Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces.

In vivo, the extracellular matrix modulates the phenotype of the connective tissue cells both through its biochemical composition and the transfer of mechanical information. In this study, the mechanical effect was investigated in collagen gels populated by skin fibroblasts maintained under tension (bound lattices (BL)) compared with free retracting lattices (FL) and monolayer on plastic. The overall proteins and collagen synthesis of human skin fibroblasts, investigated by isotopic labeling, were decreased respectively by a factor of about 20 and 40 in FL compared with monolayers and increased by a factor of 4 and 6 in BL versus FL. As assayed by the degradation of [3H]collagen type I by trypsin-activated medium conditioned by fibroblasts under the three models of culture, collagenase activity was inversely regulated and increased in lattices when compared with monolayer culture. It was four times higher in FL than in BL. The steady-state level of mRNA coding for procollagen types I, III, and VI polypeptides, fibronectin, elastin, beta-actin, and procollagenase was determined by cDNA hybridization. The mRNA coding for beta-actin as well as for the various extracellular matrix macromolecules were increased in BL when compared with FL while the level of procollagenase mRNA was lower. These data demonstrate the existence of a modulation of the function of the fibroblasts performed by mechanical forces. This regulation operates, at least in part, at a pretranslational level.     

The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts

Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-beta1 (TGF-beta1) induction of the putative fibroblast contractile marker, alpha-smooth muscle actin (alpha-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-beta1 (10 ng/ml; 6 days) increased alpha-SMA protein content by ninefold as detected by Western blots but did not affect beta-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-beta1-induced increase of alpha-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of alpha-SMA protein. Northern analyses for alpha-SMA and beta-actin indicated that TGF-beta1 selectively increased mRNA for alpha-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular alpha-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-beta1 treatment. TGF-beta1 increased alpha2 and beta1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the alpha2 and beta1 integrin subunits, TGF-beta1 failed to increase alpha-SMA protein content in all three types of collagen matrices. These data indicate that TGF-beta1-induced increases of alpha-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.     

Boundary Stiffness Regulates Fibroblast Behavior in Collagen Gels

Recent studies have illustrated the profound dependence of cellular behavior on the stiffness of 2D culture substrates. The goal of this study was to develop a method to alter the stiffness cells experience in a standard 3D collagen gel model without affecting the physiochemical properties of the extracellular matrix. A device was developed utilizing compliant anchors (0.048–0.64 N m⁻¹) to tune the boundary stiffness of suspended collagen gels in between the commonly utilized free and fixed conditions (zero and infinite stiffness boundary stiffness). We demonstrate the principle of operation with finite element analyses and a wide range of experimental studies. In all cases, boundary stiffness has a strong influence on cell behavior, most notably eliciting higher basal tension and activated force (in response to KCl) and more pronounced remodeling of the collagen matrix at higher boundary stiffness levels. Measured equibiaxial forces for gels seeded with 3 million human foreskin fibroblasts range from 0.05 to 1 mN increasing monotonically with boundary stiffness. Estimated force per cell ranges from 17 to 100 nN utilizing representative volume element analysis. This device provides a valuable tool to independently study the effect of the mechanical environment of the cell in a 3D collagen matrix.     

Mechanoregulation of valvular interstitial cell phenotype in the third dimension

A quantitative understanding of the complex interactions between cells, soluble factors, and the biological and mechanical properties of biomaterials is required to guide cell remodeling toward regeneration of healthy tissue rather than fibrocontractive tissue. In the present study, we characterized the combined effects of boundary stiffness and transforming growth factor-β1 (TGF-β1) on cell-generated forces and collagen accumulation. We first generated a quantitative map of cell-generated tension in response to these factors by culturing valvular interstitial cells (VICs) within micro-scale fibrin gels between compliant posts (0.15–1.05 nN/nm) in chemically-defined media with TGF-β1 (0–5 ng/mL). The VICs generated 100–3000 nN/cell after one week of culture, and multiple regression modeling demonstrated, for the first time, quantitative interaction (synergy) between these factors in a three-dimensional culture system. We then isolated passive and active components of tension within the micro-tissues and found that cells cultured with high levels of stiffness and TGF-β1 expressed myofibroblast markers and generated substantial residual tension in the matrix yet, surprisingly, were not able to generate additional tension in response to membrane depolarization signifying a state of continual maximal contraction. In contrast, negligible residual tension was stored in the low stiffness and TGF-β1 groups indicating a lower potential for shrinkage upon release. We then studied if ECM could be generated under the low tension environment and found that TGF-β1, but not EGF, increased de novo collagen accumulation in both low and high tension environments roughly equally. Combined, these findings suggest that isometric cell force, passive retraction, and collagen production can be tuned by independently altering boundary stiffness and TGF-β1 concentration. The ability to stimulate matrix production without inducing high active tension will aid in the development of robust tissue engineered heart valves and other connective tissue replacements where minimizing tissue shrinkage upon implantation is critical.     

