Multiscale strain analysis of tissue equivalents using a custom-designed biaxial testing device.

Mechanical signals transferred between a cell and its extracellular matrix play an important role in regulating fundamental cell behavior. To further define the complex mechanical interactions between cells and matrix from a multiscale perspective, a biaxial testing device was designed and built. Finite element analysis was used to optimize the cruciform specimen geometry so that stresses within the central region were concentrated and homogenous while minimizing shear and grip effects. This system was used to apply an equibiaxial loading and unloading regimen to fibroblast-seeded tissue equivalents. Digital image correlation and spot tracking were used to calculate three-dimensional strains and associated strain transfer ratios at macro (construct), meso, matrix (collagen fibril), cell (mitochondria), and nuclear levels. At meso and matrix levels, strains in the 1- and 2-direction were statistically similar throughout the loading-unloading cycle. Interestingly, a significant amplification of cellular and nuclear strains was observed in the direction perpendicular to the cell axis. Findings indicate that strain transfer is dependent upon local anisotropies generated by the cell-matrix force balance. Such multiscale approaches to tissue mechanics will assist in advancement of modern biomechanical theories as well as development and optimization of preconditioning regimens for functional engineered tissue constructs.

Isolating and defining cells to engineer human blood vessels.

A great deal of attention has been recently focused on understanding the role that bone marrow-derived putative endothelial progenitor cells (EPC) may play in the process of neoangiogenesis. However, recent data indicate that many of the putative EPC populations are comprised of various haematopoietic cell subsets with proangiogenic activity, but these marrow-derived putative EPC fail to display vasculogenic activity. Rather, this property is reserved for a rare population of circulating viable endothelial cells with colony-forming cell (ECFC) ability. Indeed, human ECFC possess clonal proliferative potential, display endothelial and not haematopoietic cell surface antigens, and display in vivo vasculogenic activity when suspended in an extracellular matrix and implanted into immunodeficient mice. Furthermore, human vessels derived became integrated into the murine circulatory system and eventually were remodelled into arterial and venous vessels. Identification of this population now permits determination of optimal type I collagen matrix microenvironment into which the cells should be embedded and delivered to accelerate and even pattern number and size of blood vessels formed, in vivo. Indeed, altering physical properties of ECFC-collagen matrix implants changed numerous parameters of human blood vessel formation, in host mice. These recent discoveries may permit a strategy for patterning vascular beds for eventual tissue and organ regeneration.

Collagen oligomers modulate physical and biological properties of three-dimensional self-assembled matrices.

Elucidation of mechanisms underlying collagen fibril assembly and matrix-induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how Type I collagen oligomers affect in-vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid-solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half-times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary-like network formation. This work documents the importance of oligomers as another physiologically-relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications.

Polymerization and matrix physical properties as important design considerations for soluble collagen formulations.

Despite extensive use of type I collagen for research and medical applications, its fibril-forming or polymerization potential has yet to be fully defined and exploited. Here, we describe a type I collagen formulation that is acid solubilized from porcine skin collagen (PSC), quality controlled based upon polymerization potential, and well suited as a platform polymer for preparing three-dimensional (3D) culture systems and injectable/implantable in vivo cellular microenvironments in which both relevant biochemical and biophysical parameters can be precision-controlled. PSC is compared with three commercial collagens in terms of composition and purity as well as polymerization potential, which is described by kinetic parameters and fibril microstructure and mechanical properties of formed matrices. When subjected to identical polymerization conditions, PSC showed significantly decreased polymerization times compared to the other collagens and yielded matrices with the greatest mechanical integrity and broadest range of mechanical properties as characterized in oscillatory shear, uniaxial extension, and unconfined compression. Compositional and intrinsic viscosity analyses suggest that the enhanced polymerization potential of PSC may be attributed to its unique oligomer composition. Collectively, this work demonstrates the importance of standardizing next generation collagen formulations based upon polymerization potential and provides preliminary insight into the contribution of oligomers to collagen polymerization properties.

Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo.

