Biomedical subjects

A J Banes

Publications and source records attributed to A J Banes.

At least 19 recordsLinked to original sources

PubMed2001

IL-1beta sensitizes intervertebral disc annulus cells to fluid-induced shear stress.

Chronic inflammation and altered mechanical loading are implicated as contributors to intervertebral disc degeneration. Biomechanical and biochemical factors play a role in disc degeneration but have received limited study. Mechanically, intervertebral discs are sheared during bending or twisting of the trunk. Biochemically, IL-1beta, detected in degenerative discs, promotes metalloproteinase expression. We hypothesized that disc cells might respond to shear stress and IL-1beta in a calcium signaling response. We measured the effect of single and combined stimuli on intracellular calcium concentration ([Ca2+]ic) and signaling. Cells were isolated from annulus tissue, cultured to quiescence, plated on collagen-bonded Culture Slips and incubated with Fura-2AM. Cells then were incubated in IL-1beta. Cell response to the effects of fluid flow was tested using FlexFlo, a laminar flow device. Human annulus (hAN) cells responded to laminar fluid flow with a one to three-fold increase in [Ca2+]ic. IL-1beta alone produced a small, transient stimulation. hAN cells pretreated with IL-1beta responded to shear with a more dramatic and sustained increase in [Ca2+]ic, six to ten-fold over basal level, when compared to shear then IL-1beta or shear and IL-1beta alone (P<0.001 for all comparisons). This is the first study documenting synergism of a signaling response to biomechanical and biochemical stimuli in human disc cells. IL-1beta treatment appeared to "sensitize" annulus cells to mechanical load. This increased responsiveness to mechanical load in the face of inflammatory cytokines may imply that the sensitivity of annulus cells to shear increases during inflammation and may affect initiation and progression of disc degeneration.

Adolescent↗

PubMed2001

The effects of static and intermittent compression on nitric oxide production in articular cartilage explants.

Nitric oxide (NO) production and NO synthase (NOS) expression are increased in osteoarthritis and rheumatoid arthritis, suggesting that NO may play a role in the destruction of articular cartilage. To test the hypothesis that mechanical stress may increase NO production by chondrocytes, we measured the effects of physiological levels of static and intermittent compression on NOS activity, NO production, and NOS antigen expression by porcine articular cartilage explants. Static compression significantly increased NO production at 0.1 MPa stress for 24 h (P < 0.05). Intermittent compression at 0.5 Hz for 6 h followed by 18 h recovery also increased NO production and NOS activity at 1.0 MPa stress (P < 0.05). Intermittent compression at 0.5 Hz for 24 h at a magnitude of 0.1 or 0.5 MPa caused an increase in NO production and NOS activity (P < 0.05). Immunoblot analysis showed stress-induced upregulation of NOS2, but not NOS1 or NOS3. There was no loss in cell viability following any of the loading regimens. Addition of 2 mM 1400 W (a specific NOS2 inhibitor) reduced NO production by 51% with no loss of cell viability. These findings indicate that NO production by chondrocytes is influenced by mechanical compression in vitro and suggest that biomechanical factors may in part regulate NO production in vivo.

Animals↗

PubMed2001

ATP induces Ca(2+) signaling in human chondrons cultured in three-dimensional agarose films.