Biomechanical and biochemical characteristics of a human fibroblast-produced and remodeled matrix

We report on a culture method for the rapid production of a strong and thick natural matrix by human cells for tissue engineering applications. Dermal fibroblasts were cultured for three weeks at high density on porous substrates in serum-containing or chemically defined media. The mechanical and biochemical properties of the resulting cell-derived matrix (CDM) were compared to those of standard fibroblast-populated collagen and fibrin gels and native human skin. We found that the ultimate tensile strength of CDM cultured in our chemically defined media (313+/-8.7 kPa) is significantly greater than for collagen gels (168+/-39.3 kPa), fibrin gels (133+/-8.0 kPa) and CDM cultured with serum (223+/-9.0 kPa), but less than native skin (713+/-55.2 kPa). In addition to the biomechanics, this *CDM is also biochemically more similar to native skin than the collagen and fibrin gels in terms of all parameters measured. As *CDM is produced by human cells in a chemically defined culture medium and is mechanically robust, it may be a viable living tissue equivalent for many connective tissue replacement applications requiring initial mechanical stability yet a high degree of biocompatibility.     

Effects of substratum surface topography on the organization of cells and collagen fibers in collagen gel cultures

This study investigated the orientation of fibroblasts and collagen cultured on microfabricated grooved or smooth titanium surfaces, as well as on tissue culture polystyrene, in the presence or absence of collagen gels. The gels were first added either to the confluent fibroblast culture on the surface (cell-gel condition) or to the fibroblasts were suspended within the collagen gel and then placed onto the surface (gel condition). Cells and collagen were observed with differential interference, polarization, and confocal laser scanning microscopy. Although the smooth surfaces had no effect on cell orientation in the gel for the first 2 weeks of culture, cells did orient with grooves regardless of the culture conditions. There was evidence for orthogonal multilayering of cells under the cell-gel condition at 4 weeks, and collagen alignment reflected cell alignment. The interaction of the collagen gel with the surface depended on whether the cell-gel or the gel condition was employed. In the former condition, the gel contracted toward the substratum, whereas the gel condition resulted in the formation of a ring of collagen loosely attached to the substratum. These results suggest that the order in which fibroblasts encounter substratum and extracellular matrix can influence the eventual matrix-cell interactions, and that substratum topography can influence matrix and cell orientation in zones not immediately in contact with the surface.     

Fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin: influence of surface rigidity

Open wound contraction necessitates cell and connective tissue interactions, that produce tension. Investigating fibroblast responses to tension utilizes collagen coated polyacrylamide gels with differences in stiffness. Human foreskin fibroblasts were plated on native type I collagen-coated polyacrylamide gel cover slips with different rigidities, which were controlled by bis-acrylamide concentrations. Changes in alpha smooth muscle actin (αSMA), α2β1 integrin (CD49B) and αvβ3 integrin (CD-51) were documented by immuno-histology and Western blot analysis. Cells plated on rigid gels were longer, and expressed αvβ3 integrin and αSMA within cytoplasmic stress fibers. In contrast, cells on flexible gels were shorter, expressed α2β1 integrin and had fine cytoskeletal microfilaments without αSMA. Increased tension changed the actin makeup of the cytoskeleton and the integrin expressed on the cell's surface. These in vitro findings are in agreement with the tension buildup as an open wound closes by wound contraction. It supports the notion that cells under minimal tension in early granulation tissue express α2β1 integrin, required for organizing fine collagen fibrils into thick collagen fibers. Thicker fibers create a rigid matrix, generating more tension. With increased tension cytoskeletal stress fibers develop that contain αSMA and αvβ3 integrin that replaces α2β1 integrin, consistent with cell switching from collagen to non-collagen proteins interactions.     

