Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm × 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O₂) or hypoxic (5% O₂) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10–20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (±25°; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro. This work was supported by the Swiss National Science Foundation (grant no. 3200B0-104083).     

A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage

A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.     

Dynamic compressive loading of image-guided tissue engineered meniscal constructs

This study investigated the hypothesis that dynamic compression loading enhances tissue formation and increases mechanical properties of anatomically shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2%w/v alginate, crosslinked with CaSO(4), injected into μCT based molds, and post crosslinked with CaCl(2). Samples were loaded via a custom bioreactor with loading platens specifically designed to load anatomically shaped constructs in unconfined compression. Based on the results of finite element simulations, constructs were loaded under sinusoidal displacement to yield physiological strain levels. Constructs were loaded 3 times a week for 1 h followed by 1 h of rest and loaded again for 1 h. Constructs were dynamically loaded for up to 6 weeks. After 2 weeks of culture, loaded samples had 2-3.2 fold increases in the extracellular matrix (ECM) content and 1.8-2.5 fold increases in the compressive modulus compared with static controls. After 6 weeks of loading, glycosaminoglycan (GAG) content and compressive modulus both decreased compared with 2 week cultures by 2.3-2.7 and 1.5-1.7 fold, respectively, whereas collagen content increased by 1.8-2.2 fold. Prolonged loading of engineered constructs could have altered alginate scaffold degradation rate and/or initiated a catabolic cellular response, indicated by significantly decreased ECM retention at 6 weeks compared with 2 weeks. However, the data indicates that dynamic loading had a strikingly positive effect on ECM accumulation and mechanical properties in short term culture.     

Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes

Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.     

A modular approach to creating large engineered cartilage surfaces

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Time-dependent functional maturation of scaffold-free cartilage tissue analogs

One of the most critical parameters in cartilage tissue engineering which influences the clinical success of a repair therapy is the ability to match the load-bearing capacity of the tissue as it functions in vivo. While mechanical forces are known to positively influence the development of cartilage matrix architecture, these same forces can induce long-term implant failure due to poor integration or structural deficiencies. As such, in the design of optimal repair strategies, it is critical to understand the timeline of construct maturation and how the elaboration of matrix correlates with the development of mechanical properties. We have previously characterized a scaffold-free method to engineer cartilage utilizing primary chondrocytes cultured at high density in hydrogel-coated culture vessels to promote the formation of a self-aggregating cell suspension that condenses to form a cartilage-like biomass, or cartilage tissue analog (CTA). Chondrocytes in these CTAs maintain their cellular phenotype and deposit extracellular matrix to form a construct that has characteristics similar to native cartilage; however, the mechanical integrity of CTAs had not yet been evaluated. In this study, we found that chondrocytes within CTAs produced a robust matrix of proteoglycans and collagen that correlated with increasing mechanical properties and decreasing cell-matrix ratios, leading to properties that approached that of native cartilage. These results demonstrate a unique approach to generating a cartilage-like tissue without the complicating factor of scaffold, while showing increased compressive properties and matrix characteristics consistent with other approaches, including scaffold-based constructs. To further improve the mechanics of CTAs, studies are currently underway to explore the effect of hydrodynamic loading and whether these changes would be reflective of in vivo maturation in animal models. The functional maturation of cartilage tissue analogs as described here support this engineered cartilage model for use in clinical and experimental applications for repair and regeneration in joint-related pathologies.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Shear stress magnitude and duration modulates matrix composition and tensile mechanical properties in engineered cartilaginous tissue

