Fibroblast cells are crucial in the human body for maintenance of the extracellular matrix, including synthesizing macromolecules like collagen, and they play a critical role in wound healing of soft tissues such as skin [1]. Directing fibroblast growth is an important step in tissue engineering where the focus has gone from a top-down approach of homogeneously introducing cells into a pre-formed scaffold to a bottom-up approach in which the tissue construct is built on a cell-by-cell basis with ability to manipulate specific cell environments through location, proximity, and geometry. The ability to direct cell proliferation to encourage organized tissue formation can provide tissue engineers a means of controlling the architectural and mechanical properties of soft tissue scaffolds. This approach to functional tissue engineering represents a novel direction for the development of replacement tissues. Previous attempts of directed growth have proven successful with C2C12 mouse myoblast cells. Using laser micromachined channels in agarose hydrogel lined with a basement membrane matrix, myoblast cells were guided to align and produce myotubes [2]. The objective of the current study was to apply similar principals to direct fibroblast cell growth and proliferation, ultimately leading to their growth into three-dimensional fibers, on differentially adherent substrates. Channels (widths ranging from 60 μm to 200 μm) were laser micromachined in agarose gel to explore an optimal geometry for cellular proliferation and fiber formation. The fibroblast cells used range in size from roughly 20–30 μm. Thus, the width of each channel was chosen to explore which multiple of cell width would allow for directional alignment parallel to the channel and subsequent fiber growth. The ability to direct fibroblast cells to align and produce fibers through manipulation of their environment is critical to our laboratory’s ongoing efforts to develop three-dimensional customized tissue replacement constructs to be used in many soft tissue applications such as ligament and skin grafts.

Vascular smooth muscle cells (SMC), endothelial cells (EC), and fibroblasts exist in a dynamic mechanical environment and can sense and respond to mechanical stimuli in vivo (McKnight and Frangos [1]). It is becoming more and more clear that complex dynamics not only influences vascular tone but also SMC proliferation (see Dancu et al. [2]) and extracellular matrix turnover (Cummins et al. [3]) by stimulating cell activity.

Aortic valve (AV) sclerosis is identified by irregular thickening and decreased compliance of the leaflets causing impaired cardiac function and increased mortality with age. Both mechanical and molecular factors are thought to be important in the pathogenesis of AV sclerosis. The structural integrity of the AV leaflet extracellular matrix (ECM) is maintained by the resident fibroblast AV interstitial cells (AVICs). With increasing age and loss of compliance in the aorta, a stress transfer may occur in the AV leaflets that likely subjects the AVICs to increasing strains in the circumferential direction [1].

Fibroblast cells mechanically interact with their environment through several mechanisms, including the formation of focal adhesion complexes (FACs) with adjacent ligands. A quasi-3D (Q3D) experimental device was devised to investigate chemo-mechanical stimulation of these cells. It employed a novel microscopic fixture featuring micropolymer arrays positioned just above a cellular monolayer, enabling mechanical stimulation through dorsal attachment to FACs. This protocol demonstrated the ability to preserve the normal stellate morphology of fibroblasts. Live-cell imaging during mechanical stimulation revealed robust calcium elevation in cells interacting with the Q3D device. This system provides a simple alternative to existing 3D culture methods yet allows dynamic, real-time mechanical probing of cells in a physiologically significant environment as compared to optical or magnetic tweezers.

Cardiac tissue engineering studies have demonstrated the importance of revascularization in engineered grafts for successful implantation and regeneration [1]. Understanding the myocardium’s complex cellular organization and the interactions between the major cardiac cell types (cardiomyocytes, endothelial cells, and cardiac fibroblasts) is critical for revascularization. Our previous studies have shown the importance of cardiomyocyte-endothelial interactions [2]. However, there is limited information available on endothelial-fibroblast interactions. We and others have previously observed that during capillary assembly, fibroblasts provide chemical signaling via expression of growth factors [3, 4]. In addition, fibroblasts may also regulate angiogenesis through alterations to the mechanical environment via myocardial remodeling, including matrix degradation and deposition, and tissue contraction. Changes to the extracellular mechanical enviroment may lead to changes in basic cell functions such as proliferation, apoptosis, and growth factor expression.

