Cell Signaling in Tenocytes: Response to Load and Ligands in Health and Disease.

Signaling in tenocytes during development, homeostasis and injury involves multiple and redundant pathways. Given that tendons transmit mechanical forces from muscle to bone to effect movement, a key function for tenocytes is the detection of and response to mechanical stimulation. Mechanotransduction involves matrix-integrin-cytoskeleton to nucleus signaling, gap junction intercellular communication, changes in intracellular calcium (Ca(2+)), activation of receptors and their pathways, and responses to biochemical factors such as hormones, growth factors, adenosine triphosphate (ATP) and its derivatives, and neuromodulators. The primary cilium also plays a key role in the detection of mechanical signals. During development, transforming growth factor-ß (TGF-ß), bone morphogenetic protein (BMP), and hedgehog (Hh) signaling modulate tendon differentiation and formation. The response to injury is complex and varied involving not only inflammatory mediators such as interleukin-1ß but also mechanosensing. This chapter reviews the signaling pathways tenocytes use during mechanotransduction, development and in response to injury.

Modulation of collagen gel compaction by extracellular ATP is MAPK and NF-kappaB pathways dependent.

Understanding the mechanisms that regulate mechanosensitivity in osteoblasts is important for controlling bone homeostasis and the development of new drugs to combat bone loss. It is believed that prestress or force generation (the tensile stress within the cell body) plays an important role in regulating cellular mechanosensitivity. In the present study, a three-dimensional (3D) collagen culture was used to monitor the change in prestress of the osteoblast-like cells. Collagen hydrogel compaction has been used as an indicator of the change in the degree of cell prestress. Previous results in this model demonstrated that extracellular ATP reduced the mechanosensitivity of osteoblasts by reducing cellular prestress. To elucidate the potential mechanisms involved in this process, the signaling pathways downstream of P2 purinoceptors involved in regulating the compaction of type I collagen gels were investigated. By using specific inhibitors to these signaling pathways, we found that ATP-induced reduction in collagen gel compaction rate is dependent on mitogen-activated protein kinase (MAKP) and NF-kappaB pathways. However, blocking protein kinase C with GF109203X did not change the compaction kinetics in the presence of ATPgammaS. Moreover, blocking cyclic AMP (cAMP), phosphatidylinositol-3 kinase (PI3K), calmodulin (CaM) or L-type voltage sensitive calcium channels did not affect ATP's ability to reduce collagen gel compaction. The results from the present and previous studies indicate that extracellular ATP may act as a negative feedback modulator in the mechanotransduction system since mechanical stimuli increase ATP release from stimulated cells.

A brief history of tendon and ligament bioreactors: Impact and future prospects.

Bioreactors are powerful tools with the potential to model tissue development and disease in vitro. For nearly four decades, bioreactors have been used to create tendon and ligament tissue-engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of two- and three-dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue-engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell-cell and cell-matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades.

Key developments that impacted the field of mechanobiology and mechanotransduction.

Advances in mechanobiology have evolved through insights from multiple disciplines including structural engineering, biomechanics, vascular biology, and orthopaedics. In this paper, we reviewed the impact of key reports related to the study of applied loads on tissues and cells and the resulting signal transduction pathways. We addressed how technology has helped advance the burgeoning field of mechanobiology (over 33,600 publications from 1970 to 2016). We analyzed the impact of critical ideas and then determined how these concepts influenced the mechanobiology field by looking at the citation frequency of these reports as well as tracking how the overall number of citations within the field changed over time. These data allowed us to understand how a key publication, idea, or technology guided or enabled the field. Initial observations of how forces acted on bone and soft tissues stimulated the development of computational solutions defining how forces affect tissue modeling and remodeling. Enabling technologies, such as cell and tissue stretching, compression, and shear stress devices, allowed more researchers to explore how deformation and fluid flow affect cells. Observation of the cell as a tensegrity structure and advanced methods to study genetic regulation in cells further advanced knowledge of specific mechanisms of mechanotransduction. The future of the field will involve developing gene and drug therapies to simulate or augment beneficial load regimens in patients and in mechanically conditioning organs for implantation. Here, we addressed a history of the field, but we limited our discussions to advances in musculoskeletal mechanobiology, primarily in bone, tendon, and ligament tissues. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:605-619, 2018.

