Christopher B. Raub

Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy

Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (∼200 mg ml⁻¹). Youngs modulus (E) measurements from acellular collagen gels (range 0.5-12 kPa) exhibited a power-law concentration dependence (range 3-9 mg ml⁻¹) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5-27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ∼5-200 mg ml⁻¹). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R²=0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R²=0.83, exponents of -0.7 and -1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.

Airway Epithelium Stimulates Smooth Muscle Proliferation

Communication between the airway epithelium and stroma is evident during embryogenesis, and both epithelial shedding and increased smooth muscle proliferation are features of airway remodeling. Hence, we hypothesized that after injury the airway epithelium could modulate airway smooth muscle proliferation. Fully differentiated primary normal human bronchial epithelial (NHBE) cells at an air-liquid interface were co-cultured with serum-deprived normal primary human airway smooth muscle cells (HASM) using commercially available Transwells. In some co-cultures, the NHBE were repeatedly (x4) scrape-injured. An in vivo model of tracheal injury consisted of gently denuding the tracheal epithelium (x3) of a rabbit over 5 days and then examining the trachea by histology 3 days after the last injury. Our results show that HASM cell number increases 2.5-fold in the presence of NHBE, and 4.3-fold in the presence of injured NHBE compared with HASM alone after 8 days of in vitro co-culture. In addition, IL-6, IL-8, monocyte chemotactic protein (MCP)-1 and, more markedly, matrix metalloproteinase (MMP)-9 concentration increased in co-culture correlating with enhanced HASM growth. Inhibiting MMP-9 release significantly attenuated the NHBE-dependent HASM proliferation in co-culture. In vivo, the injured rabbit trachea demonstrated proliferation in the smooth muscle (trachealis) region and significant MMP-9 staining, which was absent in the uninjured control. The airway epithelium modulates smooth muscle cell proliferation via a mechanism that involves secretion of soluble mediators including potential smooth muscle mitogens such as IL-6, IL-8, and MCP-1, but also through a novel MMP-9-dependent mechanism.

IL-13 induces a bronchial epithelial phenotype that is profibrotic

BackgroundInflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β2, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis.MethodsNormal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31.ResultsIL-13 induced increasing levels of MUC5AC protein, and TGF-β2, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β2, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β2 neutralizing antibody reversed the increase in collagen content and SHG signal.ConclusionProlonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β2 at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β2 secretion from the airway epithelium.

A novel three-dimensional model to quantify metastatic melanoma invasion

Although attempts to develop any viable chemotherapeutic approaches to combat metastatic cancers have largely failed, potential genetic targets to halt metastatic progression continue to be identified. As drugs are developed to address these targets, there is a need for high-throughput systems that accurately reproduce in vivo microenvironments to gauge their efficacy. Accordingly, we have developed a three-dimensional in vitro culture system representative of the environment present upon secondary metastasis to quantitatively measure tumor cell invasion in this setting three-dimensionally. Culturing melanomas of different metastatic capacities within the system showed that each cell type invades the matrix in a manner commensurate to its known metastatic potential in vivo. Moreover, the developed quantitative schemes were put to use to characterize the effect of microenvironmental influences (i.e., matrix components, interstitial cell presence) on planar and vertical melanoma invasion. We propose this novel, quantitative system as a useful tool to assess the effects of pharmacologic and/or microenvironmental influences on tumor cell invasion at a metastatic site. [Mol Cancer Ther 2007;6(2):552–61]

Linking optics and mechanics in an in vivo model of airway fibrosis and epithelial injury

Chronic mucosal and submucosal injury can lead to persistent inflammation and tissue remodeling. We hypothesized that microstructural and mechanical properties of the airway wall could be derived from multiphoton images. New Zealand White rabbits were intubated, and the tracheal epithelium gently denuded every other day for five days (three injuries). Three days following the last injury, the tracheas were excised for multiphoton imaging, mechanical compression testing, and histological analysis. Multiphoton imaging and histology confirm epithelial denudation, mucosal ulceration, subepithelial thickening, collagen deposition, immune cell infiltration, and a disrupted elastin network. Elastase removes the elastin network and relaxes the collagen network. Purified collagenase removes epithelium with subtle subepithelial changes. Youngs modulus [(E) measured in kiloPascal] was significantly elevated for the scrape injured (9.0+/-3.2) trachea, and both collagenase (2.6+/-0.4) and elastase (0.8+/-0.3) treatment significantly reduced E relative to control (4.1+/-0.7). E correlates strongly with second harmonic generation (SHG) signal depth decay for enzyme-treated and control tracheas (R(2)=0.77), but not with scrape-injured tracheas. We conclude that E of subepithelial connective tissue increases on repeated epithelial wounding, due in part to changes in elastin and collagen microstructure and concentration. SHG depth decay is sensitive to changes in extracellular matrix content and correlates with bulk Youngs modulus.