Developing tissue engineering approaches to generate functional vascular networks is important for improving treatments of peripheral and cardiovascular disease. Endothelial colony forming cells (ECFCs) are an endothelial progenitor cell (EPC) population defined by high proliferative potential and an ability to vascularize collagen-based matrices in vivo. Little is known regarding how physical properties of the local cell microenvironment guide vessel formation following EPC transplantation. In vitro evidence suggests that collagen matrix stiffness may modulate EPC vessel formation. The present study determined the ability of 3D collagen matrix physical properties, varied by changing collagen concentration, to influence ECFC vasculogenesis in vivo. Human umbilical cord blood ECFCs were cultured within matrices for 18 h in vitro and then fixed for in vitro analysis or implanted subcutaneously into the flank of immunodeficient mice for 14 days. We report that increasing collagen concentration significantly decreased ECFC derived vessels per area (density), but significantly increased vessel sizes (total cross sectional area). These results demonstrate that the physical properties of collagen matrices influence ECFC vasculogenesis in vivo and that by modulating these properties, one can guide vascularization.

Hyaluronan concentration within a 3D collagen matrix modulates matrix viscoelasticity, but not fibroblast response.

The use of 3D extracellular matrix (ECM) microenvironments to deliver growth-inductive signals for tissue repair and regeneration requires an understanding of the mechanisms of cell-ECM signaling. Recently, hyaluronic acid (HA) has been incorporated in collagen matrices in an attempt to recreate tissue specific microenvironments. However, it is not clear how HA alters biophysical properties (e.g. fibril microstructure and mechanical behavior) of collagen matrices or what impact these properties have on cell behavior. The present study determined the effects of varying high molecular weight HA concentration on 1) the assembly kinetics, fibril microstructure, and viscoelastic properties of 3D type I collagen matrices and 2) the response of human dermal fibroblasts, in terms of morphology, F-actin organization, contraction, and proliferation within the matrices. Results showed increasing HA concentration up to 1 mg/ml (HA:collagen ratio of 1:2) did not significantly alter fibril microstructure, but did significantly alter viscoelastic properties, specifically decreasing shear storage modulus and increasing compressive resistance. Interestingly, varied HA concentration did not significantly affect any of the measured fibroblast behaviors. These results show that HA-induced effects on collagen matrix viscoelastic properties result primarily from modulation of the interstitial fluid with no significant change to the fibril microstructure. Furthermore, the resulting biophysical changes to the matrix are not sufficient to modulate the cell-ECM mechanical force balance or proliferation of resident fibroblasts. These results provide new insight into the mechanisms by which cells sense and respond to microenvironmental cues and the use of HA in collagen-based biomaterials for tissue engineering.

Extracellular matrix (ECM) microstructural composition regulates local cell-ECM biomechanics and fundamental fibroblast behavior: a multidimensional perspective.

The extracellular matrix (ECM) provides the principal means by which mechanical information is communicated between tissue and cellular levels of function. These mechanical signals play a central role in controlling cell fate and establishing tissue structure and function. However, little is known regarding the mechanisms by which specific structural and mechanical properties of the ECM influence its interaction with cells, especially within a tissuelike context. This lack of knowledge precludes formulation of biomimetic microenvironments for effective tissue repair and replacement. The present study determined the role of collagen fibril density in regulating local cell-ECM biomechanics and fundamental fibroblast behavior. The model system consisted of fibroblasts seeded within collagen ECMs with controlled microstructure. Confocal microscopy was used to collect multidimensional images of both ECM microstructure and specific cellular characteristics. From these images temporal changes in three-dimensional cell morphology, time- and space-dependent changes in the three-dimensional local strain state of a cell and its ECM, and spatial distribution of beta1-integrin were quantified. Results showed that fibroblasts grown within high-fibril-density ECMs had decreased length-to-height ratios, increased surface areas, and a greater number of projections. Furthermore, fibroblasts within low-fibril-density ECMs reorganized their ECM to a greater extent, and it appeared that beta1-integrin localization was related to local strain and ECM remodeling events. Finally, fibroblast proliferation was enhanced in low-fibril-density ECMs. Collectively, these results are significant because they provide new insight into how specific physical properties of a cell's ECM microenvironment contribute to tissue remodeling events in vivo and to the design and engineering of functional tissue replacements.

Three-dimensional extracellular matrix substrates for cell culture.

In summary, the understanding of cell biology will be furthered as cell culture expands from 2-D to 3-D systems. In choosing which substrate, synthetic or biologically derived, is most well suited for a specific application, substrate composition and structure as well as cell type(s) must be carefully considered. In addition, optimization of seeding densities, medium conditions, growth factor supplements, and other culture parameters may be necessary. Finally, cytometric analyses of such 3-D culture systems will require concurrent innovations in 3-D imaging and methods for quantitating cell morphology, phenotype, and function.