ATP reduces gel compaction in osteoblast-populated collagen gels.

Bone remodeling is a localized process, but regulated by systemic signals such as hormones, cytokines, and mechanical loading. The mechanism by which bone cells convert these systemic signals into local signals is not completely understood. It is broadly accepted that the "prestress" in cytoskeleton of cells affects the magnitude of cellular responses to mechanical stimuli. Prestress derives from stiff cytoskeletal proteins and their connections within the cell and from cell contractility upon attaching to matrix. In an in vitro model of three-dimensional gel compaction, the relative cellular prestress levels in the same matrix environment were determined by matrix compaction rate: a greater compaction rate resulted in a higher level of prestress. In the present study, the effects of ATP on the prestress of osteoblasts were studied using mouse MC3T3-E1 cells grown in three-dimensional bioartificial tissues (BATs). ATP (> or =100 microM) reduced the compaction rate of BATs in a dose-dependent manner. ADP, 2'-(or 3')-O-(4-benzoylbenzoyl) ATP, and UTP, but not alpha,beta-methylene ATP, also reduced the compaction rate but to a lesser extent. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium did not block the effect of ATP on BAT compaction rate. These results indicate that both P2X and P2Y receptors are involved in ATP-induced reduction of BAT compaction rate. Steady fluid flow and RT-PCR results showed that ATP reduced cell attachment on type I collagen by downregulating the expression of integrin alpha(1). These results suggest a potential role for P2 receptors in matrix remodeling and repair and as a potential drug target in treatment of bone diseases.

Distributing a fixed amount of cyclic loading to tendon explants over longer periods induces greater cellular and mechanical responses.

Tendon overuse injuries are a major source of clinical concern. Cyclic loading causes material damage and induces biochemical responses in tendon. The purpose of this study was to examine the biochemical and biomechanical tendon response after applying cyclical loading over varying durations. Avian flexor digitorum profundus tendons were loaded (3 or 12 MPa) to a fixed number of cycles across either 1 or 12 days in vitro. The tendon response evaluations included biomechanical data gathered during loading and subsequent failure testing. Evaluations also included cellular viability, cell death, and proteoglycan, collagen, collagenase, and prostaglandin E(2) (PGE(2)) content measurements obtained from tissue specimens and media samples. Significant strains (up to 2%) accumulated during loading. Loading to 12 MPa significantly reduced maximum stress (33% and 27%) and energy density (42% and 50%) when applied across 1 or 12 days, respectively. Loading to 3 MPa also caused a 40% reduction in energy density, but only when applied across 12 days. Cell death and collagenase activity increased significantly with increasing magnitude and duration. However, no differences occurred in cell viability or collagen content. Glycosaminoglycan content increased 50% with load magnitude, while PGE(2) production increased 2.5-fold with loading magnitude and 11-fold with increased duration. Mechanical fatigue-induced mechanical property changes were exhibited by the tendons in response to increased loading magnitude across just 1 day. However, when the same loading was applied over a longer period, most outcomes were magnified substantially, relative to the short duration regimens. This is presumably due to the increased response time for the complex cellular response to loading. A key contributor may be the inflammatory mediator, PGE(2), which exhibited large magnitude and duration dependent increases to cyclic loading.

Comparison of cellular strain with applied substrate strain in vitro.

