Establishment of fibroblast and myofibroblast phenotypes for use in in vitro co-culture models.

Fibroblasts function to secrete and modify components of the extracellular matrix. During wound healing, fibroblasts migrate to the site of injury and differentiate into contractile myofibroblasts; this differentiation is characterised by an increased contractile capacity. Fully differentiated myofibroblasts can be distinguished from fibroblasts via the higher expression of α-smooth muscle actin as well as a denser cytoskeleton. Impaired wound healing has been characterised by a lack of myofibroblasts; as a result, tissue does not fully regain its strength and function. Under pathological conditions, this may be associated with the effect that a pro-inflammatory microenvironment has on fibroblast and skeletal muscle progenitor cell migration and differentiation. Given their distinct roles in tissue maintenance and repair, the communication between fibroblasts versus myofibroblasts with other cellular mediators of repair is likely to influence cell behaviour and the outcome of wound repair. An in vitro test model is required to investigate this intercellular influence, but the establishment of such a model is hampered by the difficulty in retaining the dedifferentiated fibroblastic phenotype under regular serum-containing cell culture conditions. We present a model that supports the establishment and retention in culture of fibroblast and myofibroblast phenotypes for use in a simple, inexpensive, yet relevant in vitro 2D assay. This model is then applied in a co-culture setting to determine whether the presence of myoblasts affects the ability of fibroblasts versus myofibroblasts to close an in vitro wound. Our results emphasize the importance of considering the impact of paracrine communication between all cells during wound healing.

A regenerative approach to the pharmacological management of hard-to-heal wounds.

A wound is considered hard-to-heal when, despite the appropriate clinical analysis and intervention, the wound area reduces by less than a third at four weeks and complete healing fails to occur within 12 weeks. The most prevalent hard-to-heal wounds are associated with underlying metabolic diseases or vascular insufficiency and include arterial, venous, pressure and diabetic foot ulcers. Their common features include an abnormal immune response and extended inflammatory phase, a subdued proliferation phase due to cellular insufficiencies and finally an almost non-existent remodeling phase. Advances in wound care technology, tested in both pre-clinical models and clinical trials, have paved the way for improved treatment options, focused on regeneration. These interventions have been shown to limit the extent of ongoing inflammatory damage, decrease bacterial load, promote angiogenesis and deposition of granulation tissue, and stimulate keratinocyte migration thereby promoting re-epithelialization in these wounds. The current review discusses these hard-to-heal wounds in the context of their underlying pathology and potential of advanced treatment options, which if applied promptly as a standard of care, could reduce morbidity, promote quality of life, and alleviate the burden on a strained health system.

Evaluating the role of nitric oxide in myogenesis in vitro.

Skeletal muscle injury activates satellite cells to proliferate as myoblasts and migrate, differentiate and fuse with existing fibres at the site of injury. Nitric oxide (NO), a free radical produced by NO synthase, is elevated and supports healing after in vivo injury. NOS-independent elevation of NO levels in vitro is possible via donors such as molsidomine (SIN-1). We hypothesized that alterations in NO levels may directly influence myogenic processes critical for skeletal muscle wound healing. This study aimed to clarify the role of NO in myoblast proliferation, migration and differentiation. Baseline NO levels were established in vitro, whereafter NO levels were manipulated during myogenesis using l-NAME (NOS inhibitor) or SIN-1. Baseline NO levels generated by myoblasts in proliferation media did not change 1 h after stimulation. Addition of a pro-proliferative dose of HGF slightly elevated NO levels 1 h post-stimulation, whereas cell numbers assessed 24 h later increased significantly; l-NAME reduced the HGF-driven increase in NO and proliferation, reducing wound closure over 16 h. In differentiation media, NO levels increased significantly within 24 h, returning to baseline over several days. Regular addition of l-NAME to differentiating cells significantly reduced NO levels and fusion. SIN-1 increased NO levels in a dose-dependent manner, reaching maximal levels 16 h post-treatment. SIN-1, added at 0, 2 and 4 days, significantly increased myofiber area (26 ± 1.8% vs 18.6 ± 3.4% in control at 5 day, p < 0.0001), without affecting proliferation or migration. In conclusion, this study demonstrates that, during skeletal muscle regeneration, increased NO specifically stimulates myoblast differentiation.

Cellular regenerative therapy for acquired noncongenital musculoskeletal disorders.

