The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds.

Biologic materials from various species and tissues are commonly used as surgical meshes or scaffolds for tissue reconstruction. Extracellular matrix (ECM) represents the secreted product of the cells comprising each tissue and organ, and therefore provides a unique biologic material for selected regenerative medicine applications. Minimal disruption of ECM ultrastructure and content during tissue processing is typically desirable. The objective of this study was to systematically evaluate effects of commonly used tissue processing steps upon porcine dermal ECM scaffold composition, mechanical properties, and cytocompatibility. Processing steps evaluated included liming and hot water sanitation, trypsin/SDS/TritonX-100 decellularization, and trypsin/TritonX-100 decellularization. Liming decreased the growth factor and glycosaminoglycan content, the mechanical strength, and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for all). Hot water sanitation treatment decreased only the growth factor content of the ECM (p ≤ 0.05). Trypsin/SDS/TritonX-100 decellularization decreased the growth factor content and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). Trypsin/Triton X-100 decellularization also decreased the growth factor content of the ECM but increased the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). We conclude that processing steps evaluated in the present study affect content, mechanical strength, and/or cytocompatibility of the resultant porcine dermal ECM, and therefore care must be taken in choosing appropriate processing steps to maintain the beneficial effects of ECM in biologic scaffolds.

Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement.

Three-dimensional culture alters cancer cell signaling; however, the underlying mechanisms and importance of these changes on tumor vascularization remain unclear. A hydrogel system was used to examine the role of the transition from 2D to 3D culture, with and without integrin engagement, on cancer cell angiogenic capability. Three-dimensional culture recreated tumor microenvironmental cues and led to enhanced interleukin 8 (IL-8) secretion that depended on integrin engagement with adhesion peptides coupled to the polymer. In contrast, vascular endothelial growth factor (VEGF) secretion was unaffected by 3D culture with or without substrate adhesion. IL-8 diffused greater distances and was present in higher concentrations in the systemic circulation, relative to VEGF. Implantation of a polymeric IL-8 delivery system into GFP bone marrow-transplanted mice revealed that localized IL-8 up-regulation was critical to both the local and systemic control of tumor vascularization in vivo. In summary, 3D integrin engagement within tumor microenvironments regulates cancer cell angiogenic signaling, and controlled local and systemic blockade of both IL-8 and VEGF signaling may improve antiangiogenic therapies.

Cyclic arginine-glycine-aspartate peptides enhance three-dimensional stem cell osteogenic differentiation.

The role of morphogens in bone regeneration has been widely studied, whereas the effect of matrix cues, particularly on stem cell differentiation, are less well understood. In this work, we investigated the effects of arginine-glycine-aspartate (RGD) ligand conformation (linear vs cyclic RGD) on primary human bone marrow stromal cell (hBMSC) and D1 stem cell osteogenic differentiation in three-dimensional (3D) culture and compared their response with that of committed MC3T3-E1 preosteoblasts to determine whether the stage of cell differentiation altered the response to the adhesion ligands. Linear RGD densities that promoted osteogenic differentiation of committed cells (MC3T3-E1 preosteoblasts) did not induce differentiation of hBMSCs or D1 stem cells, although matrices presenting the cyclic form of this adhesion ligand enhanced osteoprogenitor differentiation in 3D culture. This may be due to enhanced integrin-ligand binding. These studies indicate that biomaterial design parameters optimized for differentiated cell types may not directly translate to stem cell populations, because less-committed cells may require more instruction than differentiated cells. It is likely that design of synthetic extracellular matrices tailored to promote stem cell differentiation may enhance bone regeneration by transplanted cells.

Quantifying the relation between bond number and myoblast proliferation.

Many functions of the extracellular matrix can be mimicked by small peptide fragments (e.g., arginine-glycine-aspartic acid (RGD) sequence) of the entire molecule, but the presentation of the peptides is critical to their effects on cells. It is likely that some effects of peptide presentation from biomaterials simply relate to the number of bonds formed between cell receptors and the adhesion ligands, but a lack of tools to quantify bond number limits direct investigation of this assumption. The impact of different ligand presentations (density, affinity, and nanoscale distribution) on the proliferation of C2C12 and human primary myoblasts was first examined in this study. Increasing the ligand density or binding affinity led to a similar enhancement in proliferation of C2C12 cells and human primary myoblasts. The nanoscale distribution of clustered RGD ligands also influenced C2C12 cells and human primary myoblast proliferation, but in an opposing manner. A theological technique and a FRET technique were then utilized to quantify the number of receptor-ligand interactions as a function of peptide presentation. Higher numbers of bonds were formed when the RGD density and affinity were increased, as measured with both techniques, and bond number correlated with cell growth rates. However, the influence of the nanoscale peptide distribution did not appear to be solely a function of bond number. Altogether, these findings provide significant insight to the role of peptide presentation in the regulation of cell proliferation, and the approaches developed in this work may have significant utility in probing how adhesion regulates a variety of other cellular functions and aid in developing design criterion for cell-interactive materials.