Strain magnitudes within tenocytes undergoing substrate tensile strain are not well defined. It was hypothesized that strain magnitudes at the cellular level would reflect those of the applied substrate (equibiaxial or uniaxial) strain. A vacuum-operated device was used to apply equibiaxial or uniaxial tension to a flexible substrate upon which tenocytes were cultured in monolayer. Images of tenocytes labeled with Fura-2, to detect free intracellular calcium ions, and MitoFluor Green, to detect mitochondria, were taken prior to strain and for 20 min during application of static strain. A custom-written, texture correlation program computed strain magnitudes in the cell based on the change in pixel pattern displacements between images of non-strained and strained cells. On average, cellular strain was approximately 37+/-8% and 63+/-11% of the applied equibiaxial and uniaxial substrate strain, respectively. The largest cell strains were detected in cells oriented parallel to the direction of applied uniaxial tensile strain. However, strain magnitudes within a cell were heterogeneous. The variance in strain magnitude within and among tenocytes is dependent on cell orientation, cell stiffness, cytoskeleton organization, subcellular organelles, or placement and type of cell-substrate contacts. Results of the present study indicate that cultured tenocytes experience a moderate fraction of the applied substrate strain.

Interleukin-1beta increases elasticity of human bioartificial tendons.

Stiffness is an important mechanical property of connective tissues, especially for tissues subjected to cyclic strain in vivo, such as tendons. Therefore, modulation of material properties of native or engineered tissues is an important consideration for tissue repair. Interleukin 1-beta (IL-1beta) is a cytokine most often associated in connective tissues with induction of matrix metalloproteinases and matrix destruction. However, IL-1beta may also be involved in constructive remodeling and confer a cell survival value to tenocytes. In this study, we investigated the effects of IL-1beta on the properties of human tenocyte-populated bioartificial tendons (BATs) fabricated in a novel three-dimensional (3D) culture system. IL-1beta treatment reduced the ultimate tensile strength and elastic modulus of BATs and increased the maximum strain. IL-1beta at low doses (1, 10 pM) upregulated elastin expression and at a high dose (100 pM) downregulated type I collagen expression. Matrix metalloproteinases, which are involved in matrix remodeling, were also upregulated by IL-1beta. The increased elasticity prevented BATs from rupture caused by applied strain. The results in this study suggest that IL-1beta may act as a defense/survival factor in response to applied mechanical loading. The balance between cell intrinsic strain and external matrix strain is important for maintaining the integrity of tendons.

IL-1beta decreases the elastic modulus of human tenocytes.

Cellular responses to mechanical stimuli are regulated by interactions with the extracellular matrix, which, in turn, are strongly influenced by the degree of cell stiffness (Young's modulus). It was hypothesized that a more elastic cell could better withstand the rigors of remodeling and mechanical loading. It was further hypothesized that interleukin-1beta (IL-1beta) would modulate intracellular cytoskeleton polymerization and regulate cell stiffness. The purpose of this study was to investigate the utility of IL-1beta to alter the Young's modulus of human tenocytes. Young's modulus is the ratio of the stress to the strain, E = stress/strain = (F/A)/(deltaL/L0), where L0 is the equilibrium length, deltaL is the length change under the applied stress, F is the force applied, and A is the area over which the force is applied. Human tenocytes were incubated with 100 pM recombinant human IL-1beta for 5 days. The Young's modulus was reduced by 27-63%. Actin filaments were disrupted in >75% of IL-1beta-treated cells, resulting in a stellate shape. In contrast, immunostaining of alpha-tubulin showed increased intensity in IL-1beta-treated tenocytes. Human tenocytes in IL-1beta-treated bioartificial tendons were more tolerant to mechanical loading than were untreated counterparts. These results indicate that IL-1beta reduced the Young's modulus of human tenocytes by disrupting the cytoskeleton and/or downregulating the expression of actin and upregulating the expression of tubulins. The reduction in cell modulus may help cells to survive excessive mechanical loading that may occur in damaged or healing tendons.

Ligament cells stretch-adapted on a microgrooved substrate increase intercellular communication in response to a mechanical stimulus.