Tissue engineering provides a means to create fully functional tissue-equivalents that can grow, repair and remodel in vivo . Our laboratory’s approach to fabricating artery- and heart valve-equivalents utilizes cell-seeded fibrin gels. However, even after 4–5 weeks of static incubation, the mechanical properties of these constructs are below those of native tissue. Previous studies in our laboratory have shown a significant role of mechanical stretching in improving properties of collagen-based tissue constructs (Isenberg and Tranquillo, 2003). We examined the effects of cyclic distention (CD) of cell-seeded fibrin-based tubular constructs (TC) and valve-equivalents (VE) after five weeks of culture. We used human dermal fibroblasts and porcine valve interstitial cells as the cell sources. Circumferential strain amplitudes from 2.5% to 15% were applied to evaluate the effects of CD on remodeling of the TC. We further hypothesized that during long-term conditioning, cells adapt to CD of constant strain amplitude, diminishing the remodeling into tissue. We tested this hypothesis by applying step-wise incremental CD (ICD) from 5%–15% strain amplitude and compared this group to a set of samples subject to CD of constant strain amplitude in this range. Based on the outcome of the cyclic distension study with tubular constructs, we applied CD to VE in a novel bioreactor.

Ninety-seven percent of tendon ruptures are found in areas of chronic tendon degeneration, which is thought to be a cell-mediated process involving increased extracellular matrix turnover and remodeling [1,2]. The degenerative aspects seen within regions of tendon degeneration include collagen matrix disorganization, collagen fibril thinning, cellular hyperplasia, and neovascularization [1–3]. The etiology of tendon degeneration is unclear at this point. One theory involves the introduction of multiple mechanical insults (mechanical overuse) that act to trigger a degenerative pathway of increased matrix degradation by matrix metalloproteinases (MMP). Inflammation is not seen within regions of degenerative tendon. However, inflammatory mediators such as prostaglandin-E 2 (PGE 2 ) may have a role as they have been shown to be upregulated by fibroblasts as a result of mechanical over-stimulation in culture [4]. Multiple injections of PGE 2 within the midsubstance of the rabbit patellar tendon (PT) also produced collagen fibril disorganization and thinning [5].

The architecture of the vascular wall is highly intricate and requires unique biomechanical properties in order to function properly. Native artery is composed of a mix of collagens, elastin, endothelial cells (ECs), smooth muscle cells (SMC), fibroblasts, and proteoglycans arranged into three distinct layers: the intima, media, and adventitia. Throughout artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery while undergoing pulsatile deformations [1]. The low-strain mechanical response of artery to blood flow is dominated by the elastic behavior, of elastin, which prevents pulsatile energy from being dissipated as heat [2]. A higher amount of energy loss indicates a decrease in recoverability, which could lead to eventual disruption of blood flow. An effective way to quantify recoverability is through hysteresis and compliance measurement. The hypothesis of this study was that the fabrication of a multi-layered electrospun tissue engineering scaffold composed of polycaprolactone (PCL), elastin, and collagen would demonstrate dynamic mechanical properties indicative of a highly elastic material, similar to the three distinct layers of native arterial tissue, while remaining conducive to tissue regeneration. PCL was chosen, in this case, to provide mechanical integrity and elasticity, while elastin and collagen would provide further elasticity and bioactivity [3,4].

Mechanical cues arising from extracellular matrices greatly affect cellular properties, and hence, are of significance in designing biomaterials. Similar to many other cell types, including fibroblasts and hepatocytes, central nervous system (CNS) neurons have been found to exhibit distinct responses to the stiffness of the substrates they reside on [1]. There is an increasing awareness that mechanical properties also play a key role in successful utilization of scaffolds for those tissues whose major functions are not load-bearing, such as the spinal cord. In light of this, there is a growing interest in incorporating mechanical cues in biomaterial design for neural tissue engineering applications, including spinal cord injury.

Load-bearing tissues owe their mechanical properties to the presence of highly-organized arrays of collagen fibrils. Aligned lamellae in cornea and aligned fascicles in tendon are the best examples of collagen fibrillar organization at the macroscopic level. The process by which collagen is organized in the extracellular matrix (ECM) is still unclear. But it is generally thought to be facilitated locally via “fibripositors” or cell surface “crypts”. According to this theory, fibroblasts create bounded “compartments” in the ECM through which they deposit organized groups of fibrils (in the form of lamellae in the cornea and in the form of fascicles in the tendon) [1, 2]. An alternative hypothesis proposed by Marie Giraud-Guille suggests that fibroblasts concentrate collagen monomers to form cholesteric liquid crystalline patterns that resemble those found in collagenous matrices in vivo [3–8]. Such organization has been demonstrated in vitro using extracted collagen monomers. However, the data presented in these studies focuses principally on the alignment of the collagen molecules and not on the organization and resulting morphology of condensed collagen fibrils. Considering that matrix mechanical properties in vivo are the result of the fibrillar alignment and not the alignment of individual molecules, further investigation of cholesterically organized condensed fibrils and their morphology is necessary.

