Biomedical subjects

James H-C Wang

Publications and source records attributed to James H-C Wang.

At least 19 recordsLinked to original sources

PubMed2007

Cell traction force and measurement methods.

Cell traction forces (CTFs) are crucial to many biological processes such as inflammation, wound healing, angiogenesis, and metastasis. CTFs are generated by actomyosin interactions and actin polymerization and regulated by intracellular proteins such as alpha-smooth muscle actin (alpha-SMA) and soluble factors such as transforming growth factor-beta (TGF-beta). Once transmitted to the extracellular matrix (ECM) through stress fibers via focal adhesions, which are assemblies of ECM proteins, transmembrane receptors, and cytoplasmic structural and signaling proteins (e.g., integrins), CTFs direct many cellular functions, including cell migration, ECM organization, and mechanical signal generation. Various methods have been developed over the years to measure CTFs of both populations of cells and of single cells. At present, cell traction force microscopy (CTFM) is among the most efficient and reliable method for determining CTF field of an entire cell spreading on a two-dimensional (2D) substrate surface. There are currently three CTFM methods, each of which is unique in both how displacement field is extracted from images and how CTFs are subsequently estimated. A detailed review and comparison of these methods are presented. Future research should improve CTFM methods such that they can automatically track dynamic CTFs, thereby providing new insights into cell motility in response to altered biological conditions. In addition, research effort should be devoted to developing novel experimental and theoretical methods for determining CTFs in three-dimensional (3D) matrix, which better reflects physiological conditions than 2D substrate used in current CTFM methods.

Algorithms↗

PubMed2007

A new approach to study fibroblast migration.

This paper presents a new approach to study cell migration. Human tendon fibroblasts were plated on silicone membranes coated with 10 microg/ml ProNectin-F. The silicone surfaces were micro-fabricated with parallel microgrooves, with 10 microm ridge and groove width, and 3 microm groove depth. Fibroblasts grown in the microgrooves had an elongated shape and oriented along the microgroove direction. They also moved along the same direction instead of "random walk" when cells migrate on smooth culture surfaces. In response to TGF-beta1 (5 ng/ml) treatment, these fibroblasts on the microgrooved surfaces were differentiated into myofibroblasts, as judged by an elevated expression of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts. Moreover, these myofibroblasts were found to be approximately 30% less motile compared to that of untreated fibroblasts. Thus, use of microgrooved surface may be an effective approach to detect difference in cell motility because cell migration on the microgrooved surface is one dimensional and hence easier to be quantified than two-dimensional random movement on conventional smooth culture surfaces.

Actins↗

PubMed2007

Alpha-smooth muscle actin expression enhances cell traction force.

Using an established corneal stromal cell differentiation model, we manipulated alpha-smooth muscle actin (alpha-SMA) protein expression levels in fibroblasts by treating them with TGF-beta1, bFGF, TGF-beta type I receptor inhibitor (SB-431542), and siRNA against alpha-SMA. The corresponding cell traction forces (CTFs) were determined by cell traction force microscopy. With all these treatments, we found that alpha-SMA is not required for CTF induction, but its expression upregulates CTF. This upregulation involves the modification of stress fibers but does not appear to relate to non-muscle myosin II expression or beta-actin expression. Moreover, there exists a linear relationship between alpha-SMA protein expression level and CTF magnitude. Finally, CTFs were found to vary among a population of myofibroblasts, suggesting that alpha-SMA protein expression levels of individual cells also vary.

Actins↗

PubMed2006

RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts.

A novel technique for conjugating Arg-Gly-Asp (RGD) peptides to poly(dimethylsiloxane) (PDMS) surfaces as well as its application to cell culture is presented in this paper. This technique performs RGD conjugation to PDMS through photochemical immobilization of functional NHS groups to PDMS surface followed with linking RGD peptide to the surface via coupling reaction with NHS. A bifunctional photolinker, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), was used to conjugate RGD peptide to the surface. Compared to existing methods for peptide conjugation to PDMS, this technique is convenient, efficient, and free of organic contamination to PDMS surfaces. It can also be used to conjugate other peptides or proteins to most polymeric materials. In addition, cell culture studies showed that the RGD-conjugated PDMS surfaces promoted the adhesion, proliferation, and collagen production of human skin fibroblasts (HSFs). Finally, the RGD-conjugated PDMS surfaces are resistant to autoclaving and UV irradiation, which enables them to be repeatedly used in cell culture studies.

Biocompatible Materials↗

PubMed2006

EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1 beta treatment.

Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.