An in vitro model was used to investigate the effect of mechanical stimuli on adaptation to load and calcium signaling in aligned medial collateral ligament cells (MCL). This model used a patterned silicone membrane to align the cells parallel with the direction of the microgrooves. Alignment created an architecture that simulated a degree of cell orientation in native ligament tissue. It was hypothesized that aligned ligament cells would be more efficient at calcium wave propagation than cells that were randomly oriented. It was further hypothesized that calcium wave propagation would be greater among cells that were both aligned and subjected to mechanical stretch compared to cells that were aligned but not stretched. Rat MCL cells were loaded with Fura-2AM, a calcium-binding dye, and mechanically indented using a micropipette tip. A ratio-imaging fluorescence technique was used to quantitate the calcium (Ca2+) response. It was concluded that stretching ligament cells prior to stimulation increased their sensitivity to load and their ability to propagate a calcium wave. However, the ability of aligned cells to propagate this wave was not significantly different when compared to nonaligned cells. Treatment of cultures with inhibitors such as apyrase and suramin significantly reduced the number of cells recruited in the calcium response. Hence, it was concluded that ATP released from mechanically stimulated cells was a principal mediator responsible for the rise in intracellular calcium in ligament cells. Further, purinoceptor activation may amplify the signal to alert and recruit more cells in a response to mechanical stimulation.

Tendon mechanobiology: Current knowledge and future research opportunities.

Tendons mainly function as load-bearing tissues in the muscloskeletal system; transmitting loads from muscle to bone. Tendons are dynamic structures that respond to the magnitude, direction, frequency, and duration of physiologic as well as pathologic mechanical loads via complex interactions between cellular pathways and the highly specialized extracellular matrix. This paper reviews the evolution and current knowledge of mechanobiology in tendon development, homeostasis, disease, and repair. In addition, we review several novel mechanotransduction pathways that have been identified recently in other tissues and cell types, providing potential research opportunities in the field of tendon mechanobiology. We also highlight current methods, models, and technologies being used in a wide variety of mechanobiology research that could be investigated in the context of their potential applicability for answering some of the fundamental unanswered questions in this field. The article concludes with a review of the major questions and future goals discussed during the recent ORS/ISMMS New Frontiers in Tendon Research Conference held on September 10 and 11, 2014 in New York City.

Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production.

The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P<0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro.

Novel system for engineering bioartificial tendons and application of mechanical load.

Cells cultured in three-dimensional collagen gels express a more native state phenotype because they form a syncytial network that can be mechanically loaded. Moreover, cells remodel their matrix by eliminating water, and by reorganizing and aligning the collagen fibrils. Last, the ability to subject cells to mechanical loading in a native matrix is desirable because cells, in tissues as well as the matrix, bear strains and alter their expression profile consistent with either immobilization, moderate activity, or repetitive loading. This is the first report of a model bioreactor system to fabricate and culture tendon cell-populated, linear, tethered matrix constructs that can be mechanically loaded by a computer-driven, pressure-controlled system. Bioartificial tissues (BATs) as tendon constructs were molded in a novel, rubber bottom Tissue Train culture plate bearing nonwoven nylon mesh anchors at the east and west poles of each culture well. Mechanical loading was achieved by placing an Arctangle loading post (an Arctangle is a rectangle with curved short ends) beneath each well of the six-well culture plate and using vacuum to displace the flexible membrane downward, resulting in uniaxial strain on the BAT. BATs populated with avian flexor tendon cells expressed collagen genes I, III, and XII as well as aggrecan, fibronectin, prolyl hydroxylase, and tenascin, consistent with expression levels of cells grown on collagen-bonded two-dimensional surfaces or in native, whole, avian flexor tendon. Likewise, cells in BATs established a morphology of linearly arranged cells aligned with the principal strain direction as in fasicles of whole tendons. Last, BATs that were mechanically loaded had an ultimate tensile strength that was nearly 3-fold greater than that of nonloaded BATs in the first week of culture. Taken together, these results indicate that tendon cells fabricated in a mechanically loaded, linear collagen gel construct assume a phenotype that is similar to that of a native tendon in terms of appearance and expression and are stronger than nonexercised counterparts yet far weaker than native adult tendons. This technique represents a novel approach to culturing cells in a mechanically active, three-dimensional culture environment that can be readily used for the fabrication of tissue simulates for drug testing or tissue engineering.