Biocompatible nanofiber matrices for the engineering of a dermal substitute for skin regeneration

Natural and synthetic biodegradable nanofibers are extensively used for biomedical applications and tissue engineering. Biocompatibility and a well-established safety profile for polycaprolactone (PCL) and collagen represent a favorable matrix for preparing a dermal substitute for engineering skin. Collagen synthesized by fibroblasts is a good surface active agent and demonstrates its ability to penetrate a lipid-free interface. During granulation tissue formation, fibronectin provides a temporary substratum for migration and proliferation of cells and provides a template for collagen deposition, which increases stiffness and tensile strength of this healing tissues. The objective of this study was to fabricate nanofiber matrices from novel biodegradable PCL and collagen to mimic natural extracellular matrix (ECM) and to examine the cell behavior, cell attachment, and interaction between cells and nanofiber matrices. Collagen nanofiber matrices show a significant (p < 0.001) level of fibroblast proliferation and increase up to 54% compared with control tissue culture plate (TCP) after 72 h. The present investigation shows that PCL-coated collagen matrices are suitable for fibroblast growth, proliferation, and migration inside the matrices. This novel biodegradable PCL and collagen nanofiber matrices support the attachment and proliferation of human dermal fibroblasts and might have potential in tissue engineering as a dermal substitute for skin regeneration.     

Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension

Human dermal fibroblasts entrapped in fibrin gels cast in cross-shaped (cruciform) geometries with 1:1 and 1:0.5 ratios of arm widths were studied to assess whether tension and alignment of the cells and fibrils affected ECM deposition. The cruciforms of contrasting geometry (symmetric vs. asymmetric), which developed different fiber alignment patterns, were harvested at 2, 5, and 10 weeks of culture. Cruciforms were subjected to planar biaxial testing, polarimetric imaging, DNA and biochemical analyses, histological staining, and SEM imaging. As the cruciforms compacted and developed fiber alignment, fibrin was degraded, and elastin and collagen were produced in a geometry-dependent manner. Using a continuum mechanical model that accounts for direction-dependent stress due to cell traction forces and cell contact guidance with aligned fibers that occurs in the cruciforms, the mechanical stress environment was concluded to influence collagen deposition, with deposition being the greatest in the narrow arms of the asymmetric cruciform where stress was predicted to be the largest.     

Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities

With the rapid development of tissue engineering and gene therapy, collagen-based biomaterials frequently are used as cell transplant devices. In this study we determined the behavior of mouse fibroblasts cultured for up to 6 weeks in control sponges treated by severe dehydration and used commercially as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by diphenylphosphorylazide, a method developed in our laboratory. Growth capacity, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblasts in monolayer culture on plastic and in floating or attached collagen gels. Control sponges with and without seeded mouse fibroblasts showed rapid partial denaturation or contraction, weight loss, and severe calcification (13-18% Ca) after 6 weeks. In contrast, the crosslinked sponges showed only slightly decreased size and weight, and the calcification was inhibited (0.2% Ca) in the presence of cells. Mouse fibroblasts seeded on the crosslinked sponge surface at 50,000-200,000 cells/cm(2) progressively penetrated the matrix and proliferated to give the same constant cell density after 3 weeks (around 600,000 cells/sponge). A specific, two- to threefold decrease in collagen synthesis was observed between 1 and 3 or 6 weeks, due mainly to a decrease in the fraction secreted into the medium (25-30% instead of 45-50%). No collagenase 3 activity was detected in the culture medium under any condition or time whereas 25% gelatinase A was found by gelatin zymography to be in an active form in cultures within sponges as compared with less than 10% in monolayers and more than 50% in floating collagen gel. A small amount of gelatinase B was observed after 1 week in sponge cultures and was completely absent thereafter. These results show that the biosynthetic and proteolytic behavior of mouse fibroblasts cultured in crosslinked collagen scaffolds is different from that in monolayers or in floating collagen gels and more similar to that previously described in attached collagen gels.     