The goal of this study was to investigate the kinetics of NIH 3T3 fibroblast cell adhesion and their actin cytoskeleton organization on different geometry electrospun polycaprolactone nanofibrous scaffolds. Cell adhesion kinetics was measured by MTS assay. Cells on beaded scaffolds, which are not uniform fibrous scaffolds, showed the lowest adhesion rate and the smallest cytoskeleoton organization in all experiments. For uniform fiber scaffolds, cell adhesion rate was a function of scaffold specific surface area (SSA). Cell adhesion rate increased with increasing SSA when scaffold SSA was higher than 7.13 μm −1 . Cell adhesion in high serum concentration culture medium was higher than in low serum concentration medium for all scaffolds. Results indicate that from 0–5% serum concentrations, cell adhesion at 4 or 8 hours were not significantly different between different geometry scaffolds. F-actin maximum projected area was quantified to indicate mature cell adhesion. Results indicate higher area for cells cultured in high serum concentration medium on 18.79 μm −1 (SSA) scaffolds, and had higher values when cultured for 8 hrs compared to 24 hrs. 10% serum concentration and 18.79 μm −1 (SSA) scaffolds are considered to be an optimum combination for enhanced cell adhesion.

Recent studies utilizing compliant culture substrates clearly demonstrate that many cell behaviors such as migration and contraction are strongly influenced by the stiffness of the local environment. The goal of this work was to develop a method for studying stiffness-dependent biology within 3D cell-populated gels without altering the biochemical environment of the gel. A device was developed utilizing compliant anchors to tune the boundary stiffness of suspended collagen gels. Human fibroblasts compacted the gels to a greater extent as the stiffness of the anchors was decreased. Further, the force generated by the cells increased with increased boundary stiffness, and the response of the cells to TGF-β1 was accentuated as the stiffness increased. This new method represents a promising tool for the controlled study of stiffness-dependent biology within tissuelike 3D environments.

Female athletes have significantly higher incidence of anterior cruciate ligament (ACL) injuries than males participating in similar sports [1]. To date, no clear explanation has emerged for this disparity. However, hormonal differences may provide an explanation because some ACL injuries have been linked to physiologic fluctuations in estrogen levels over the menstrual cycle [2]. Receptors for estrogen have been identified in rabbit and human ACLs and medial collateral ligaments (MCLs) [3]. Increased estrogen levels caused decreased fibroblast proliferation and collagen synthesis in cell cultures from human and rabbit ACLs [4]. Since fibroblasts maintain collagen production and degradation in ligaments and collagen is the major load-bearing component of ligaments, estrogen may affect knee ligament mechanical properties.

The intervertebral disc (IVD) is a unique orthopaedic tissue consisting of at least two cell types: fibroblast-like annulus fibrosus (AF) cells and chondrocyte-like nucleus pulposus (NP) cells. Culture of cells in 3D gel matrices (such as alginate or agarose), maintains the normal morphology and ECM molecule production of chondrocytes for extended periods of time and also allows the application of various forms of mechanical stimulation, such as hydrostatic or compressive loading. In vivo studies have shown IVD cells to be responsive to frequency, duration, and amplitude of mechanical load [1]. IVD literature on mechanobiology uses varying methodologies to apply dynamic loads (compression, hydrostatic forces), with different times of mechanical stimulation, differences in model systems (in vivo, tissue culture, cell culture), species, and ages, and an optimal loading protocol to stimulate extracellular matrix protein accumulation is unknown. The overall goal of this work is to evaluate the potential, and perhaps even feasibility, of mechanical stimulation for extracellular matrix (ECM) regeneration using intervertebral disc cells. Also of interest is whether cells from mature tissue are capable of serving as a potential cell source for future IVD regeneration [2,3].

In this paper, we discuss the results arising from using a finite-element model [1] of cell deformation to study the optical stretching [2,3] of normal and malignantly transformed fibroblast cells. The key feature of our model is the use of a constitutive viscoelastic fluid element [4] whose parameters are both spatially and temporally varying so as to mimic the experimentally-observed spatiotemporal heterogeneity of cellular material properties. First, we show that normal fibroblast cells can undergo active cellular response by increasing their cellular viscosity when optically stretched for loading times of between 0.2s and 2.5s. Second, we show that, under similar optical conditions, cells of a smaller radius will experience more stretching compared to cells of a larger radius. This may explain why malignantly transformed cells experience higher strains than normal cells. Third, we compute the extent of the propagation of stress in the cytoplasm, and show that, for malignantly transformed cells, the maximal stress does propagate into the nuclear region whereas for normal cells, the maximal stress does not. We discuss how this may impact the transduction of cancer signalling pathways.