Stretch and interleukin-1beta induce matrix metalloproteinases in rabbit tendon cells in vitro.

Little is known about the factors that initiate and propagate tendon overuse injuries, but chronic inflammation and matrix destruction have been implicated. The purpose of this study was to evaluate the production of cyclooxygenase II (COX-2) and matrix metalloproteinases (MMPs) by tendon cells exposed to cyclic strain and inflammatory cytokines in vitro. Rabbit Achilles tendon cells were subjected to a stretching protocol with 5% elongation at 0.33 Hz for 6 h, or treated with 1000 pM interleukin-1beta (IL-1beta), or exposed to IL-1beta and stretching together. Gene expression was evaluated by RT-PCR and production of stromelysin was quantified with an ELISA. IL-1beta induced the expression of the collagenase-1 and stromelysin-1 genes. Production of stromelysin proenzyme by cells stimulated with IL-1beta was 17 times higher than production by control cells. Cells exposed to IL-1beta and stretching produced 20 times more stromelysin than control cells. Cells subjected to stretching alone did not produce more stromelysin than control cells. The synergistic effect of IL-1beta and stretching was observed at doses of IL-1beta ranging from 10 to 1000 pM. These data suggest that mechanical load and inflammatory cytokines can initiate a matrix destructive pathway in tendon that is more pronounced than with mechanical loading or inflammation alone.

Rabbit tendon cells produce MMP-3 in response to fluid flow without significant calcium transients.

Forces applied to tendon during movement cause cellular deformation, as well as fluid movement. The goal of this study was to test the hypothesis that rabbit tendon fibroblasts detect and respond to fluid-induced shear stress. Cells were isolated from the paratenon of the rabbit Achilles tendon and then subjected to fluid flow at 1 dyn/cm(2) for 6h in a specially designed multi-slide flow device. The application of fluid flow led to an increased expression of the collagenase-1 (MMP-1), stromelysin-1 (MMP-3), cyclooxygenase II (COX-2) and interleukin-1beta (IL-1beta) genes. The release of proMMP-3 into the medium exhibited a dose-response with the level of fluid shear stress. However, not all cells aligned in the direction of flow. In other experiments, the same cells were incubated with the calcium-reactive dye FURA-2 AM, then subjected to laminar fluid flow in a parallel plate flow chamber. The cells did not significantly increase intracellular calcium concentration when exposed to fluid shear stress levels of up to 25 dyn/cm(2). These results show that gene expression in rabbit tendon cells is sensitive to fluid flow, but that signal transduction is not dependent on intracellular calcium transients. The upregulation of the MMP-1, MMP-3 and COX-2 genes shows that fluid flow could be an important mechanical stimulus for tendon remodelling or injury.

Nandrolone decanoate and load increase remodeling and strength in human supraspinatus bioartificial tendons.