Cartilage tissue‐engineering strategies aim to produce a functional extracellular matrix similar to that of the native tissue. However, none of the myriad approaches taken have successfully generated a construct possessing the structure, composition, and mechanical properties of healthy articular cartilage. One possible approach to modulating the matrix composition and mechanical properties of engineered tissues is through the use of bioreactor‐driven mechanical stimulation. In this study, we hypothesized that exposing scaffold‐free cartilaginous tissue constructs to 7 days of continuous shear stress at 0.001 or 0.1 Pa would increase collagen deposition and tensile mechanical properties compared to that of static controls. Histologically, type II collagen staining was evident in all construct groups, while a surface layer of type I collagen increased in thickness with increasing shear stress magnitude. The areal fraction of type I collagen was higher in the 0.1‐Pa group (25.2 ± 2.2%) than either the 0.001‐Pa (13.6 ± 3.8%) or the static (7.9 ± 1.5%) group. Type II collagen content, as assessed by ELISA, was also higher in the 0.1‐Pa group (7.5 ± 2.1%) compared to the 0.001‐Pa (3.0 ± 2.25%) or static groups (3.7 ± 3.2%). Temporal gene expression analysis showed a flow‐induced increase in type I and type II collagen expression within 24 h of exposure. Interestingly, while the 0.1‐Pa group showed higher collagen content, this group retained less sulfated glycosaminoglycans in the matrix over time in bioreactor culture. Increases in both tensile Young's modulus and ultimate strength were observed with increasing shear stress, yielding constructs possessing a modulus of nearly 5 MPa and strength of 1.3 MPa. This study demonstrates that shear stress is a potent modulator of both the amount and type of synthesized extracellular matrix constituents in engineered cartilaginous tissue with corresponding effects on mechanical function. Biotechnol. Bioeng. 2009; 104: 809–820 © 2009 Wiley Periodicals, Inc.     

Protein formulation and lyophilization cycle design: prevention of damage due to freeze-concentration induced phase separation

Isolated equine chondrocytes, from juveniles and adults, were cultured in resorbable polyglycolic acid meshes for up to 5 weeks with semicontinuous feeding using a custom-made system to intermittently compress the regenerating tissue. Assays of the tissue constructs indicate that intermittent compression at 500 and 1000 psi (3.44 and 6.87 MPa, respectively) stimulated the production of extracellular matrix, enhancing the rate of de novo chondrogenesis. Constructs derived from juvenile cells contained concentrations of extracellular matrix components at levels more like that of native tissue than did constructs derived from adult cells. With intermittent pressurization, however, even adult cells were induced to increase the production of extracellular matrix. At both levels of intermittent pressure, the concentration of sulfated glycosaminoglycan in constructs from juvenile cells was found to be up to ten times greater than concentrations in control (nonpressurized) and adult cell-derived constructs. Whereas collagen concentrations in the 500 psi and control constructs were not significantly different for either juvenile or adult cell-derived constructs, intermittent pressurization at 1000 psi enhanced the production of collagen, suggesting that there may be a minimum level of pressure necessary to stimulate collagen formation.     

Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).     

Cyclic mechanical compression increases mineralization of cell-seeded polymer scaffolds in vivo

Despite considerable documentation of the ability of normal bone to adapt to its mechanical environment, very little is known about the response of bone grafts or their substitutes to mechanical loading even though many bone defects are located in load-bearing sites. The goal of this research was to quantify the effects of controlled in vivo mechanical stimulation on the mineralization of a tissue-engineered bone replacement and identify the tissue level stresses and strains associated with the applied loading. A novel subcutaneous implant system was designed capable of intermittent cyclic compression of tissue-engineered constructs in vivo. Mesenchymal stem cell-seeded polymeric scaffolds with 8 weeks of in vitro preculture were placed within the loading system and implanted subcutaneously in male Fisher rats. Constructs were subjected to 2 weeks of loading (3 treatments per week for 30 min each, 13.3 N at 1 Hz) and harvested after 6 weeks of in vivo growth for histological examination and quantification of mineral content. Mineralization significantly increased by approximately threefold in the loaded constructs. The finite element method was used to predict tissue level stresses and strains within the construct resulting from the applied in vivo load. The largest principal strains in the polymer were distributed about a modal value of -0.24% with strains in the interstitial space being about five times greater. Von Mises stresses in the polymer were distributed about a modal value of 1.6 MPa, while stresses in the interstitial tissue were about three orders of magnitude smaller. This research demonstrates the ability of controlled in vivo mechanical stimulation to enhance mineralized matrix production on a polymeric scaffold seeded with osteogenic cells and suggests that interactions with the local mechanical environment should be considered in the design of constructs for functional bone repair.     

