Application Of Dynamic Compression Research Articles

Engineering zonal cartilaginous tissue by modulating oxygen levels and mechanical cues through the depth of infrapatellar fat pad stem cell laden hydrogels.

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad-derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC-laden hydrogels (either 2mm or 4mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC-laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96MPa to 1.45MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint-like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd.

The effects of dynamic compression on the development of cartilage grafts engineered using bone marrow and infrapatellar fat pad derived stem cells

Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d−1) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important role in the development of phenotypically stable constructs.

Exploring the roles of integrin binding and cytoskeletal reorganization during mesenchymal stem cell mechanotransduction in soft and stiff hydrogels subjected to dynamic compression

The objective of this study was to explore how the response of mesenchymal stem cells (MSCs) to dynamic compression (DC) depends on their pericellular environment and the development of their cytoskeleton. MSCs were first seeded into 3% agarose hydrogels, stimulated with the chondrogenic growth factor TGF-β3 and exposed to DC (~10% strain at 1Hz) for 1h on either day 7, 14, or 21 of culture. At each time point, the actin, vimentin and tubulin networks of the MSCs were assessed using confocal microscopy. Similar to previous results, MSCs displayed a temporal response to DC; however, no dramatic changes in gross cytoskeletal organization were observed with time in culture. Vinculin (a membrane-cytoskeletal protein in focal adhesions) staining appeared more intense with time in culture. We next aimed to explore how changes to the pericellular environment, independent of the duration of exposure to TGF-β3, would influence the response of MSCs to DC. To this end, MSCs were encapsulated into either ‘soft’ or ‘stiff’ agarose hydrogels that are known to differentially support pericellular matrix (PCM) development. The application of DC led to greater relative increases in the expression of chondrogenic marker genes in the stiffer hydrogels, where the MSCs were found to have a more well developed PCM. These increases in gene expression were not observed following the addition of RGDS, an integrin blocker, suggesting that integrin binding plays a role in determining the response of MSCs to DC. Microtubule organization in MSCs was found to adapt in response to DC, but this effect was not integrin mediated, as this cytoskeletal reorganization was also observed in the presence of RGDS. In conclusion, although the PCM, integrin binding, and cytoskeletal reorganization are all involved in mechanotransduction of DC, none of these factors in isolation was able to completely explain the temporal mechanosensitivity of MSCs to dynamic compression.

Modulating Gradients in Regulatory Signals within Mesenchymal Stem Cell Seeded Hydrogels: A Novel Strategy to Engineer Zonal Articular Cartilage

Engineering organs and tissues with the spatial composition and organisation of their native equivalents remains a major challenge. One approach to engineer such spatial complexity is to recapitulate the gradients in regulatory signals that during development and maturation are believed to drive spatial changes in stem cell differentiation. Mesenchymal stem cell (MSC) differentiation is known to be influenced by both soluble factors and mechanical cues present in the local microenvironment. The objective of this study was to engineer a cartilaginous tissue with a native zonal composition by modulating both the oxygen tension and mechanical environment thorough the depth of MSC seeded hydrogels. To this end, constructs were radially confined to half their thickness and subjected to dynamic compression (DC). Confinement reduced oxygen levels in the bottom of the construct and with the application of DC, increased strains across the top of the construct. These spatial changes correlated with increased glycosaminoglycan accumulation in the bottom of constructs, increased collagen accumulation in the top of constructs, and a suppression of hypertrophy and calcification throughout the construct. Matrix accumulation increased for higher hydrogel cell seeding densities; with DC further enhancing both glycosaminoglycan accumulation and construct stiffness. The combination of spatial confinement and DC was also found to increase proteoglycan-4 (lubricin) deposition toward the top surface of these tissues. In conclusion, by modulating the environment through the depth of developing constructs, it is possible to suppress MSC endochondral progression and to engineer tissues with zonal gradients mimicking certain aspects of articular cartilage.