OBJECTIVE: In vivo, chondrocytes are surrounded by an extracellular matrix, preventing direct cell-to-cell contact. Consequently, intercellular communication through gap junctions is unlikely. However, signaling at a distance is possible through extracellular messengers such as nitric oxide (NO) and nucleotides and nucleosides, adenosine triphosphate (ATP), uridine triphosphate (UTP), or adenosine diphosphate (ADP). We hypothesized that chondrons, chondrocytes surrounded by their native pericellular matrix, increase their intracellular calcium concentration ([Ca(2+)]ic) in response to ATP and other signaling molecules and that the source of Ca(2+) is from intracellular stores. The objectives of this study were to determine if chondrons in a 3-D gel respond to ATP by increasing [Ca(2+)]ic through a purinoceptor mechanism and to test whether chondrons in whole tissue samples would respond to ATP in a similar fashion. DESIGN: Human chondrons, cultured in a three-dimensional agarose gel or in whole cartilage loaded with Fura-2AM, a calcium sensitive dye, were stimulated with 1, 5 and 10 microM ATP. A ratio-imaging fluorescence technique was used to quantitate the [Ca(2+)]ic. RESULTS: ATP-stimulated chondrons increased their [Ca(2+)]ic from a basal level of 60 nM to over 1000 nM. Chondrons incubated in calcium-free medium also increased their [Ca(2+)]ic in response to ATP, indicating the source of Ca(2+) was not extracellular. ATP-induced calcium signaling was inhibited in chondrons pre-treated with suramin, a generic purinoceptor blocker. In addition, UTP and adenosine 5'-O-(3-thiotriphosphate) (ATPgammas) induced a calcium response, but 2-methylthio-ATP (2-MeSATP), ADP, and adenosine did not induce a significant increase in [Ca(2+)]ic, substantiating that the P2Y2 purinoceptor was dominant. Chondrons in whole cartilage increased [Ca(2+)]ic in response to ATP. CONCLUSIONS: We conclude that chondrons in 3-D culture respond to ATP by increasing [Ca(2+)]ic via P2Y2 receptor activation. Thus, ATP can pass through the agarose gel and the pericellular matrix, bind purinoceptors and increase intracellular Ca(2+) in a signaling response.

Adenosine Diphosphate↗

PubMed2000

ATP release by mechanically loaded porcine chondrons in pellet culture.

OBJECTIVE: To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures. METHODS: Chondron pellets were formed from 1.6 x 10(6) cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of gamma32P-ATP to resting cultures. RESULTS: Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures. CONCLUSION: The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases.

Adenosine Triphosphate↗

PubMed2000

Time-dependent increases in type-III collagen gene expression in medical collateral ligament fibroblasts under cyclic strains.

Numerous studies have demonstrated the capacity of mechanical strains to modulate cell behavior through several different signaling pathways. Understanding the response of ligament fibroblasts to mechanically induced strains may provide useful knowledge for treating ligament injury and improving rehabilitation regimens. Biomechanical studies that quantify strains in the anterior cruciate and medial collateral ligaments have shown that these ligaments are subjected to 4-5% strains during normal activities and can be strained to 7.7% during external application of loads to the knee joint. The objective of this study was to characterize the expression of types I and III collagen in fibroblast monolayers of anterior cruciate and medial collateral ligaments subjected to equibiaxial strains on flexible growth surfaces (0.05 and 0.075 strains) by quantifying levels of mRNA encoding these two proteins. Both cyclic strain magnitudes were studied under a frequency of 1 Hz. The results indicated marked differences in responses to strain regimens not only between types I and III collagen mRNA expression within each cell type but also in patterns of expression between anterior cruciate and medial collateral ligament cells. Whereas anterior cruciate ligament fibroblasts responded to cyclic strains by expression of higher levels of type-I collagen message with almost no significant increases in type-III collagen, medial collateral ligament fibroblasts exhibited statistically significant increases in type-III collagen mRNA at all time points after initiation of strain with almost no significant increases in type-I collagen. Furthermore, differences in responses by fibroblasts from the two ligaments were detected between the two strain magnitudes. In particular, 0.075 strains induced a time-dependent increase in type-III collagen mRNA levels in medial collateral ligament fibroblasts whereas 0.05 strains did not. The strain-induced changes in gene expression of these two collagens may have implications for the healing processes in ligament tissue. The differences may explain, in part, the healing differential between the anterior cruciate and medial collateral ligaments in vivo.

Adult↗

PubMed2000

Insulin-like growth factor-I is expressed by avian flexor tendon cells.

