Mechanical regulation of chondrogenesis.

Mechanical factors play a crucial role in the development of articular cartilage in vivo. In this regard, tissue engineers have sought to leverage native mechanotransduction pathways to enhance in vitro stem cell-based cartilage repair strategies. However, a thorough understanding of how individual mechanical factors influence stem cell fate is needed to predictably and effectively utilize this strategy of mechanically-induced chondrogenesis. This article summarizes some of the latest findings on mechanically stimulated chondrogenesis, highlighting several new areas of interest, such as the effects of mechanical stimulation on matrix maintenance and terminal differentiation, as well as the use of multifactorial bioreactors. Additionally, the roles of individual biophysical factors, such as hydrostatic or osmotic pressure, are examined in light of their potential to induce mesenchymal stem cell chondrogenesis. An improved understanding of biomechanically-driven tissue development and maturation of stem cell-based cartilage replacements will hopefully lead to the development of cell-based therapies for cartilage degeneration and disease

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients

Measurement of intracellular strain on deformable substrates with texture correlation.

Mechanical stimuli are important factors that regulate cell proliferation, survival, metabolism and motility in a variety of cell types. The relationship between mechanical deformation of the extracellular matrix and intracellular deformation of cellular sub-regions and organelles has not been fully elucidated, but may provide new insight into the mechanisms involved in transducing mechanical stimuli to biological responses. In this study, a novel fluorescence microscopy and image analysis method was applied to examine the hypothesis that mechanical strains are fully transferred from a planar, deformable substrate to cytoplasmic and intranuclear regions within attached cells. Intracellular strains were measured in cells derived from the anulus fibrosus of the intervertebral disc when attached to an elastic silicone membrane that was subjected to tensile stretch. Measurements indicated cytoplasmic strains were similar to those of the underlying substrate, with a strain transfer ratio (STR) of 0.79. In contrast, nuclear strains were much smaller than those of the substrate, with an STR of 0.17. These findings are consistent with previous studies indicating nuclear stiffness is significantly greater than cytoplasmic stiffness, as measured using other methods. This study provides a novel method for the study of cellular mechanics, including a new technique for measuring intranuclear deformations, with evidence of differential magnitudes and patterns of strain transferred from the substrate to cell cytoplasm and nucleus

Composition of the pericellular matrix modulates the deformation behaviour of chondrocytes in articular cartilage under static loading

The aim was to assess the role of the composition changes in the pericellular matrix (PCM) for the chondrocyte deformation. For that, a three-dimensional finite element model with depth-dependent collagen density, fluid fraction, fixed charge density and collagen architecture, including parallel planes representing the split-lines, was created to model the extracellular matrix (ECM). The PCM was constructed similarly as the ECM, but the collagen fibrils were oriented parallel to the chondrocyte surfaces. The chondrocytes were modelled as poroelastic with swelling properties. Deformation behaviour of the cells was studied under 15% static compression. Due to the depth-dependent structure and composition of cartilage, axial cell strains were highly depth-dependent. An increase in the collagen content and fluid fraction in the PCMs increased the lateral cell strains, while an increase in the fixed charge density induced an inverse behaviour. Axial cell strains were only slightly affected by the changes in PCM composition. We conclude that the PCM composition plays a significant role in the deformation behaviour of chondrocytes, possibly modulating cartilage development, adaptation and degeneration. The development of cartilage repair materials could benefit from this information

Energy recovery in individuals with knee osteoarthritis.

OBJECTIVE: Pathological gaits have been shown to limit transfer between potential (PE) and kinetic (KE) energy during walking, which can increase locomotor costs. The purpose of this study was to examine whether energy exchange would be limited in people with knee osteoarthritis (OA). METHODS: Ground reaction forces during walking were collected from 93 subjects with symptomatic knee OA (self-selected and fast speeds) and 13 healthy controls (self-selected speed) and used to calculate their center of mass (COM) movements, PE and KE relationships, and energy recovery during a stride. Correlations and linear regressions examined the impact of energy fluctuation phase and amplitude, walking velocity, body mass, self-reported pain, and radiographic severity on recovery. Paired t-tests were run to compare energy recovery between cohorts. RESULTS: Symptomatic knee OA subjects displayed lower energetic recovery during self-selected walking speeds than healthy controls (P = 0.0018). PE and KE phase relationships explained the majority (66%) of variance in recovery. Recovery had a complex relationship with velocity and its change across speeds was significantly influenced by the self-selected walking speed of each subject. Neither radiographic OA scores nor subject self-reported measures demonstrated any relationship with energy recovery. CONCLUSIONS: Knee OA reduces effective exchange of PE and KE, potentially increasing the muscular work required to control movements of the COM. Gait retraining may return subjects to more normal patterns of energy exchange and allow them to reduce fatigue

Meniscus-derived matrix scafolds promote the integrative repair of meniscal defects

Meniscal tears have a poor healing capacity, and damage to the meniscus is associated with significant pain, disability, and progressive degenerative changes in the knee joint that lead to osteoarthritis. Therefore, strategies to promote meniscus repair and improve meniscus function are needed. The objective of this study was to generate porcine meniscus-derived matrix (MDM) scaffolds and test their effectiveness in promoting meniscus repair via migration of endogenous meniscus cells from the surrounding meniscus or exogenously seeded human bone marrow-derived mesenchymal stem cells (MSCs). Both endogenous meniscal cells and MSCs infiltrated the MDM scaffolds. In the absence of exogenous cells, the 8% MDM scaffolds promoted the integrative repair of an in vitro meniscal defect. Dehydrothermal crosslinking and concentration of the MDM influenced the biochemical content and shear strength of repair, demonstrating that the MDM can be tailored to promote tissue repair. These findings indicate that native meniscus cells can enhance meniscus healing if a scaffold is provided that promotes cellular infiltration and tissue growth. The high affinity of cells for the MDM and the ability to remodel the scaffold reveals the potential of MDM to integrate with native meniscal tissue to promote long-term repair without necessarily requiring exogenous cells