Cells, Cultured↗

PubMed2006

Determining substrate displacement and cell traction fields--a new approach.

This paper presents a new approach for the traction force microscopy (TFM) method which determines traction forces exerted by adherent cells on a thin, elastic polyacrylamide gel embedded with fluorescent microbeads. In this enhanced TFM method, a pattern recognition technique is first applied to match the pair of microbead embedded images before and after deformation, which subsequently provides the displacement field of the elastic substrate. Once the displacement field is obtained, the 3-D finite element method (FEM) is used to compute cell traction forces. The new TFM has been applied to determine traction forces of human tendon fibroblasts. Compared to existing TFM methods, the present method has the following advantages: (1) its displacement field obtained is associated with microbead movements; (2) it considers the finite thickness of the thin polyacrylamide gel and is therefore free from the infinite half-space approximation adopted by existing TFM methods; and (3) its computation procedure for determining cell traction forces is fast.

Biomechanical Phenomena↗

PubMed2006

Healing and normal fibroblasts exhibit differential proliferation, collagen production, alpha-SMA expression, and contraction.

This study determines the differences in proliferation, collagen production, alpha-smooth muscle actin (alpha-SMA) expression, and contraction between healing and normal fibroblasts. Transected and sham-operated rat medial collateral ligaments (MCL) were used to obtain healing and normal fibroblasts, respectively. It was found that healing fibroblasts in monolayer culture proliferated 1.4-fold faster at 48 h and had 1.7-fold greater protein expression of alpha-SMA than normal fibroblasts. In addition, it was noted that the proliferation of healing fibroblasts in collagen gels was not significantly different from that of normal fibroblasts at 24 h, but it was at 48 h. Furthermore, in collagen gels, healing fibroblasts produced more type I collagen than normal fibroblasts and generated 1.6- and 1.7-fold larger contractile forces at 15 and 20 h, respectively, than their normal counterparts. Taken together, the results of this study show that healing fibroblasts possess a differential proliferation, alpha-SMA protein expression, and contraction than normal fibroblasts.

Actins↗

PubMed2006

Leukotriene B4 at low dosage negates the catabolic effect of prostaglandin E2 in human patellar tendon fibroblasts.

Tendinopathy often involves inflammation and matrix degeneration. The inflammatory mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are implicated in the development of tendinopathy. Therefore, the purpose of this study was to determine the effect of PGE2 and LTB4 on the proliferation of human patellar tendon fibroblasts (HPTFs), the gene expression of collagen type I, MMP-1 and MMP-3, as well as the protein secretion of these gene products by the cells. The results showed that LTB4 at low doses (0.1 and 1 nM) significantly increased cell proliferation compared to controls and LTB4 at 0.1 nM negated the PGE2-induced decrease in cell proliferation. In addition, PGE2 at 100 ng/ml significantly increased the expression of MMP-1 and MMP-3 at both mRNA and protein levels. These stimulatory effects were significantly diminished by co-treatment with LTB4 at 0.1 nM. Finally, neither PGE2 nor LTB4 treatment affected collagen type I gene expression. These results suggest that low levels of LTB4 counterbalance the negative effects mediated by PGE2 on tendon fibroblast proliferation and MMP production, which may lead to matrix degradation. Thus, our findings suggest that although LTB4 is generally thought to be pathogenic, low levels of LTB4 are actually beneficial in maintaining tendon tissue homeostasis.

Cell Proliferation↗

PubMed2006

Vanadate ingestion enhances the organization and collagen fibril diameters of rat healing medical collateral ligaments.

Although an injured medial collateral ligament (MCL) will naturally heal, the quality of healing tissue is inferior to the uninjured MCL tissue. Previous studies have shown promising results of sodium orthovanadate (vanadate) in enhancing the quality of rat skin wounds. This study therefore investigated whether vanadate enhances the quality of the rat healing MCL in terms of the collagen fibril organization and diameter. Six mature male Sprague-Dawley rats, with weight ranges of 475-505 g and ages of 25 weeks, were used in this study. Three rats in the experimental group received vanadate (0.2 mg/ml) in their saline drinking water (150 mM NaCl), whereas three rats in the control group were only given saline water. Three weeks after transection, the rat MCLs were harvested for hematoxylin and eosin (H&E) staining and transmission electron microscopy. It was found that vanadate promoted organization of collagen fibrils and significantly increased the diameters of collagen fibrils by 14% in healing MCL (P<0.001). These results indicate that application of vanadate may be a promising tissue engineering approach to enhance the quality of healing tissues such as injured MCLs.