Cells in normal tendon are in a resting G0 state, performing maintenance functions. However, traumatic injury introduces growth factors such as platelet-derived growth factor and insulin-like growth factor from blood as well as activates endogenous growth factors. These factors stimulate migration and proliferation of tendon cells at the wound area. Tendon cells require growth-promoting factors to transit the cell cycle. To evaluate the contribution of endogenous growth factors in tendon, extracts of the epitenon and internal compartment of avian flexor tendon as well as medium of cultured cells from the epitenon (tendon surface cells) and internal tendon (tendon internal fibroblasts) were collected to assess their ability to stimulate DNA synthesis. Acid-ethanol extracts of tissues and medium were chromatographed on a P-30 molecular sieve column and assayed for mitogenic activity by quantitating [3H]thymidine incorporation into tendon cell DNA. The extract from the internal tendon compartment was more stimulatory for DNA synthesis than that from the epitenon, particularly when tested on tendon internal fibroblasts. However, conditioned medium fractions from surface epitenon cells stimulated DNA synthesis to a high degree on both tendon surface cells and tendon internal fibroblasts. Conditioned medium from tendon internal fibroblasts was also stimulatory. An anti-insulin-like growth factor-I antibody ablated most of the mitogenic activity present in both tissues and conditioned medium. The levels of acid-extractable insulin-like growth factor-I in tendon were determined by competitive radioimmunoassay as 1.48+/-0.05 ng/g tissue for the epitenon and 3.83+/-0.03 ng/g tissue for the internal compartment. Results of Western immunoblots of conditioned medium revealed insulin-like growth factor-I at the 7.5 kDa position. Cultured tendon surface cells and tendon internal fibroblasts as well as cells in intact flexor tendon expressed insulin-like growth factor-I mRNA detected by reverse transcriptase-polymerase chain reaction. In situ hybridization histochemistry positively identified insulin-like growth factor-I mRNA in tendons from 52-day-old chickens. Platelet-derived growth factor was not detected at the protein or message levels. Furthermore, tendon surface cells and tendon internal fibroblasts both expressed receptors for insulin-like growth factor-I detected by flow cytometry. These data suggest that tendon cells express insulin-like growth factor-I mRNA and synthesize insulin-like growth factor-I in both the epitenon and the internal compartment of tendon, which is present in an inactive form, most likely bound to insulin-like growth factor-binding proteins.

Animals↗

PubMed1999

Mechanical load stimulates expression of novel genes in vivo and in vitro in avian flexor tendon cells.

OBJECTIVE: Our experiments were designed to test the hypothesis that tendon cells might respond differently to applied strain in vitro than in vivo. DESIGN: We tested cells in whole tendons from exercised chickens and from isolated surface (TSC) and internal tendon (TIF) in vitro that were subjected to mechanical strain. We hypothesized that tendon cells differentially express genes in response to mechanical loading in vivo and in vitro. METHODS: We utilized an in-vivo exercise model in which chickens were run on a treadmill in an acute loading regime for 1 h 45 min with the balance of time at rest to 6 h total time. Gene expression was analyzed by a differential display technique. In addition, isolated avian flexor digitorum profundus TSC and TIF cells were subjected to cyclic stretching at 1 Hz, 5% average elongation for 6 h, +/- PDGF-BB, IGF-I, TGF-beta 1, PTH, estrogen, PGE2, or no drug and/or no load. mRNA was then collected and samples were subjected to differential display analysis. CONCLUSIONS: Load with or without growth factor and hormone treatments induced expression of novel genes as well as some known genes that were novel to tendon cells. We conclude that the study of gene expression in mechanically loaded cells in vivo and in vitro will lead to the discovery of novel and important marker proteins that may yield clues to positive and negative cell strain responses that are protective under one set of conditions and destructive under another.

Animals↗

PubMed1999

Gap junctions regulate responses of tendon cells ex vivo to mechanical loading.