Stem cells have an inherent capacity to facilitate regeneration; this has led to unprecedented growth in their experimental use in various clinical settings, particularly in patients with diseases with few alternative treatment options. However, their approved clinical use has to date been restricted largely to haematological diseases and epidermal transplantation to treat severe burns. After thorough searching of two databases, this review illuminates the role of stem cell therapy for treatment of musculoskeletal diseases. Research suggests that successful application of stem cells as regenerative mediators is in all likelihood dependent on the ability of endogenous tissue-resident reparative mediators to respond to paracrine signals provided by the applied stem cells. Therefore, an understanding of how the pathological environment influences this process is crucial for the ultimate success of stem cell therapies. The current review presents both the progress and limitations of stem cells as regenerative mediators in the context of musculoskeletal disorders.

Rapid quantification of cellular proliferation and migration using ImageJ.

Cellular proliferation and migration are crucial during development, regeneration and disease. Methods to quantify these processes are available; however, many are time consuming and require specialized equipment and costly reagents. Simple cell counts (proliferation analysis) and the scratch assay (migration analysis) are favorable methods due to their simplicity and cost-effectiveness; however, they rely on subjective and labor-intensive manual analysis, resulting in low throughput. We have developed optimized protocols to rapidly and accurately quantify adherent cell number and wound area using ImageJ, an open-source image processing program. Notably, these adaptable protocols facilitate quantification with significantly greater accuracy than manual identification.

Cellular regenerative therapy for acquired noncongenital musculoskeletal disorders

tem cells have an inherent capacity to facilitate regeneration; this has led to unprecedented growth in their experimental use in various clinical settings, particularly in patients with diseases with few alternative treatment options. However, their approved clinical use has to date been restricted largely to haematological diseases and epidermal transplantation to treat severe burns. After thorough searching of two databases, this review illuminates the role of stem cell therapy for treatment of musculoskeletal diseases. Research suggests that successful application of stem cells as regenerative mediators is in all likelihood dependent on the ability of endogenous tissue-resident reparative mediators to respond to paracrine signals provided by the applied stem cells. Therefore, an understanding of how the pathological environment influences this process is crucial for the ultimate success of stem cell therapies. The current review presents both the progress and limitations of stem cells as regenerative mediators in the context of musculoskeletal disorders

Cellular alignment and fusion: Quantifying the effect of macrophages and fibroblasts on myoblast terminal differentiation.

Successful skeletal muscle wound repair requires the alignment and fusion of myoblasts to generate multinucleated myofibers. In vitro, the accurate quantification of cellular alignment remains a challenge. Here we present the application of ImageJ and ct-FIRE to quantify muscle cell orientation by means of an alignment index (AI). Our optimised method, which does not require programming skills, allows the alignment of myoblasts in vitro to be determined independently of a predefined reference point. Using this method, we demonstrate that co-culture of myoblasts with macrophages, but not fibroblasts, promotes myoblast alignment in a cell density-dependent manner. Interestingly, myoblast fusion was significantly decreased in response to co-culture with macrophages, while the effect of fibroblasts on fusion was density-dependent. At lower numbers, fibroblasts significantly increased myoblast fusion, whereas at higher numbers a significant decrease was observed. Finally, triple co-culture revealed that the effect of macrophages on myoblast alignment and fusion is unaltered by the additional presence of fibroblasts. Application of our optimised method has therefore revealed quantitative differences in the roles of macrophages versus fibroblasts during alignment and fusion: while successful myoblast alignment is promoted by increasing macrophage numbers, regenerative fusion coincides with a decreasing macrophage population and initial rise in fibroblast numbers.

The extracellular matrix regulates the effect of decorin and transforming growth factor beta-2 (TGF-ß2) on myoblast migration.