Three-dimensional imaging of extracellular matrix and extracellular matrix-cell interactions.

In summary, noninvasive and nondestructive imaging modalities such as reflection and autofluorescence can readily be used in conjunction with the 3-D optical sectioning capabilities of confocal and multiphoton microscopy to investigate biological processes within living systems. The elimination of specimen fixation and extensive processing reduces the possibility of structural artifacts and facilitates repeat observations within a single sample. Therefore, information representing up to four dimensions (x, y, z, and time) can be readily collected and reconstructed for purposes of visualization and/or quantitative analysis. An advantage of using the techniques described in this chapter is the possibility of performing quantitative measurement of cell size, surface area, volume, depth (in matrix), orientation, receptor density, as well as fluorescence-based indicators of phenotype and function. At present, we are effectively utilizing these techniques to study collagen fibrillogenesis and ECM assembly, structural aspects of ECM-based biomaterials, as well as cell interactions within 3-D matrices (e.g., migration). New insights provided by these techniques regarding ECM and ECM-cell signaling will further the understanding of tissue structure and function and contribute to the development of new and improved strategies for tissue repair, replacement, and maintenance.

Improved islet survival and in vitro function using solubilized small intestinal submucosa.

In vitro proliferation of isolated pancreatic islets has become an area of great interest given the scarcity of clinical islet donors and the islet mass requirements for clinical islet transplantation. Small intestinal submucosa (SIS), a naturally occurring extracellular matrix, has been investigated to promote wound healing, tissue remodeling and cell growth. This study evaluated recovery and function of isolated canine pancreatic islets following in vitro tissue culture. Pancreatic islets were isolated from mongrel dogs using standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and EuroFicoll purification. Groups of purified islets were cultured in a humidified atmosphere of 95% air and 5% CO(2) for 48 hours in standard islet culture conditions of CMRL 1066 tissue culture media (Gibco) which had been supplemented with 25microM HEPES, penicillin/streptomycin and either 10% heat inactivated fetal calf serum (FCS, Gibco) or solubilized SIS solution (Cook Biotech, Inc., West Lafayette, IN). The mean recovery of islets following the culture period was determined by sizing duplicate counts of a known volume and viability was assessed by static incubation with low glucose (2.8 mM), high glucose (20 mM) and high glucose solution supplemented with 50 microm IBMX solution. Remaining islets were embedded histologically. From a consecutive series of six culture experiments, a significantly higher (p < 0.05) recovery of islets co-cultured with SIS was observed when compared to controls. Mean islet recovery was 84.5 +/- 2.9% (mean +/- SEM) from the SIS cultured group compared with 64.7 +/- 4.5% from the control group cultured in FCS (p < 0.05, n=6). Islets from the SIS treated group exhibited a significantly higher (p <, 0.05) insulin response to the high glucose stimulus than islets cultured in the standard FCS cultured solution. The calculated stimulation index was 12.3 +/- 3.4 for the SIS-treated group compared with 5.6 +/- 1.8 for the standard cultured group (p < 0.05). The overall mean numbers of islets recovered following in vitro culture was also higher in the SIS-treated group. The proportion of islets with a mean diameter >150 microm increased from 24% to 31% in the SIS-treated group, whereas the same proportion decreased to 18% from 22% in the control (FCS-treated) group. Histological evaluation of fixed tissue samples collected following the culture period identified insulin and glucagon-secreting cells in the SIS and FCS treated groups, however a higher frequency of insulin positive cells were detected consistently in the SIS treated group. A proliferation marker (PCNA) identified positive cells within both groups as well. This study suggests that co-culture of freshly isolated canine islets in medium supplemented with solubilized SIS can improve the post-culture recovery and in vitro islet function. Future investigations will focus on the cellular interactions of SIS, both in vitro and in vivo.

Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro.