Novel synovial fluid recovery method allows for quantification of a marker of arthritis in mice

SummaryObjectiveWe evaluated three methodologies – a calcium sodium alginate compound (CSAC), polyacrylate beads (PABs), and Whatman paper recovery (WPR) – for the ability to recover synovial fluid (SF) from mouse knees in a manner that facilitated biochemical marker analysis.MethodsPilot testing of each of these recovery vehicles was conducted using small volumes of waste human SF. CSAC emerged as the method of choice, and was used to recover and quantify SF from the knees of C57BL/6 mice (n=12), six of which were given left knee articular fractures. SF concentrations of cartilage oligomeric matrix protein (COMP) were measured by enzyme-linked immunosorbent assay.ResultsThe mean concentration ratio [(COMPleft knee)/(COMPright knee)] was higher in the mice subjected to articular fracture when compared to the non-fracture mice (P=0.026). The mean total COMP ratio (taking into account the quantitative recovery of SF) best discriminated between fracture and non-fracture knees (P=0.004).ConclusionsOur results provide the first direct evidence of accelerated joint tissue turnover in a mouse model responding to acute joint injury. These data strongly suggest that mouse SF recovery is feasible and that biomarker analysis of collected SF samples can augment traditional histological analyses in mouse models of arthritis

Alterations in the mechanical properties of the human chondrocyte pericellular matrix with osteoarthritis

Introduction: In articular cartilage, chondrocytes are surrounded by a pericellular matrix (PCM), which together with the chondrocyte have been termed the chondron [1]. This region is characterized by the presence of type VI collagen and increased proteoglycan concentration relative to the extracellular matrix. While the specific function of the PCM is not fully understood, it has been hypothesized to play important roles in regulating the biomechanical, biophysical, and biochemical signals that the chondrocyte perceives during normal joint activity [1,5,7]. A more thorough understanding of the mechanical properties of the PCM would provide important insights into the potential biomechanical function of the chondron as a discrete entity in articular cartilage. Furthermore, previous studies have reported changes in the distribution and amount of collagen VI in osteoarthritic (OA) cartilage, suggesting that the biomechanical function of the PCM may be altered with disease. The goal of this study was to test the hypothesis that the biomechanical properties of the PCM vary with depth from the cartilage surface and are altered with OA. Using a newly developed microaspiration technique, chondrons were extracted from normal and osteoarthritic cartilage. The Young's modulus of the PCM was determined using the micropipette aspiration technique in combination with a recently developed theoretical model that represents the chondron as an elastic, compressible layer overlying an elastic half-space ( Methods: Chondrons were mechanically isolated from full thickness articular cartilage of human femoral heads at the time of joint replacement surgery (N=73 chondrons from 13 donors, ages: 19-75 yr). Chondrons were extracted from two different zones (surface and middle/deep) by applying suction pressure to the cartilage surface with a glass pipette. Chondrons were classified as osteoarthritic ('OA') or nonosteoarthritic ('non-OA') based on a semi-quantitative histology grading scale from 0 (normal) to 20 (OA) of the cartilage from which they were extracted. The ave rage grades for non-OA and OA cartilage were 4.5±3.1 and 15.8 ±2.1, respectively. The elastic properties of chondrons were measured using a new axisymetric layered elastic half-space model for the micropipette aspiration technique Results: The mean Young's modulus of the PCM of chondrons from non-OA cartilage was Enon-OA=66.5±23.3 kPa. With OA, the Young's modulus of the PCM was significantly reduced to EOA=41.3±21.1 kPa (p<0.05). No zonal variation was found in the Young's modulus of PCM of chondrons from OA or non-OA cartilage In contrast to previous studies on enzymatically isolated chondrons [6] which reported a mean Young's modulus of ~1.5 kPa for the PCM [7], our findings suggest that the Young's modulus of the mechanically isolated PCM is nearly 50 times larger than that of the enzymatically isolated chondrons. This difference is most likely attributable to the effects of enzymatic isolation on the properties of the PCM. These findings are consistent with previous studies examining the deformation behavior of enzymatically and mechanically isolated chondrons embedded in an agarose matrix [8], and suggest that the Young's modulus of the mechanically isolated chondrons is greater than the Young's modulus of enzymatically isolated chondrons and in excess of the Young's modulus of the agarose (25kPa). A unique aspect of this study was the development of a new chondron isolation technique, which is based on extraction of chondrons directly from the cartilage by applying suction pressure using a small diameter pipette. This technique requires minimal tissue preparation, and can be used to extract chondrons from precise sites (i.e., zones) of the cartilage. Compared with the homogenization technique [1], our method is fast and easy and yields a large number of chondrons with a smaller amount of debris. Increasing evidence suggests that the chondron is a distinct functional compartment in articular cartilage and serves to regulate the mechanical environment of the chondrocytes [5,8]. The mechanical properties of the PCM determined in this study can be applied in several models of articular cartilage to further examine the mechanical microenvironment of chondrocytes. In addition, the development of an easy chondron extraction technique will further facilitate the investigation of this unique structure in articular cartilage. Acknowledgments: The authors would like to thank Clint Walker from Duke University for his excellent assistance