Avian digital flexor tendons were used with a device to apply load ex vivo to study the effects on deoxyribonucleic acid and collagen synthesis when cell to cell communication is blocked. Flexor digitorum profundus tendons from the middle toe of 52-day-old White Leghorn chickens were excised and used as nonloaded controls, or clamped in the jaws of a displacement controlled tissue loading device and mechanically loaded for 3 days at a nominal 0.65% elongation at 1 Hz for 8 hours per day with 16 hours rest. Tendon samples were radiolabeled during the last 16 hours with 3H-thymidine to monitor deoxyribonucleic acid synthesis or with 3H-proline to radiolabel newly synthesized collagen. Cyclic loading of whole avian flexor tendons stimulated deoxyribonucleic acid and collagen synthesis, which could be blocked with octanol, a reversible gap junction blocker. Cells from human digital flexor tendon were used to populate a rectangular, three-dimensional, porous, polyester foam that could be deformed cyclically in vitro. Together, these results support the hypothesis that tendon cells must communicate to sustain growth and matrix expression and that an engineered three-dimensional construct can be used to study responses to mechanical load in vitro.

Animals↗

PubMed1998

Loading paradigms--intentional and unintentional--for cell culture mechanostimulus.

Recent interest in the response of cells or tissues to mechanical stimuli has led to the introduction of a variety of laboratory devices designed to deliver quantified mechanical inputs to culture systems. Such devices commonly rely upon distention of a flexible culture substrate, achieved either by direct platen abutment or by transmural pressure differentials. Unfortunately, the substrate distentions in such systems are often unintentionally nonuniform, and typically also induce motions in the overlying liquid nutrient medium--motions which in turn exert unintended reactive stresses upon the culture layer. In order to characterize the nature of these reactive fluid stresses, computer models have been developed for the nutrient medium flow fields (ie, the velocity and pressure distributions) for three established contemporary cell culture mechanostimulus systems. Temporal and spatial distributions of reactive normal and shear stresses are reported for typical duty cycles in these respective instruments.

Cell Culture Techniques↗

PubMed1997

Effect of mechanical loading on vascular alpha 1D- and alpha 1B-adrenergic receptor expression.

Heterogeneous distribution and function of alpha 1-adrenergic receptor subtypes on arterial and venous vessels, together with evidence for altered alpha-adrenergic receptor expression in hypertension, led us to examine whether mechanical load influences expression of alpha 1B- and alpha 1D-adrenergic receptors in rat aortic smooth muscle cells (SMCs). We used RNase protection and radioligand binding assays to measure mRNA and alpha 1-adrenergic receptor density. In the first model, SMCs were subjected to phasic loading using flexible culture plates. As a positive control for the load stimulus, postconfluent, quiescent passage 5 cells demonstrated the expected load-dependent morphological realignment. However, no changes were detected in expression of either alpha 1D- or alpha 1B-adrenergic receptor mRNAs or receptor density after 24 to 48 hours of loading. beta-Actin and SMC-specific alpha-actin mRNA, as well as cell number and per-cell total RNA and protein, were also unaffected. In a second model, intact thoracic aortas, in either the presence or absence of endothelial cells, were cultured for 48 hours under tonic load. Like cultured cells, 48 hours of load did not affect SMC expression of alpha 1-adrenergic receptor mRNAs. We used suprarenal aortic coarctation to examine effects of increased pressure in vivo. As with the previous in vitro and in situ models, hypertension (30 days) had no effect on expression of alpha 1B- and alpha 1D-adrenergic receptor mRNAs in the suprarenal aorta compared with sham coarctation. To separate pressure per se from humoral influences, we also measured mRNAs in the subrenal, normotensive aorta, alpha 1B mRNA levels decreased to 68 +/- 14% of sham-coarcted controls in subrenal aorta exposed to normal blood pressure but also to systemic humoral changes induced by coarctation. As a positive control for a load effect, SMC-specific alpha-actin mRNA increased for loaded aorta in organ culture and in hypertensive aorta in vivo, whereas expression of beta-actin mRNA was unaffected. These results from cell culture, organ culture, and in vivo models suggest that pressure (load) alone has no effect on alpha 1B- and alpha 1D-adrenergic receptor expression. In coarctation hypertension, smooth muscle protected from the hypertension showed a decline in alpha 1B mRNA that may be due to a humoral factor or factors.