Integrin-adhesion ligand bond formation of preosteoblasts and stem cells in three-dimensional RGD presenting matrices.

Cell-interactive polymers have been widely used as synthetic extracellular matrices to regulate cell function and promote tissue regeneration. However, there is a lack of quantitative understanding of the cell-material interface. In this study, integrin-adhesion ligand bond formation of preosteoblasts and D1 stem cells with RGD presenting alginate matrices were examined using FRET and flow cytometry. Bond number increased with adhesion ligand density but did not change with RGD island spacing for both cell types. Integrin expression varied with cell type and substrate in 2D culture, but the integrin expression profiles of both cell types were similar when cultured in 3D RGD presenting substrates and distinct from 2D culture. In summary, combining a FRET technique to quantify bond formation with flow cytometry to elucidate integrin expression can define specific cell-material interactions for a given material system and may be useful for informing biomaterial design strategies for cell-based therapies.

AFM imaging of RGD presenting synthetic extracellular matrix using gold nanoparticles.

Several high-resolution imaging techniques such as FESEM, TEM and AFM are compared with respect to their application on alginate hydrogels, a widely used polysaccharide biomaterial. A new AFM method applicable to RGD peptides covalently conjugated to alginate hydrogels is described. High-resolution images of RGD adhesion ligand distribution were obtained by labeling biotinylated RGD peptides with streptavidin-labeled gold nanoparticles. This method may broadly provide a useful tool for sECM characterization and design for tissue regeneration strategies.

Differentiation stage alters matrix control of stem cells.

Cues from the material to which a cell is adherent (e.g., adhesion ligand presentation, substrate elastic modulus) clearly influence the phenotype of differentiated cells. However, it is currently unclear if stem cells respond similarly to these cues. This study examined how the overall density and nanoscale organization of a model cell adhesion ligand (arginine-glycine-aspartic acid [RGD] containing peptide) presented from hydrogels of varying stiffness regulated the proliferation of a clonally derived stem cell line (D1 cells) and preosteoblasts (MC3T3-E1). While the growth rate of MC3T3-E1 preosteoblasts was responsive to nanoscale RGD ligand organization and substrate stiffness, the D1 stem cells were less sensitive to these cues in their uncommitted state. However, once the D1 cells were differentiated towards the osteoblast lineage, they became more responsive to these signals. These results demonstrate that the cell response to material cues is dependent on the stage of cell commitment or differentiation, and these findings will likely impact the design of biomaterials for tissue regeneration.

Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix.

There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg-Gly-Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation.

Regulation of chondrocyte differentiation level via co-culture with osteoblasts.

The close apposition of osteoblasts and chondrocytes in bone and their interaction during bone development and regeneration suggest that they may each regulate the other's growth and differentiation. In these studies, osteoblasts and chondrocytes were co-cultured in vitro, with both direct and indirect contact. Proliferation of the co-cultured chondrocytes was enhanced using soluble factors produced from the osteoblasts, and the differentiation level of the osteoblasts influenced the differentiation level of the chondrocytes. In addition, the chondrocytes regulated differentiation of the co-cultured osteoblasts using soluble factors and direct contact. These data support the possibility of direct, reciprocal instructive interactions between chondrocytes and osteoblasts in a variety of normal processes and further suggest that it may be necessary to account for this signaling in the regeneration of complex tissues comprising cartilage and mineralized tissue.

Multi-scale modeling to predict ligand presentation within RGD nanopatterned hydrogels.

The adhesion ligand RGD has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters cell adhesion, proliferation, and differentiation. However, elucidating the impact of nanopattern parameters on cellular responses has been stymied by a lack of understanding of the actual ligand presentation within these systems. We have developed a multi-scale predictive modeling approach to characterize the adhesion ligand nanopatterns within an alginate hydrogel matrix. The models predict the distribution of ligand islands, the spacing between ligands within an island and the fraction of ligands accessible for cell binding. These model predictions can be used to select pattern parameter ranges for experiments on the effects of individual parameters on cellular responses. Additionally, our technique could also be applied to other polymer systems presenting peptides or other signaling molecules.