Muscular injuries that destroy the basal lamina result in poor functional recovery of skeletal muscle. This is due, in part, to the deposition of structural fibrotic proteins such as fibronectin and collagen by fibroblasts and other cells. Transforming growth factor-ß (TGF-ß) promotes fibrosis, whereas the proteoglycan decorin is known to act as an anti-fibrotic agent, in part via the binding and neutralization of TGF-ß. We have previously established that decorin can alter the migratory response of skeletal muscle myoblasts to the extracellular matrix (ECM) factor collagen, but not fibronectin. We have also shown that TGF-ß reduces myoblast migration. In the current study we demonstrate that decorin can dramatically alter the inhibitory role of TGF-ß on human myoblast migration and go on to shown that the extracellular matrix can significantly modify this effect. Decorin and TGF-ß2 in combination were observed to significantly increase the rate of human myoblast migration, despite the inhibitory effect of TGF-ß2 on its own. Furthermore, in the presence of fibronectin, TGF-ß2 and decorin no longer acted synergistically to promote migration; while in the presence of collagen I, TGF-ß2 failed to inhibit migration. These studies show, for the first time, that decorin can alter the bioactivity of TGF-ß2 on human myoblast migration and emphasize the crucial regulatory role of the extracellular matrix in determining the response of skeletal muscle myoblasts to migratory cues.

MMP-14 in skeletal muscle repair.

MMP-14 (also known as MT1-MMP) is a membrane-bound collagenase and member of the Matrix Metalloprotease (MMP) family known to target a broad range of extracellular matrix (ECM) proteins. Remodelling of the ECM is of particular importance following skeletal muscle injury involving myofiber necrosis, when satellite cells are activated to facilitate myogenesis and regeneration. Myogenesis (broadly encompassed by the processes of satellite cell activation, proliferation, migration, differentiation and fusion) requires the myoblast to move either on or through a changing milieu of ECM components. The ECM composition, and especially the degree of fibrosis, influences ability of satellite cells to mediate a successful regenerative program. As a result, MMP activity is central to this regeneration; its activity increases following skeletal muscle injury, while inhibition of MMP reduces regeneration in this tissue. Besides its direct effect on matrix invasion, MMP-14 itself can affect this regeneration via activation of other MMPs (MMP-2, -9 and -13) as well as cytokines, chemokines and growth factors. Indeed recent research suggests that MMP-14 is necessary for the migration of human myoblasts into a collagen I matrix. Here we provide a current review on MMP-14 in the context of its role as a critical mediator of skeletal muscle regeneration.

ROCK-2 is associated with focal adhesion maturation during myoblast migration.

Satellite cell migration is critical for skeletal muscle growth and regeneration. Controlled cell migration is dependent on the formation of mature focal adhesions between the cell and the underlying extracellular matrix (ECM). These cell-ECM interactions trigger the activation of signalling events such as the Rho/ROCK pathway. We have previously identified a specific role for ROCK-2 during myoblast migration. In this study we report that ROCK inhibition with Y-27632 increases C2C12 myoblast velocity, but at the expense of directional migration. In response to Y-27632 an increased number of smaller focal adhesions were distributed across adhesion sites that in turn were clearly larger than sites in untreated cells, suggesting a reduction in focal adhesion maturation. We also confirm ROCK-2 localisation to the focal adhesion sites in migrating myoblasts and demonstrate a change in the distribution of these ROCK-2 containing adhesions in response to Y-27632. Taken together, our observations provide further proof that ROCK-2 regulates directional myoblast migration through focal adhesion formation and maturation.

Satellite cell count, VO(2max) , and p38 MAPK in inactive to moderately active young men.

Satellite cells (SCs) are responsible for muscle repair following strenuous exercise or injury. SC responses to intervention have been studied, but most studies do not discuss or take into account the substantial variability in SC number among young individuals. We hypothesized that an active lifestyle reflected in higher VO(2max) may be associated with greater SC number. As training alters basal p38-mitogen-activated protein kinase (MAPK) activity, which is associated with SC proliferation, SC count may also correlate with this stress signaling kinase. Muscle biopsies from vastus lateralis of eight male participants were analyzed for fiber type, myogenin, and p38/phospho-p38 MAPK using SDS-PAGE and Western blotting. Immunofluorescence was used to detect Pax7(+) SCs. Two weeks following the biopsy, subjects underwent an incremental treadmill test to determine VO(2max) . A strong positive correlation (P = 0.0087) was found between the number of Pax7(+) nuclei and VO(2max) . Pax7(+) cell number correlated negatively with phospho-p38/p38 MAPK (P = 0.0006), but had no correlation with fiber type or myogenin. SC number is proportional to VO(2max) , and hence it can be postulated that higher levels of physical activity activate SC proliferation but not fusion, underlining the relevance of exercise in stimulating SC pool size even without injury.

Decorin modulates collagen I-stimulated, but not fibronectin-stimulated, migration of C2C12 myoblasts.