Animals↗

PubMed1997

Changes in the amplitude of cyclic load biphasically modulate endothelial cell DNA synthesis and division.

Several physical factors, including shear stress and cyclic load, modulate the ability of endothelial cells to respond to injury. The objective of these experiments was to test the hypothesis that cyclic mechanical load stimulates endothelial cell DNA synthesis and division in vitro. Rabbit aortic endothelial cells were cultured on Flex I flexible-bottomed culture plates, and subjected to load amplitudes of increasing magnitude (0, 0.18, 0.24 and 0.27 load at 1 Hz) using a Flexercell strain unit. Cells were harvested enzymatically and cell numbers determined on days 1, 3 and 5 after initiating the load regimen. DNA synthesis was quantified after trichloroacetic acid precipitation of [3H]thymidine-labeled cells from: (1) whole culture wells and (2) areas of minimum and maximum strain in culture cells. Data were analyzed using analysis of variance and a Tukey's test (n = 6 observations/strain regimen per day in triplicate). Results from analysis of endothelial cells in whole, subconfluent cultures showed that cells subjected to strains of 0.18 had a decreased rate of cell division (76% of control) and DNA synthesis (63% of control), while cells subjected to strains of 0.24 and 0.27 had an increased rate of cell division (108 and 83% increase, respectively, compared with control; p < 0.001) and DNA synthesis (39 and 172% increase, respectively, compared with control; p < 0.001 for 0.27) on day 3 when compared with control cells. The results indicate that endothelial cells respond to various physiologic levels of cyclic load in a biphasic manner to initiate DNA synthesis and cell division. These data suggest that endothelial cell mitogenesis may be modulated by specific levels of cyclic load.

Animals↗

PubMed1996

Increased proliferation in keloid fibroblasts wounded in vitro.

A keloid is a pathological overgrowth of scar expanding beyond the boundaries of the initiating skin wound. Ultimately, this expansive scar is a result of excess collagen synthesized by fibroblasts within the wound. The processes that lead to this collagen excess remain unknown. An in vitro wound model was developed to test the hypothesis that fibroblasts isolated from keloid tissue and wounded in vitro might proliferate more rapidly that similarly wounded normal dermal fibroblasts. Keloid fibroblasts (KF) and normal human dermal fibroblasts (NDF) were grown to confluence and quiescence in flexible-bottomed culture plates. Wounds were created in a standardized fashion using a specially designed jig. The jig utilized a 25 gauge needle to reproducibly ablate 16-20% of cells from confluent cell sheets. Wounded and nonwounded cells were labeled with 3H-thymidine at 24, 48, 72 and 96 hr postwounding to measure DNA synthesis. Wounded KF and NDF demonstrated increased 3H-thymidine incorporation compared to nonwounded control cultures, and wounded KF demonstrated significantly higher levels of 3H-thymidine incorporation than wounded NDF both 24 and 48 hr after wounding. A similar trend was seen in cell counts. The wounded KF also showed a statistically greater labeling index quantitated by autoradiography than did wounded NDF. The increased commitment to DNA synthesis in response to wounding in vitro in keloid fibroblasts correlates with pathology seen in vivo. Keloid fibroblasts may have a lower inherent threshold for S phase entry than do normal fibroblasts contributing to the increased proliferation of keloid fibroblasts in response to wounding in vitro.

Autoradiography↗

PubMed1996

Hailey-Hailey disease keratinocytes: normal assembly of cell-cell junctions in vitro.