The development of the next generation of biomaterials for restoration of tissues and organs (i.e., tissue engineering) requires a better understanding of the extracellular matrix (ECM) and its interaction with cells. Extracellular matrix is a macromolecular assembly of natural biopolymers including collagens, glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins. Interestingly, several ECM components have the ability to form three-dimensional (3D), supramolecular matrices (scaffolds) in vitro by a process of self-directed polymerization, "self-assembly". It has been shown previously that 3D matrices with distinct architectural and biological properties can be formed from either purified type I collagen or a complex mixture of interstitial ECM components derived from intestinal submucosa. Unfortunately, many of the imaging and analysis techniques available to study these matrices either are unable to provide insight into 3D preparations or demand efforts that are often prohibitory to observations of living, dynamic systems. This is the first report on the use of reflection imaging at rapid time intervals combined with laser-scanning confocal microscopy for analysis of structural properties and kinetics of collagen and ECM assembly in 3D. We compared time-lapse confocal reflection microscopy (TL-CRM) with a well-established spectrophotometric method for determining the self-assembly properties of both purified type I collagen and soluble interstitial ECM. While both TL-CRM and spectrophotometric techniques provided insight into the kinetics of the polymerization process, only TL-CRM allowed qualitative and quantitative evaluation of the structural parameters (e.g., fibril diameter) and 3D organization (e.g., fibril density) of component fibrils over time. Matrices formed from the complex mixture of soluble interstitial ECM components showed an increased rate of assembly, decreased opacity, decreased fibril diameter, and increased fibril density compared to that of purified type I collagen. These results suggested that the PG/GAG components of soluble interstitial ECM were affecting the polymerization of the component collagens. Therefore, the effects of purified and complex mixtures of PG/GAG components on the assembly properties of type I collagen and interstitial ECM were evaluated. The data confirmed that the presence of PG/GAG components altered the kinetics and the 3D fibril morphology of assembled matrices. In summary, TL-CRM was demonstrated to be a new and useful technique for analysis of the 3D assembly properties of collagen and other natural biopolymers which requires no specimen fixation and/or staining.

Enhanced bone regeneration using porcine small intestinal submucosa.

Small intestinal submucosa (SIS) is an easily produced material that has been used experimentally for tissue engineering. To evaluate the ability of SIS to facilitate bone growth within a long-bone defect, a segment of the radius was surgically removed in adult, female Sprague-Dawley rats. The defect was either left unfilled or implanted with SIS, demineralized cortical bone (DMCB), or ovalbumin. The defect was evaluated radiographically and histologically after 3, 6, 12, and 24 weeks. Tissue remodeling within the defect was evident by week 3 in SIS- and DMCB-treated rats. Filling was characterized initially by infiltration of mononuclear cells and extracellular material in SIS-implanted rats and multifocal remodeling bone particles and cartilage formation in DMCB-implanted rats. Cartilage was observed as early as 3 weeks and bone as early as 6 weeks in SIS-implanted rats. Filling of the defect arose from multiple foci in DMCB-implanted rats, but was contiguous with and parallel to the ulnar shaft in SIS-implanted rats, suggesting that defect repair by SIS may be conductive rather than inductive. Rats in which the defect was left unfilled demonstrated slow but progressive filling of the defect, characterized by mononuclear cell infiltrates and fibrous extracellular material. In summary, SIS facilitated rapid filling of a long-bone defect. These results suggest that SIS may be useful as a bone repair material.

Application and evaluation of the alamarBlue assay for cell growth and survival of fibroblasts.

Cell proliferation assays are essential to developing an understanding of the molecular mechanisms that modulate cell growth and differentiation. In this paper, we describe the application of alamarBlue, a new and versatile metabolic dye, for the detection of Swiss 3T3 fibroblast proliferation and/or survival. As a redox indicator, alamarBlue is reduced by reactions innate to cellular metabolism and, therefore, provides an indirect measure of viable cell number. Various assay parameters were optimized for a 96-well format to achieve a detectable range of fibroblast cell number from 100 to 20,000 cells/well, which is similar to that obtained with traditional (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and [3H]thymidine assay techniques. Standard (reference) curves generated with a known fibroblast stimulator were used to facilitate quantitation and comparison of unknown test substances. The alamarBlue assay offers the advantages of technical simplicity, freedom from radioisotopes, versatility in detection, no extraction, and excellent reproducibility and sensitivity. We anticipate that this simple and versatile alamarBlue assay, when used alone or in conjunction with other bioassays, will be a useful tool for investigating the complex mechanisms of cellular proliferation.

Impairment of human neutrophil oxidative burst by polychlorinated biphenyls: inhibition of superoxide dismutase activity.