Tenocytic extract and mechanical stimulation in a tissue‐engineered tendon construct increases cellular proliferation and ECM deposition

Chemical and mechanical stimulation, when properly utilized, positively influence both the differentiation of in vitro cultured stem cells and the quality of the deposited extracellular matrix (ECM). This study aimed to find if cell‐free extract from primary tenocytes can positively affect the development of a tissue‐engineered tendon construct, consisting of a human umbilical vein (HUV) seeded with mesenchymal stem cells (MSCs) subjected to cyclical mechanical stimulation. The tenocytic cell‐free extract possesses biological material from tendon cells that could potentially influence MSC tenocytic differentiation and construct development. We demonstrate that the addition of tenocytic extract in statically cultured tendon constructs increases ECM deposition and tendon‐related gene expression of MSCs. The incorporation of mechanical stimulation (2% strain for 30 min/day at 0.5 cycles/min) with tenocytic extract further improved the MSC seeded HUV constructs by increasing cellularity of the construct by 37% and the ultimate tensile strength by 33% compared to the constructs with only mechanical stimulation after 14 days. Furthermore, the addition of mechanical stimulation to the extract supplementation produced longitudinal ECM fibril alignment along with dense connective tissue, reminiscent of natural tendon.     

Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds

Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

A finite element model of cell-matrix interactions to study the differential effect of scaffold composition on chondrogenic response to mechanical stimulation

Mechanically induced cell deformations have been shown to influence chondrocyte response in 3D culture. However, the relationship between the mechanical stimulation and cell response is not yet fully understood. In this study a finite element model was developed to investigate cell-matrix interactions under unconfined compression conditions, using a tissue engineered encapsulating hydrogel seeded with chondrocytes. Model predictions of stress and strain distributions within the cell and on the cell boundary were shown to exhibit space-dependent responses that varied with scaffold mechanical properties, the presence of a pericellular matrix (PCM), and the cell size. The simulations predicted that when the cells were initially encapsulated into the hydrogel scaffolds, the cell size hardly affected the magnitude of the stresses and strains that were reaching the encapsulated cells. However, with the inclusion of a PCM layer, larger cells experienced enhanced stresses and strains resulting from the mechanical stimulation. It was also noted that the PCM had a stress shielding effect on the cells in that the peak stresses experienced within the cells during loading were significantly reduced. On the other hand, the PCM caused the stresses at the cell-matrix interface to increase. Based on the model predictions, the PCM modified the spatial stress distribution within and around the encapsulated cells by redirecting the maximum stresses from the periphery of the cells to the cell nucleus. In a tissue engineered cartilage exposed to mechanical loading, the formation of a neo-PCM by encapsulated chondrocytes appears to protect them from initially excessive mechanical loading. Predictive models can thus shed important insight into how chondrocytes remodel their local environment in order to redistribute mechanical signals in tissue engineered constructs.     

Metabolic responses induced by compression of chondrocytes in variable-stiffness microenvironments

Cells sense and respond to mechanical loads in a process called mechanotransduction. These processes are disrupted in the chondrocytes of cartilage during joint disease. A key driver of cellular mechanotransduction is the stiffness of the surrounding matrix. Many cells are surrounded by extracellular matrix that allows for tissue mechanical function. Although prior studies demonstrate that extracellular stiffness is important in cell differentiation, morphology and phenotype, it remains largely unknown how a cell’s biological response to cyclical loading varies with changes in surrounding substrate stiffness. Understanding these processes is important for understanding cells that are cyclically loaded during daily in vivo activities (e.g. chondrocytes and walking). This study uses high-performance liquid chromatography – mass spectrometry to identify metabolomic changes in primary chondrocytes under cyclical compression for 0–30 minutes in low- and high-stiffness environments. Metabolomic analysis reveals metabolites and pathways that are sensitive to substrate stiffness, duration of cyclical compression, and a combination of both suggesting changes in extracellular stiffness in vivo alter mechanosensitive signaling. Our results further suggest that cyclical loading minimizes matrix deterioration and increases matrix production in chondrocytes. This study shows the importance of modeling in vivo stiffness with in vitro models to understand cellular mechanotransduction.     

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.