The blisters in the inherited disorder, Hailey-Hailey disease, may be caused by defective epidermal junctional complexes. We evaluated these structural complexes in vivo and in vitro. We induced a vesicular lesion in the apparently normal skin of a patient with Hailey-Hailey disease and studied a biopsy of this lesion by transmission electron microscopy. To determine whether acantholysis was related to a defect in the number or assembly of intercellular junctions, we cultured Hailey-Hailey disease keratinocytes in medium containing 0.1 mM Ca2+ and increased the [Ca2+] to 1.1 mM in order to induce assembly of cell-cell junctions. Keratinocytes were examined by double immunofluorescence with antibodies to the desmosome protein, desmoplakin, and the adherens junction protein, vinculin, at intervals after the increase in [Ca2+]. Characteristic Hailey-Hailey disease histopathology was observed by electron microscopy of the patient's skin after trauma, but we found no splitting of desmosomes. Based on the location, intensity, and rate of change of immunofluorescent staining, Hailey-Hailey and normal keratinocytes did not differ in their ability to assemble desmosomes and adherens junctions. Furthermore, we observed no significant morphologic differences between normal and Hailey-Hailey keratinocytes cultured in low and high [Ca2+]-containing media; Hailey-Hailey cells contained abundant normal-appearing desmosomes in 1.1 mM [Ca2+]. Since Hailey-Hailey disease keratinocytes can assemble normal-appearing adherens junctions and desmosomes in vitro, the functional defect may not lie in assembly of cell-cell adhering junctions, or additional perturbation may be required to expose the defect.

Adult↗

PubMed1996

Plastic surgery research.

Several areas of plastic surgery research, both in and out of the basic science laboratory, have been highlighted. Progress is being made in our ability to produce biologic structures to replace destroyed or damaged ones in vitro, our ability to manipulate the genetic structure of cells, and our ability to define and potentially manipulate the wound healing process in normal and abnormal healing states. More is also being learned about the biology of nerve repair, the physiologic response to silicone implants, and the mechanism of reperfusion injury. Virtual surgery may become a major part of the planning, performance, and teaching of surgical procedures in the future. Outcomes need to be better defined to prove the value of what plastic surgeons do to increasingly skeptical third parties, and progress is being made in this area as well. These areas represent only a few aspects of what is new in plastic surgery research, but they at least provide a window into the field and demonstrate the increasing sophistication of work being done in all areas. This work, as well as work in other areas, will undoubtedly affect clinical practices in the near future.

Cell Transplantation↗

PubMed1996

Tendon cells in vivo form a three dimensional network of cell processes linked by gap junctions.

Tendons respond to mechanical load by modifying their extracellular matrix. The cells therefore sense mechanical load and coordinate an appropriate response to it. We show that tendon cells have the potential to communicate with one another via cell processes and gap junctions and thus could use direct cell/cell communication to detect and/or coordinate their load responses. Unfixed cryosections of adult rat digital flexor tendons were stained with the fluorescent membrane dye DiI to demonstrate cell shape. Similar sections were immunolabelled with monoclonal antibodies to rat connexin 32 or connexin 43 to demonstrate gap junctions and counterstained with propidium iodide to show nuclei, or the membrane stain DiOC7 to show cell membranes. Sections were examined with a laser scanning confocal microscope and 3-dimensional reconstructions were prepared from optical section series to demonstrate cell shape and the position of connexin immunolabel. Cells had a complex interconnected morphology with gap junctions at points of contact with other cells. Cell bodies contained the nucleus and extended broad flat lateral cell processes that enclosed collagen bundles and interacted with similar processes from adjacent cells. They also had long thin longitudinal processes interacting with the cell process network further along the tendon. Connexin 43 occurred where cell processes met and between cell bodies, whereas connexin 32 was only found between cell bodies. The results indicate the presence of a 3-dimensional communicating network of cell processes within tendons. The intimate relationship between cell processes and collagen fibril bundles suggests that the cell process network could be involved in load sensing and coordination of response to load. The presence of 2 different types of connexins suggests that there could be at least 2 distinct communicating networks.

Animals↗

PubMed1995

PDGF-BB, IGF-I and mechanical load stimulate DNA synthesis in avian tendon fibroblasts in vitro.

