP) filler, the copolymer matrix derived from the polymerization of a resin system and the corresponding ACP composite was analyzed utilizing cell culture techniques. The photo cured polymer was derived from an activated resin comprised of an ethoxylated bisphenol A dimethacrylate, urethane dimethacrylate, triethylene glycol dimethacrylate, and 2-hydroxyethyl methacrylate. The resin was admixed with a zirconia-ACP filler to prepare the composite. Specimens were extracted in media overnight and then MC3T3-E1 osteoblast-like cells were cultured in the extracts for 3 days. Cytoxicity was evaluated by phase contrast microscopy and an enzymatic assay for mitochondrial dehydrogenase activity (Wst-1). Cellular response to the experimental ACP composite was compared to the cellular response of commercially available light-cure orthodontic adhesive. In addition to the cytotoxicity testing the ion release profiles of ACP composites was determined. Furthermore, a degree of vinyl conversion (DVC) attained in the experimental composite and in the commercial control was compared. No adverse response regarding cell morphology and/or viability was observed with ACP composites compared to the unfilled copolymers or to the commercial adhesives. Sustained release of potentially remineralizing calcium and phosphate ions and favorable DVC of these composites confirms their value in a variety of dental and possibly orthopedic applications where anti-demineralizing/remineralizing efficacy is the primary goal.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-β and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation.

Cell adhesion to borate glasses by colloidal probe microscopy

The adhesion of osteoblast-like cells to silicate and borate glasses was measured in cell growth medium using colloidal probe microscopy. The probes consisted of silicate and borate glass spheres, 25-50 μm in diameter, attached to atomic force microscope cantilevers. Variables of the study included glass composition and time of contact of the cell to the glasses. Increasing the time of contact from 15 to 900 s increased the force of adhesion. The data could be plotted linearly on a log-log plot of adhesive force versus time. Of the seven glasses tested, five had slopes close to 0.5, suggesting a square root dependence of the adhesive force on the contact time. Such behavior can be interpreted as a diffusion limited process occurring during the early stages of cell attachment. We suggest that the rate limiting step in the adhesion process is the diffusion of integrins resident in the cell membrane to the area of cell attachment. Data presented in this paper support the hypothesis of Hench et al. that strong adhesion depends on the formation of a calcium phosphate reaction layer on the surfaces of the glass. Glasses that did not form a calcium phosphate layer exhibited a weaker adhesive force relative to those glasses that did form a calcium phosphate layer.