Animals↗

PubMed2006

Biomechanical basis for tendinopathy.

Tendinopathy affects millions of people in athletic and occupational settings and is a nemesis for patients and physicians. Mechanical loading is a major causative factor for tendinopathy; however, the exact mechanical loading conditions (magnitude, frequency, duration, loading history, or some combinations) that cause tendinopathy are poorly defined. Exercise animal model studies indicate that repetitive mechanical loading induces inflammatory and degenerative changes in tendons, but the cellular and molecular mechanisms responsible for such changes are not known. Injection animal model studies show that collagenase and inflammatory agents (inflammatory cytokines and prostaglandin E1 and E2) may be involved in tendon inflammation and degeneration; however, whether these molecules are involved in the development of tendinopathy because of mechanical loading remains to be verified. Finally, despite improved treatment modalities, the clinical outcome of treatment of tendinopathy is unpredictable, as it is not clear whether a specific modality treats the symptoms or the causes. Research is required to better understand the mechanisms of tendinopathy at the tissue, cellular, and molecular levels and to develop new scientifically based modalities to treat tendinopathy more effectively.

Biomechanical Phenomena↗

PubMed2005

Repetitive mechanical stretching modulates IL-1beta induced COX-2, MMP-1 expression, and PGE2 production in human patellar tendon fibroblasts.

While mechanical loading is known to be essential in maintaining tendon homeostasis, repetitive mechanical loading has also been implicated in the etiology of tendon overuse injuries. The purpose of this study was to determine whether cyclic mechanical stretching regulates inflammatory responses induced by interleukin-1beta (IL-1beta) treatment in human patellar tendon fibroblasts (HPTFs). HPTFs were grown in microgrooved silicone dishes, where they became elongated in shape and aligned with the microgrooves, which is similar to the shape and organization of tendon fibroblasts in vivo. Cyclic uniaxial stretching was then applied to silicone culture dishes with a 4% or 8% stretch at a stretching frequency of 0.5 Hz for a duration of 4 h in the presence or absence of 10 pM IL-1beta treatment. Non-stretched cells in the presence or absence of IL-1beta were used for controls, respectively. The expression of cyclooxygenase-2 (COX-2), matrix metalloproteinase-1 (MMP-1), and the production of prostaglandin E2 (PGE2) were measured. In the absence of stretching, it was found that 10 pM of IL-1beta markedly induced higher levels of COX-2, MMP-1 gene expression, and PGE2 production than non-treated cells. Furthermore, cells with 4% stretching decreased the COX-2 and MMP-1 gene expression and PGE2 production that were stimulated by IL-1beta, whereas cells with 8% stretching further increased these gene products and/or expression levels in addition to the effects of IL-1beta stimulation. Thus, the results suggest that repetitive, small-magnitude stretching is anti-inflammatory, whereas large-magnitude stretching is pro-inflammatory. Therefore, moderate exercise may be beneficial to reducing tendon inflammation.

Adolescent↗

PubMed2005

Mechanobiology of tendon.

Tendons are able to respond to mechanical forces by altering their structure, composition, and mechanical properties--a process called tissue mechanical adaptation. The fact that mechanical adaptation is effected by cells in tendons is clearly understood; however, how cells sense mechanical forces and convert them into biochemical signals that ultimately lead to tendon adaptive physiological or pathological changes is not well understood. Mechanobiology is an interdisciplinary study that can enhance our understanding of mechanotransduction mechanisms at the tissue, cellular, and molecular levels. The purpose of this article is to provide an overview of tendon mechanobiology. The discussion begins with the mechanical forces acting on tendons in vivo, tendon structure and composition, and its mechanical properties. Then the tendon's response to exercise, disuse, and overuse are presented, followed by a discussion of tendon healing and the role of mechanical loading and fibroblast contraction in tissue healing. Next, mechanobiological responses of tendon fibroblasts to repetitive mechanical loading conditions are presented, and major cellular mechanotransduction mechanisms are briefly reviewed. Finally, future research directions in tendon mechanobiology research are discussed.

Animals↗

PubMed2005

Decreasing inflammatory response of injured patellar tendons results in increased collagen fibril diameters.