Mechanical forces affect the behavior of a variety of cell types including fibroblasts, chondrocytes, osteoblasts, smooth muscle cells, and endothelial cells. In vitro cellular orientation is primarily achieved through cell-generated mechanical forces or contact guidance. Endothelial cell (EC) cords align both normal to the stretch direction as well as along pre-tensioned collagen matrices. Confluent ECs orient parallel to grooved surfaces while tractional forces generated by ECs contract the matrix aligning collagen fibers, which in turn are considered a guiding scaffold. The influence of mechanical forces and boundary conditions on the growth and proliferation of microvessels during the process of angiogenesis is unknown. A better understanding of these interactions is essential both from a basic science standpoint as well as in the engineering of artificial vascularized matrices with specific orientation of vessels and endothelial cells. The objective of this study was to examine the role of internal and external mechanical forces in microvessel orientation during angiogenesis using an in vitro model of angiogenesis.

Cells translate environmental mechanical stimuli into biochemical reactions that govern a range of cellular processes such as proliferation, death and tissue matrix remodeling. Mechanical activation of individual focal adhesions formed between the cell and its environment directly correspond to several internal responses. Intracellular calcium concentration, [Ca 2+ ] in , has been shown to profoundly change during force sensing. In order to understand this dynamic in cells, we compared calcium mobilization resulting from chemical stimulation and that resulting from mechanical stimulation. We have analyzed the response of fibroblasts to membrane displacements of over 5 μm resulting in eventual spikes in [Ca 2+ ] in . Our data initially indicates that fibroblasts may process mechanical calcium events in unique manner in comparison to other cell types. This finding has implications in a range of fields including mechanobiology and magnetics based activation.

Vascular remodeling plays a key role in many physiological and pathophysiological processes, as well as the success or failure of many clinical interventions; examples include vascular development and aging, hypertension and atherosclerosis, and restenosis of vascular grafts. Despite the explosion of information on vascular remodeling, from the molecular level to the tissue level, attempts at integrating these data into a predictive multi-scale model are still in their infancy. Humphrey and Rajagopal said well that in order to capture the salient features of these remodeling processes ‘one must track local balances or imbalances in the continual production, removal, [and remodeling] of individual constituents, the mechanical state in which the constituents are formed, and how these constituents are organized’. Abdominal aortic aneurysms (AAA’s) provide a good illustration of the need for a multi-scaled microstructurally-motivated mathematical model. During progression of AAA’s, circumferential expansion, vessel wall thinning and axial lengthening are coincident with a progressive loss of elastin and smooth muscle and decrease in glycosaminoglycans, with mature aneurysms consisting primarily of collagen and fibroblasts; thus, AAA’s experience spatial and temporal variations in their geometry, microstructural content and organization, and applied loads. To develop a predictive model for vascular remodeling, the complex interplay between evolving material behavior (via changes in microstructural content and organization) and applied loads, which determine the local mechanical environment and the mechano-biological response to this changing mechanical environment, must be incorporated.

Every cell is composed of a combination of proteins and lipids that define the cell. The biomolecular composition of a cell can yield a unique IR spectral profile, termed chemical “fingerprint”, in the FTIR. Even though it provides overall information on the state and composition, the challenge is in obtaining a more sensitive and mechanistically relevant “fingerprint” of a cell particularly in the protein regime where numerous detection assays are now based [1]. Heat induced denaturation of proteins in a cell, which can be termed as thermal “fingerprint”, offers detailed and mechanistically relevant information for identification and characterization of different cells. For this study, the chemical and the thermal “fingerprint” of four different cell types are characterized and compared — human dermal fibroblasts (HDFs), LNCaP prostate tumor cells, smooth muscle cells (SMCs) and microvascular endothelial cells (MVECs). Multiple protein denaturation peaks were observed during heating from room temperature to 90°C at 1°C/min that are cell specific. A correlation coefficient (r) was used to compare responses between the cell types (higher ‘r’ implies closer resemblance). For the chemical “fingerprint”, r values of cells compared to HDFs are 0.97 for MVECs, 0.91 for LNCaPs and 0.70 for SMCs. Similarly, for the thermal “fingerprint”, r values of cells compared to HDFs are 0.86 for MVECs, 0.73 for LNCaPs and 0.54 for SMCs. The deviation of ‘r’ values from 1.00 is wider in the case of the thermal “fingerprint” than the chemical “fingerprint”. This result demonstrates that thermal “fingerprinting”, based on protein denaturation, offers a mechanistic basis for enhanced differentiation between cells as compared to chemical “fingerprinting”. Extensive applications ranging from rapid disease diagnostics, forensics, cell culture quality control, cell sorting techniques etc. may benefit from this approach.

Mechanical cues have been found to play an important role in the cell decision making process, as manifested in survival, adhesion, growth, proliferation, differentiation and functioning. Due to their resemblance to the natural tissues, polyacrylamide hydrogels have been used in the studies of mechanobiology and particularly cell-substrate interactions for various cell types including fibroblasts, hepatocytes and neurons [1]. As the initial investigation, we have successfully cultured rat spinal cord neurons on bis-crosslinked gels with large range of stiffnesses [2].