The Response of Bone Marrow-Derived Mesenchymal Stem Cells to Dynamic Compression Following TGF-β3 Induced Chondrogenic Differentiation

The objective of this study was to investigate the hypothesis that the application of dynamic compression following transforming growth factor-beta3 (TGF-beta3) induced differentiation will further enhance chondrogenesis of mesenchymal stem cells (MSCs). Porcine MSCs were encapsulated in agarose hydrogels and cultured in a chemically defined medium with TGF-beta3 (10 ng/mL). Dynamic compression (1 Hz, 10% strain, 1 h/day) was initiated at either day 0 or day 21 and continued until day 42 of culture; with TGF-beta3 withdrawn from some groups at day 21. Biochemical and mechanical properties of the MSC-seeded constructs were evaluated up to day 42. The application of dynamic compression from day 0 inhibited chondrogenesis of MSCs. This inhibition of chondrogenesis in response to dynamic compression was not observed if MSC-seeded constructs first underwent 21 days of chondrogenic differentiation in the presence of TGF-beta3. Spatial differences in sGAG accumulation in response to both TGF-beta3 stimulation and dynamic compression were observed within the constructs. sGAG release from the engineered construct into the surrounding culture media was also dependent on TGF-beta3 stimulation, but was not effected by dynamic compression. Continued supplementation with TGF-beta3 appeared to be a more potent chondrogenic stimulus than the application of 1 h of daily dynamic compression following cytokine initiated differentiation. In the context of cartilage tissue engineering, the results of this study suggest that MSC seeded constructs should be first allowed to undergo chondrogenesis in vitro prior to implantation in a load bearing environment.

Chondrogenesis of Human Bone Marrow Mesenchymal Stem Cells in Fibrin–Polyurethane Composites Is Modulated by Frequency and Amplitude of Dynamic Compression and Shear Stress

In this study, human bone marrow mesenchymal stem cell (hMSC)-seeded fibrin-polyurethane composite scaffolds were subjected to various mechanical load protocols to determine the effect of compression and surface rotation frequency and axial compression magnitude on the induction of chondrocyte-specific gene expression and protein synthesis. After 7 days of preculture and 7 days of load, application of dynamic compression and surface shear 1 h daily enhanced chondrogenesis of hMSCs compared with the nonloaded control samples. Higher load frequency and higher compression amplitude induced higher glycosaminoglycan synthesis, higher chondrocytic gene expression, higher chondrocytic:fibroblastic, chondrocytic:hypertrophic, and chondrocytic:osteoblastic gene expression ratios, as well as higher transforming growth factor beta1 (TGFB1) and TGFB3 gene expression. The chondrogenesis level of hMSCs was positively related to the TGFB1 and TGFB3 gene expression level, which was determined by various load regimes. In conclusion, chondrogenesis of human MSCs induced by mechanical stimulation can be further enhanced by modifying frequency and compression.

Rho kinase–dependent CCL20 induced by dynamic compression of human chondrocytes

Mechanical stimulation of cartilage affects tissue homeostasis and chondrocyte function. The chondrocyte phenotype is dependent on cell shape, which is largely determined by the actin cytoskeleton. Reorganization of the actin cytoskeleton results from Rho GTPase activation. The purpose of this study was to examine the roles of both actin and Rho in mechanotransduction in chondrocytes. We embedded human articular chondrocytes in 2 x 6-mm agarose discs at 5 x 10(6) cells/ml and subjected the discs to unconfined dynamic compression at 0.5 Hz. By comparing samples with and without dynamic compression, we identified Rho activation according to the GTP-bound active RhoA measured in cell lysates. We identified rearrangements in filamentous actin structures using fluorescence-labeled phalloidin and confocal microscopy of fixed samples. We identified altered gene expression using TaqMan quantitative reverse transcription-polymerase chain reaction analysis. We tested for a requirement for Rho signaling by performing the dynamic compression in the presence of Rho kinase inhibitors. RhoA activation occurred within 5-10 minutes of dynamic compression. Rho kinase-dependent actin reorganization occurred within 20 minutes after application of dynamic compression and was apparent as "punctate" F-actin structures that were visible under confocal microscopy. We identified early-phase mechanoresponsive genes (CCL20 and inducible nitric oxide synthase) that were highly up-regulated within 1 hour of dynamic compression in a Rho kinase-dependent and actin-dependent manner. Together, these results are the first demonstration that the Rho-Rho kinase pathway and actin cytoskeletal reorganization are required for changes in the expression of genes involved in human chondrocyte mechanotransduction.

Dynamic compression influences interleukin-1β-induced nitric oxide and prostaglandin E2 release by articular chondrocytes via alterations in iNOS and COX-2 expression

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.