We report evidence of a novel mechanism by which polychlorinated biphenyls might act as potent inducers of inflammation. Aroclor 1242 (A1242), a polychlorinated biphenyl mixture, and 2,2',4,4'-tetrachlorobiphenyl (PCB47), a constituent of A1242 that produces the same patterns of effects, impaired the oxidative burst of human neutrophils by inhibiting the antioxidant enzyme superoxide dismutase, which converts O2- to H2O2. Pre-incubation of neutrophils with A1242 or PCB47 before stimulation with phorbol 12-myristate 13-acetate heightened the respiratory burst, producing a significant increase in intracellular O2- production along with a significant decrease in H2O2 production compared with unexposed agonist-stimulated neutrophils. This was also evident in a physiologically relevant situation in which neutrophils pre-incubated with A1242 were subsequently stimulated with a combination of N-formyl-L-methionyl-L-leucyl-L-phenylalanine and tumor necrosis factor-alpha. Incubation of bovine copper-zinc superoxide dismutase (EC 1.15.1.1) with A1242 or PCB47 in a cell-free system reversed the enzyme-mediated inhibition of 6-hydroxydopamine autoxidation, indicating that polychlorinated biphenyls inhibited superoxide dismutase activity. Low superoxide dismutase activity in neutrophils leads to imbalances between production of free radicals and antioxidant defense mechanisms, which can in turn induce tissue damage and hasten the onset of neutrophil apoptosis.

Identification of extractable growth factors from small intestinal submucosa.

When implanted as a biomaterial for tissue replacement, selected submucosal layers of porcine small intestine induce site-specific tissue remodeling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix material. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodeling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesize DNA and proliferate. Each of the four different extracts of small intestinal submucosa had measurable cell-stimulating activity when analyzed in both a whole cell proliferation assay (alamarBlue dye reduction) and a DNA synthesis assay ([3H]-thymidine incorporation). Proteins extracted from SIS with 2 M urea induced activity profiles in the two assays which were very similar to the activity profiles of basic fibroblast growth factor (FGF-2) in the assays. As well, the changes in cell morphology in response to the extracted proteins mimicked the changes induced by FGF-2. Neutralization experiments with specific antibodies to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% of the fibroblast-stimulating activity of the urea extract of small intestinal submucosa. Western blot analysis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence of FGF-2. Cell stimulating activity of proteins extracted from SIS with 4 M guanidine was neutralized by an antibody specific for transforming growth factor beta (TGF beta). Changes in the morphology of the fibroblasts exposed to this extract were nearly identical to changes induced by TGF beta. Although no reactive protein band was detected at 25 kDa in nonreduced western blot analysis, several bands were reactive at higher molecular weight. The identity of this TGF beta-related component of small intestinal submucosa is unknown. Identification of FGF-2 and TGF beta-related activities in SIS, two growth factors known to significantly affect critical processes of tissue development and differentiation, provides the opportunity to further elucidate the mechanisms by which this extracellular matrix biomaterial modulates wound healing and tissue remodeling.

Glycosaminoglycan content of small intestinal submucosa: a bioscaffold for tissue replacement.

Small intestinal submucosa (SIS) is a resorbable biomaterial that induces tissue remodeling when used as a xenogeneic tissue graft in animal models of vascular, urologic, dermatologic, neurologic, and orthopedic injury. Determination of the composition and structure of naturally occurring biomaterials such as SIS that promote tissue remodeling is necessary for the greater understanding of their role in wound healing. Since glycosaminoglycans (GAGs) are important components of extracellular matrix (ECM) and SIS is primarily an ECM-based material, studies were performed to identify the species of glycosaminoglycans present in SIS. Porcine SIS was chemically extracted and the extracts were analyzed for uronic acid. The extractable uronic acid content was determined to be 47.7 micromol/g (approximately 21 microg GAG/mg) of the dry weight of the SIS tissue. Using electrophoretic separation of GAGs on cellulose acetate membranes, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate A, and dermatan sulfate were identified. Digestion of specific GAGs with selective enzymes confirmed the presence of these GAG species. Two GAGs common to other tissues with large basement membrane ECM components, keratan sulfate and chondroitin sulfate C, were not detected in the SIS extracts. Identification of specific GAGs in the composition of the ECM-rich SIS provides a starting point toward a more comprehensive understanding of the structure and function of this naturally occurring biomaterial with favorable in vivo tissue remodeling properties.