BACKGROUND: To date, no studies document the effect of anabolic steroids on rotator cuff tendons. STUDY DESIGN: Controlled laboratory study. HYPOTHESIS: Anabolic steroids enhance remodeling and improve the biomechanical properties of bioartificially engineered human supraspinatus tendons. METHODS: Bioartificial tendons were treated with either nandrolone decanoate (nonload, steroid, n = 18), loading (load, nonsteroid, n = 18), or both (load, steroid, n = 18). A control group received no treatment (nonload, nonsteroid [NLNS], n = 18). Bioartificial tendons' remodeling was assessed by daily scanning, cytoskeletal organization by staining, matrix metalloproteinase-3 levels by ELISA assay, and biomechanical properties by load-to-failure testing. RESULTS: The load, steroid group showed the greatest remodeling and the best organized actin cytoskeleton. Matrix metallo-proteinase-3 levels in the load, steroid group were greater than those of the nonload, nonsteroid group (P <.05). Ultimate stress and ultimate strain in the load, steroid group were greater than those of the nonload, nonsteroid and nonload, steroid groups (P <.05). The strain energy density in the load, steroid group was greater when compared to other groups (P <.05). CONCLUSIONS: Nandrolone decanoate and load acted synergistically to increase matrix remodeling and biomechanical properties of bioartificial tendons. CLINICAL RELEVANCE: Data suggest anabolic steroids may enhance production of bioartificial tendons and rotator cuff tendon healing in vitro. More research is necessary before such clinical use is recommended.

Vibratory loading decreases extracellular matrix and matrix metalloproteinase gene expression in rabbit annulus cells.

BACKGROUND CONTEXT: Whole body vibration is an important factor contributing to low back and radicular pain. Vibratory loading as a mechanical stimulus is transferred to connective tissues as energy from ground reaction forces, as well as a direct input from the use of motorized tools and vehicles. Extracellular matrix degradation parallels increased age and mechanical stimuli resulting in disc degeneration and eventual spinal deformity. PURPOSE: The objective of this study was to investigate the relationship between vibratory loading and extracellular matrix expression in cultured rabbit annulus cells. STUDY DESIGN/SETTING: An in vitro rabbit model using cultured annulus fibrosis cells isolated from normal intervertebral disc was used to study matrix and metalloproteinase expression in response to vibration. METHODS: Annulus fibrosis cells were isolated by collagenase digestion from New Zealand White rabbits. Vibratory stimulation was applied to annulus cells in vitro, using an oscillating platform to deliver 0.1 x gravity load at 6 Hz for 2, 4, 6 or 8 hours. Gene expression was assessed by reverse transcriptase polymerase chain reaction. RESULTS: Aggrecan, collagen Type III and matrix metalloproteinase-3 gene expression was suppressed by vibratory loading in rabbit annulus cells. Suppression of the aggrecan gene might lead to a decrease in proteoglycan synthesis. CONCLUSIONS: These data suggest that vibratory load may play an important role in extracellular matrix metabolism of intervertebral disc cells, especially in the gene expression pathway of proteoglycans. It has been proposed that vibratory loading increases production of matrix-degrading, proteolytic enzymes. We have demonstrated that gene expression for key matrix messages and matrix metalloproteinase is decreased by vibration. In conclusion, we believe that study of the roles between extracellular matrix gene suppression and mechanical stress may clarify the pathomechanism of disc degeneration, such as disc herniation or degeneration.

Out of Academics: Education, Entrepreneurship and Enterprise.

The author started a niche biotech company in 1985 called Flexcell® to distribute an enabling technology, mechanobiology devices, to the field. He was the first University of North Carolina faculty member to start a company and stay with it as he pursued his career in academics. That was an unpopular route at that time, but a path he was driven to navigate. Those interests, merged with his training, led to the design and manufacture of mechanobiology devices such as the Flexercell® Strain Unit and the BioFlex® flexible bottom culture plates to study fundamental responses of cells to strain. Principles in these devices were also incorporated into bioreactors for tissue engineering, which are standard in the marketplace today. In this article, the major roadblocks will be chronicled that were overcome to help build the field of mechanobiology and create a small biotechnology company. Through example, the author's formula for achieving milestones will be discussed including, the DRIVE it takes to get there ["DRIVE": Determination (Confidence), Research and Development (R&D) and Risk-Taking, Innovation (Imagination) and Intellectual Property, achieving Victory, and Enterprise].

Primary cilia: the chemical antenna regulating human adipose-derived stem cell osteogenesis.

Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.