Resident cells in the surface epitenon and internal compartment of flexor tendons are subjected to cyclic mechanical load as muscle contracts to move limbs or digits. Tendons are largely tensile load bearing tissues and are highly matrix intensive with nondividing cells providing maintenance functions. However, when an injury occurs, tendon cells are stimulated to divide by activated endogenous growth factors and those from platelets and plasma. We hypothesize that tendon cells detect mechanical load signals but do not interpret such signals as mitogenic unless an active growth factor is present. We have used an in vitro mechanical load model, application of cyclic strain to cells cultured on flexible bottomed culture plates, to test the hypothesis that tendon cells require platelet-derived growth factor (PDGF-BB) and insulin-like growth factor-I (IGF-I) in addition to mechanical load to stimulate DNA synthesis. In addition, we demonstrate that in avian tendon cells, load and growth factors stimulate phosphorylation of tyrosine residues in multiple proteins, including pp60src, a protein kinase that phosphorylates receptor protein tyrosine kinases. A lack of mitogenic responsiveness to mechanical load alone by tendon cells may be a characteristic of a regulatory pathway that modulates cell division.

Animals↗

PubMed1994

Fibronectin in the tendon-synovial complex: quantitation in vivo and in vitro by ELISA and relative mRNA levels by polymerase chain reaction and northern blot.

An enzyme-linked immunosorbent assay was used to quantitate fibronectin (Fn) levels in the outer synovia (epitenon) and internal fibrous portion (endotenon) of chicken flexor tendon and sheath. Primary cell cultures from these tissues and their secretions also were assayed for Fn levels. The polymerase chain reaction (PCR) was used to determine relative steady-state levels of Fn mRNA in primary cultures of synovial and internal fibroblasts from chicken tendon, and Northern blot analysis was performed to verify relative levels of the Fn message. The epitenon contained 3.8-fold more Fn than did the endotenon, and the sheath synovium contained 21-fold more Fn than did the internal fibrous portion of sheath. Cells cultured from the epitenon produced 9.3 and 13-fold more cell-associated and secreted Fn, respectively, than did cultured endotenon fibroblasts. Sheath synovial cells produced 17 and 3.2-fold more cell-associated and secreted Fn, respectively, than did sheath internal fibroblasts. Levels of Fn mRNA, as measured by PCR and Northern blot, were 1.6 and 1.8-fold greater, respectively, in tendon synovial cells compared with tendon internal fibroblasts. The biologic reason for increased Fn in tendon synovium is not known. We theorize that Fn may stabilize tendon synovium to shear stress and may play a role in the modulation of synovial rheology in the normal tendon. In the injured tendon, Fn may be involved in the organization of collagen deposition or may act through association with growth factors to aid healing.

Animals↗

PubMed1994

Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro.

Cells in the body are constantly subjected to cyclic mechanical deformation involving tension, compression, or shear strain or all three. A mechanical loading system which deforms cultured cells in vitro was analyzed in order to quantify the deformation or strain to which the cells are subjected. The dynamic system utilizes vacuum pressure to deform a circular silicone rubber substrate on which cells are cultured. These thick circular growth surfaces or plates are formed in the bottoms of the wells of 6-well culture plates. An axisymmetric model was formulated and analyzed using rectangular hyperelastic elements in a finite element analysis (FEA) software package. The thick circular plate has some disadvantages such as difficulty in observing cells and a nonhomogeneous strain profile which is maximum at the periphery and minimal at the center. A thinner circular surface (a thin plate) was also investigated in order to provide a more homogeneous strain profile. The radial strain on the thick circular plate, as determined by FEA, was nonlinear with a peak strain value of 0.30 (vacuum pressure of 22 kPa) about three-quarters of the distance from the center to the edge. In contrast, the radial strain of the thin circular plate was moderately constant across the surface. The circumferential strain for both of these models was less than the radial strain except for the center where they are equal. Avian tendon cells were cultured on the surface of a thick plate and exposed to cyclic strains for 24 h at a rate of 0.17 Hz and observed for cellular alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