Tissue inflammation is essential in the healing process, but its effect on the quality of the healing tissue is not clear. This study determines the effect of decreasing early inflammation during wound healing in genetic deficient mice on collagen fibril diameter. Two strains of mice were used: three C3H/HeJ mice and three C3H/HeN mice for each of two time points (7 and 14 days postinjury). C3H/HeJ mice have a genetic deficiency in the production of tumor necrosis factor by macrophages and other cytokines in response to endotoxin, and C3H/HeN mice have no genetic deficiency. The right patellar tendon of both mouse strains was transversely transected, whereas the left patellar tendon was left intact for control. After 7 and 14 days, both right and left patellar tendons were harvested, and tendon samples were examined with transmission electron microscopy. We found that at 7 days, transected tendons of C3H/HeJ mice exhibited on average 1.6 times larger collagen fibril diameters than transected C3H/HeN tendons, whereas at 14 days, collagen fibril diameters of the C3H/HeJ mice were 1.3 times that of C3H/HeN mice. Also, at both 7 days and 14 days, collagen fibrils in C3H/HeJ mice appeared more organized than C3H/HeN mice. In addition, control tendons in both mouse strains showed no significant differences in collagen fibril diameter and organization. Therefore, these results suggest that decreasing the inflammatory response in the early stages of tendon wound healing enhances the quality of the healing tendon through increased collagen fiber diameter and better organization.

Animals↗

PubMed2005

Repeated exposure of tendon to prostaglandin-E2 leads to localized tendon degeneration.

OBJECTIVE: To determine whether repeated exposure of rabbit patellar tendon to prostaglandin-E(2) leads to degenerative changes in the tendon. SETTING: Laboratory animal study. MAIN OUTCOME MEASURES: Intratendinous changes including cellularity, matrix organization, collagen fibril packing, and diameter. METHODS: Skeletally mature New Zealand White rabbits (n = 10) were transcutaneously injected in the midsubstance of the patellar tendon with prostaglandin-E(2) (PGE(2); 50 ng or 500 ng). The contralateral tendons were used as 3 different controls (no injection, saline injection, and needlestick only). The injection was repeated once a week for 4 weeks, and the rabbits were killed 1 week after the last injection. The patellar tendons were harvested and examined using hematoxylin and eosin staining and transmission electron microscopy. RESULTS: Compared with the control groups, tendons exposed to PGE(2) by injection showed focal areas of hypercellularity, loss of normal tissue architecture, and focal areas of tendon disorganization and degeneration. Tendons injected with PGE(2) exhibited loosely organized collagen fibrils and had thinner collagen fibril diameter compared with control tendons (P < 0.005). Tendons injected with 500 ng PGE(2) appeared to be more disorganized and degenerated than those injected with 50 ng PGE(2). CONCLUSIONS: Repeated exposure of the tendon to PGE(2) leads to degenerative changes within the tendon. CLINICAL RELEVANCE: It is known that human tendon fibroblasts produce PGE(2) in vitro and in vivo in response to repetitive mechanical loading. This study demonstrates that repetitive exposure of the tendon to PGE(2) can result in degenerative changes within the tendon. Therefore, PGE(2) produced by tendon fibroblasts in response to repetitive mechanical loading in vivo might contribute to the development of exercise-induced tendinopathy.

Animals↗

PubMed2004

Fibroblast responses to cyclic mechanical stretching depend on cell orientation to the stretching direction.

Fibroblasts in intact tendons align with stretching direction, but they tend to orient randomly in healing tendons. Therefore, a question arises: Do fibroblast responses to mechanical stretching depend on their orientation? To address this question, human patellar tendon fibroblasts were grown in custom-made silicone dishes that possess microgrooved culture surfaces. The direction of the microgrooves was either parallel or normal to the direction of cyclic uniaxial stretching. Fibroblasts grown in these microgrooves had a polar morphology and oriented along the direction of the microgrooves regardless of the stretching conditions. Tendon fibroblasts expressed higher levels of alpha-smooth muscle actin when they were oriented parallel to the stretching direction than when they were oriented normal to the stretching direction. Also, cyclic stretching of the fibroblasts perpendicular to their orientation induced a higher activity level of secretory phospholipase A(2) compared with stretching of the cells parallel to their orientation. Thus, these results show that fibroblast responses to mechanical stretching depend on cell orientation to the stretching direction.

Actins↗

PubMed2004

Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions.