Detection and monitoring of early airway injury effects of half-mustard (2-chloroethylethylsulfide) exposure using high-resolution optical coherence tomography

Optical coherence tomography (OCT) is a noninvasive, high-resolution imaging technology capable of delivering real-time, near-histologic images of tissues. Mustard gas is a vesicant-blistering agent that can cause severe and lethal damage to airway and lungs. The ability to detect and assess airway injury in the clinical setting of mustard exposure is currently limited. The purpose of this study is to assess the ability to detect and monitor progression of half-mustard [2-chloroethylethylsulfide (CEES)] airway injuries with OCT techniques. A ventilated rabbit mustard exposure airway injury model is developed. A flexible fiber optic OCT probe is introduced into the distal trachea to image airway epithelium and mucosa in vivo. Progression of airway injury is observed over eight hours with OCT using a prototype time-domain superluminescent diode OCT system. OCT tracheal images from CEES exposed animals are compared to control rabbits for airway mucosal thickening and other changes. OCT detects the early occurrence and progression of dramatic changes in the experimental group after exposure to CEES. Histology and immunofluorescence staining confirms this finding. OCT has the potential to be a high resolution imaging modality capable of detecting, assessing, and monitoring treatment for airway injury following mustard vesicant agent exposures.

Glycosaminoglycan and Collagen Remodeling During In Vitro Dynamic Compression of Articular Cartilage: Experiments and Finite Element Modeling

The biomechanical properties of articular cartilage (AC) can be altered by chemical and mechanical stimuli. Dynamic unconfined compression (UCC) has been shown to increase biosynthesis at moderate strain amplitudes (1–4%) and frequencies from 0.01Hz. to 0.1Hz [1]. Furthermore, interstitial fluid velocity and maximum principle strain have been proposed as candidates for controlling glycosaminoglycan (GAG) and collagen (COL) remodeling, respectively [2,3]. The goal of this study was to integrate in vitro growth data, including biochemical and microstructural properties, into a computational continuum mixture model to elucidate potential mechanical triggers for AC tissue remodeling.Copyright

Integrating qPLM and biomechanical test data with an anisotropic fiber distribution model and in vitro regulation of articular cartilage fiber modulus

Articular cartilage (AC) metabolism and mechanical properties are regulated by in vitro culture with transforming growth factor–beta 1 (TGF-β1) and insulin-like growth factor–1 (IGF-1) [1]. In general, TGF-β1 maintains tissue size accompanied by a maintenance or increase in tensile and compressive moduli and a maintenance or decrease of compressive Poisson’s ratios while IGF-1 produces significant tissue expansion at the expense of reduced tensile and compressive moduli and increased compressive Poisson’s ratios [1,2]. The goal of this study was to integrate experimental data including AC mechanical properties, biochemical contents including overall collagen (COL) volume fraction, and micro structural measures of COL fiber distribution with a continuum mixture model to predict how COL fiber modulus changes in vitro with TGF-β1 and IGF-1 treatment.Copyright

Regulation of Articular Cartilage Volumetric and Compressive Properties by Sequential Application of IGF-1 and TGF-β1 During In Vitro Growth

Articular cartilage (AC) is a load bearing material that provides a low friction wear resistant interface in synovial joints. Naturally-occurring and stimulated intrinsic repair of damaged AC is challenging [1]. Thus, there is a need to guide the formation of tissue of specific size and mechanical properties for AC repair. Previous studies [2,3] with immature cartilage have shown that culture with medium containing TGF-β1 results in enhanced mechanical integrity while culture with IGF-1 results in diminished mechanical integrity. Conversely, culture with medium containing IGF-1 results in substantial volumetric growth while culture with TGF-β 1 results in little or no volumetric growth.Copyright

Correlations between second harmonic signal, microstructure, and mechanics of contracting collagen gels

Second harmonic generation (SHG) from collagen provides an optical signal that can yield detailed information about collagen microstructure when imaged with laser scanning microscopy, from both collagen-based engineered tissue and connective tissues from animals. Therefore SHG images may provide information that correlates with bulk tissue mechanical properties, or at least a component of those properties resulting from collagen. In order to probe these correlations, we used multiphoton microscopy to gather SHG signal intensity and depth decay information from fibroblast-seeded contracting collagen hydrogels. These gels were polymerized at pH 6 to engineer a tissue with large diameter collagen fibers and large pores between fibers, and pH 9 to produce smaller diameter collagen fibers with smaller pores. Both gels initially contained 4 mg/ml collagen; after 16 days of floating culture, the pH 6-polymerized gels had contracted to 4.4 ± 0.6% of their original volume, and the pH 9-polymerized gels to 10.7 ± 2.7%. During this time period, the bulk compressive moduli (CM) of the gels increased ~9.2-fold and ~1.4-fold for the pH 6 and pH 9 polymerization conditions, respectively. Correspondingly, the SHG signal at the tissue surface increased ~25-fold and ~19-fold for the pH 6 and pH 9 gels, respectively; whereas the effective SHG attenuation coefficient increased ~4.5 and ~5.8-fold, respectively. Meaningful linear correlations only existed between the CM and surface SHG signal and the CM and SHG attenuation coefficient for pH 6-polymerized gels, indicating a possible influence of fibroblast activity on the CM of the pH-9 polymerized gels.