Evaluation of nanostructured composite collagen--chitosan matrices for tissue engineering

The development of suitable three-dimensional matrices for the maintenance of cellular viability and differentiation is critical for applications in tissue engineering and cell biology. The structure and composition of the extracellular matrix (ECM) has been shown to modulate cell behavior with respect to shape, movement, proliferation, and differentiation. Although collagen and chitosan have separately been proposed as in vitro ECM materials, the influence of chitosan--collagen composite matrices on cell morphology, differentiation, and function is not well studied. To this end, gel matrices of different proportions of collagen and chitosan were examined ultrastructurally and characterized for their ability to regulate cellular activity. A three-chamber system with circulating hydraulic fluids was used to evaluate the gel stability under fluid force. Results indicated that overall matrix integrity increased with the proportion of chitosan. Scanning electron microscopy indicated that the addition of chitosan greatly influences ultrastructure and changes collagen fiber cross-linking, reinforcing the structure and increasing pore size. K562 cells cultured in three-dimensional gels were examined for cell proliferation and differentiation. Although cell proliferation was inhibited with an increasing proportion of chitosan, cell function based on cytokine-release was greatly augmented. Results suggest that a hybrid chitosan--collagen matrix may have potential biological and mechanical benefits for use as a cellular scaffold.     

The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes

Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell–matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young's modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates.     

Design of biomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen

In tissues, collagen forms the scaffold for cell attachment and migration, and it modulates cell differentiation and morphogenesis by mediating the flux of chemical and mechanical stimuli. We are constructing biomimetic environments by immobilizing a collagen-derived high-affinity cell-binding peptide P-15 in three-dimensional (3-D) templates. The cell-binding peptide can be expected to transduce mechanical forces. In their physiological environment, periodontal ligament fibroblasts (PDLF) are subject to significant mechanical forces. We have examined the behavior of human PDLF in culture on particulate bovine anorganic bone mineral (ABM) coated with P-15 (ABM-P-15). Greater numbers of cells associated with ABM-P-15 compared to ABM alone. Higher levels of incorporation of radiolabeled precursors in DNA and protein were consistent with the presence of larger numbers of cells on ABM-P-15 compared to ABM cultures. Scanning electron microscopic examination showed that cultures on ABM-P-15 generated highly oriented 3-D colonies of elongated cells and formed copious amounts of fibrous as well as membranous matrix reminiscent of ligamentous structures. PDLF cultured on ABM formed sparse monolayers with little order and a meager matrix. Alizarin Red stained the matrix of particle associated cells and inter-particle cellular bridges in P-15-associated cultures, indicating mineralization. 3-D colony formation and ordering of cells along with increased mineralization suggests that the coupling of cells to the ABM matrix through P-15 may provide a biomimetic environment permissive for cell differentiation and morphogenesis. Our studies suggest that ABM-P-15 templates may be effective as endosseous grafts, and, when seeded with PDLF, these matrices may serve as tissue engineered substitutes for autologous bone grafts.     

Novel porous aortic elastin and collagen scaffolds for tissue engineering

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.     

Influence of chondroitin sulfate and hyaluronic acid on structure, mechanical properties, and glioma invasion of collagen I gels

To mimic the extracellular matrix surrounding high grade gliomas, composite matrices composed of either acid-solubilized (AS) or pepsin-treated (PT) collagen and the glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are prepared and characterized. The structure and mechanical properties of collagen/CS and collagen/HA gels are studied via confocal reflectance microscopy (CRM) and rheology. CRM reveals that CS induces fibril bundling and increased mesh size in AS collagen but not PT collagen networks. The presence of CS also induces more substantial changes in the storage and loss moduli of AS gels than of PT gels, in accordance with expectation based on network structural parameters. The presence of HA significantly reduces mesh size in AS collagen but has a smaller effect on PT collagen networks. However, both AS and PT collagen network viscoelasticity is strongly affected by the presence of HA. The effects of CS and HA on glioma invasion is then studied in collagen/GAG matrices with network structure both similar to (PT collagen-based gels) and disparate from (AS collagen-based gels) those of the corresponding pure collagen matrices. It is shown that CS inhibits and HA has no significant effect on glioma invasion in 1.0?mg/ml collagen matrices over 3 days. The inhibitory effect of CS on glioma invasion is more apparent in AS than in PT collagen gels, suggesting invasive behavior in these environments is affected by both biochemical and network morphological changes induced by GAGs. This study is among the few efforts to differentiate structural, mechanical and biochemical effects of changes to matrix composition on cell motility in 3D.     

Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices

Interactions between cells and the extracellular matrix are at the core of tissue engineering and biology. However, most studies of these interactions have used traditional two-dimensional (2D) tissue culture, which is less physiological than three-dimensional (3D) tissue culture. In this study, we compared cell behavior in four types of commonly used extracellular matrix under 2D and 3D conditions. Specifically, we quantified parameters of cell adhesion and migration by human foreskin fibroblasts in cell-derived matrix or hydrogels of collagen type I, fibrin, or basement membrane extract (BME). Fibroblasts in 3D were more spindle shaped with fewer lateral protrusions and substantially reduced actin stress fibers than on 2D matrices; cells failed to spread in 3D BME. Cell-matrix adhesion structures were detected in all matrices. Although the shapes of these cell adhesions differed, the total area per cell occupied by cell-matrix adhesions in 2D and 3D was nearly identical. Fibroblasts migrated most rapidly in cell-derived 3D matrix and collagen and migrated minimally in BME, with highest migration directionality in cell-derived matrix. This identification of quantitative differences in cellular responses to different matrix composition and dimensionality should help guide the development of customized 3D tissue culture and matrix scaffolds for tissue engineering.     

A system for monitoring the response of uniaxial strain on cell seeded collagen gels

The success of cell seeded constructs for the repair of collagenous tissues may be improved by the use of mechanical stimulation in vitro. A mechanical loading apparatus, termed the cell straining system, was developed according to a set of design criteria, to enable cell seeded constructs to be cyclically loaded in tension. A suitable cell seeded collagen gel model system was used to characterise the apparatus. These gels were subjected to a cyclic strain of 10% superimposed on two separate tare loads of 2 and 10 mN, while being maintained in cell culture conditions. The computer controlled apparatus was shown to be capable of monitoring the individual loads on six specimens simultaneously, to an accuracy of 0.02 mN. Results indicated a wide variability between individual specimens. Following cyclic loading, the cell seeded collagen gels exhibited an increase in structural stiffness compared with the unloaded controls. This novel and versatile apparatus will provide a means of enhancing structural and mechanical integrity of tissue engineered repair systems.     

Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels

Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications.     

Mesenchymal stem cell-seeded collagen matrices for bone repair: effects of cyclic tensile strain, cell density, and media conditions on matrix contraction in vitro

Type I collagen is the most abundant extracellular matrix protein in bone and contains arginine- glycine-aspartic acid sequences that promote cell adhesion and proliferation. We have previously shown that human mesenchymal stem cells (hMSCs) seeded in three-dimensional (3D) collagen gels upregulate BMP-2 mRNA expression in response to tensile strain, indicative of osteogenesis. Therefore, collagen could be a promising scaffold material for functional bone tissue engineering using hMSCs. However, high contraction of the collagen gels by hMSCs poses a challenge to creating large, tissue-engineered bone constructs. The effects of cyclic tensile strain, medium (with and without dexamethasone), and hMSC seeding density on contraction of collagen matrices have not been investigated. hMSCs were seeded in 3D collagen gels and subjected to cyclic tensile strain of 10% or 12% for 4 h/day at a frequency of 1 Hz in osteogenic-differentiating or complete MSC growth media for up to 14 days. Viability of hMSCs was not affected by strain or media conditions. While initial seeding density affected matrix contraction alone, there was a high interdependence of strain and medium on matrix contraction. These findings suggest a correlation between hMSC proliferation and osteogenic differentiation on collagen matrix contraction that is affected by media, cell-seeding density, and cyclic tensile strain. It is vital to understand the effects of culture conditions on collagen matrix contraction by hMSCs in order to consider hMSC-seeded collagen constructs for functional bone tissue engineering in vitro.