We studied the effect of cyclic mechanical stretching on the proliferation and collagen mRNA expression and protein production of human patellar tendon fibroblasts under serum-free conditions. The role of transforming growth factor-beta1 (TGF-beta1) in collagen production by cyclically stretched tendon fibroblasts was also investigated. The tendon fibroblasts were grown in microgrooved silicone dishes, where the cells were highly elongated and aligned with the microgrooves. Cyclic uniaxial stretching with constant frequency and duration (0.5 Hz, 4 h) but varying magnitude of stretch (no stretch, 4%, and 8%) was applied to the silicone dishes. Following the period of stretching, the cells were rested for 20 h in stretching-conditioned medium to allow for cell proliferation. In separate experiments, the cells were stretched for 4h and then rested for another 4 h. Samples of the medium, total cellular RNA and protein were used for analysis of collagen and TGF-beta1 gene expression and production. It was found that there was a slight increase in fibroblast proliferation at 4% and 8% stretch, compared to that of non-stretched fibroblasts, where at 8% stretch the increase was significant. It was also found that the gene expression and protein production of collagen type I and TGF-beta1 increased in a stretching-magnitude-dependent manner. And, levels of collagen type III were not changed, despite gene expression levels of the protein being slightly increased. Furthermore, the exogenous addition of anti-TGF-beta1 antibody eliminated the increase in collagen type I production under cyclic uniaxial stretching conditions. The results suggest that mechanical stretching can modulate proliferation of human tendon fibroblasts in the absence of serum and increase the cellular production of collagen type I, which is at least in part mediated by TGF-beta1.

Adolescent↗

PubMed2004

A multi-station dynamic-culture force monitor system to study cell mechanobiology.

To study mechanobiological responses of cells, a dynamic-culture force monitor (D-CFM) system has been developed. The D-CFM extends our previous work to measure contractile forces of a cell-populated collagen gel (CPCG) using a cantilever beam with semiconductor strain gauges. Linear actuators are used in the system and are computer controlled using a LabVIEW interface to independently apply precise motion waveforms to multiple CPCGs. The feasibility tests showed that the new system can detect the differences in force patterns resulting from different motion waveforms imparted to the CPCG. This new system will facilitate the study of the effects of dynamic mechanical loading on cells, remodeling of extracellular matrix, and cell-matrix interactions in vitro.

Cell Culture Techniques↗

PubMed2004

The use of porcine small intestinal submucosa to enhance the healing of the medial collateral ligament--a functional tissue engineering study in rabbits.

INTRODUCTION: Small intestinal submucosa (SIS) from porcine has been successfully used as a collagen scaffold for the repair of various tissues, including those of the human vascular, urogenital, and musculoskeletal systems. The objective of this study was to evaluate whether SIS can be used to enhance the healing process of a medial collateral ligament (MCL) with a gap injury in a rabbit model. METHODS: A 6 mm wide gap was surgically created in the right MCL of 20 skeletally mature, female New Zealand White rabbits. In 10 rabbits, a strip of SIS was sutured onto the two ends of the MCL, while for the other 10 animals their injured MCL remained untreated and served as a non-treated group. The left MCL of all animals was exposed and undermined serving as the sham-operated side. At 12 weeks post-healing, eight hind limbs from each group were used for mechanical testing. The cross-sectional areas (CSA) of the MCLs were measured. The femur-MCL-tibia complex (FMTC) was tensile tested to failure. The load-elongation curves representing the structural properties of the FMTC and the stress-strain curves representing the mechanical properties of the healing MCL were obtained. The remaining two animals from each group were prepared for histological evaluation. RESULTS: The CSA between the SIS-treated and non-treated groups were not significantly different (p>0.05). Both treatment groups appeared to increase by nearly 40% compared to the sham-operated side, although statistical significance was not found for the non-treated group (p>0.05). The stiffness of the FMTC from the SIS-treated group was 56% higher than the non-treated group (45.7+/-13.3 N/mm vs. 29.2+/-9.2 N/mm, respectively, p<0.05) and the ultimate load also nearly doubled (117.434.5 N vs. 66.4+/-31.4 N, respectively, p<0.05). These values were lower compared to the sham-operated side (89.7+/-15.3 N/mm and 332.0+/-50.8 N, respectively). The tangent modulus of the healing MCL (279.7+/-132.1 MPa vs. 149.0+/-76.5 MPa, respectively) and stress at failure (15.7+/-4.1 MPa vs. 10.2+/-3.9 MPa, respectively) both increased by more than 50% with SIS treatment (p<0.05). Yet, each remained lower compared to the sham-operated side (936.3+/-283.6 MPa and 75.6+/-14.2 MPa, respectively). Blinded histological comparisons between the SIS-treated MCL and the non-treated control demonstrated qualitatively that the SIS treated group had increased cellularity, greater collagen density, and improved collagen fiber alignment. CONCLUSION: Healing of a gap MCL injury was significantly enhanced with SIS. The improved mechanical properties and histological appearance of the MCL suggest that SIS treatment improves the quality of tissue and renders the possibility for future studies investigating functional tissue engineering of healing ligaments.

Animals↗