Extracellular matrix factors, specifically fibronectin and collagen I, are essential for structural support during muscle regeneration. Decorin has been identified as an anti-fibrotic agent with binding sites located on both fibronectin and collagen I. Upon injury, activated myoblasts are required to migrate through the extracellular matrix factors deposited by the myofibroblasts to facilitate skeletal muscle regeneration. In this study we looked at the effects decorin on fibronectin- and collagen I-stimulated myoblast migration. Dose response studies demonstrated 10 µg/ml, 5 µg/ml and 25 µg/ml as the optimal stimulatory concentrations of decorin (1.2 fold increase), fibronectin (3.5 fold increase) and collagen I (2.4 fold increase), when compared with control respectively. A synergistic effect was identified when decorin and collagen I were added in combination; this effect was not evident when decorin was added with fibronectin. The effects of these factors on the ROCK signalling pathway were also analyzed. ROCK-2 was identified as the key Rho-activated kinase isoform involved in migration, due to its higher expression levels and localisation to focal points within migrating C2C12 myoblasts. Decorin and collagen I in combination stimulated an increase in the number of ROCK-2 localized focal points when compared with control, decorin and collagen I added separately. Fibronectin did not show any increase in ROCK-2 focal points when compared with control. These results show for the first time that decorin can modify collagen I-stimulated, but not fibronectin-stimulated myoblast migration in vitro. Furthermore, the synergistic, rather than additive, effect observed suggests a direct modification of collagen I signalling by decorin mediated, at least in part, by ROCK-2 rather than ROCK-1.

Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis.

Myoblast migration is essential for muscle development, regeneration, and repair. Various assays are available for the study of migration; however, many are not suitable for the analysis of myoblast migration during skeletal muscle wound repair. Furthermore, versatility (e.g., to investigate the effect of the extracellular matrix on cell movement) is limited within these methods. Here we report the optimization of the scratch assay specifically for two-dimensional myoblast migration and discuss the advantages of this assay in comparison with other commercially available methods.

Adipose depot-specific expression of cIAP2 in human preadipocytes and modulation of expression by serum factors and TNFalpha.

BACKGROUND: Visceral obesity is more closely associated with many deleterious metabolic sequelae than obesity per se. The identification of properties that distinguish fat cells of the omentum from adipocytes situated elsewhere in the body may lead to the development of therapeutic strategies targeting visceral obesity. We have previously demonstrated that cIAP2 mRNA is significantly overexpressed in omental (Om) compared with subcutaneous (Sc) adipocytes. This molecule is involved in the TNFalpha signalling pathway and may inhibit apoptosis. OBJECTIVE: To examine the effect of serum agents and TNFalpha upon cIAP2 mRNA expression in human primary culture preadipocytes. DESIGN: Paired omental and subcutaneous adipose tissue biopsies were obtained from 11 patients, nine female and two male, with ages ranging from 29 to 82. These were cultured in either serum containing medium or serum-free medium with or without the addition of TNFalpha for 4 h and mRNA levels analysed by quantitative reverse-transcription polymerase chain reaction. RESULTS: When human preadipocytes were cultured in a defined medium containing foetal calf serum the Om cells had a greater level of expression of cIAP2 mRNA than Sc cells from the same individual (mean 3.5-fold higher Sc>Om; P<0.01). However, when serum was removed from this media for a transitory period the level of cIAP2 mRNA decreased in the omental depot such that Sc preadipocytes had greater cIAP2 expression than their Om counterparts. Addition of TNFalpha induced a large increase in mRNA levels of cIAP2 (mean 20-fold). CONCLUSIONS: These results demonstrate that cIAP2 is expressed in a depot-specific manner in human preadipocytes and that levels of expression are regulated by serum factors and TNFalpha. Thus there may be intrinsic differences between preadipocyte cells from different adipose depots and this may play a role in the regulation of body fat distribution via the modulation of fat cell apoptosis.

Comparison of anti-apoptotic signalling by the insulin receptor and IGF-I receptor in preadipocytes and adipocytes.

We compared the effectiveness of insulin receptor (IR) and type I insulin-like growth factor (IGF) receptor (IGFR) cytoplasmic domains in mediating anti-apoptotic effects in 3T3-L1 preadipocytes and adipocytes. We used TrkC/IR and TrkC/IGFR chimeras, stably expressed in these cells and stimulated with neurotrophin-3 (NT-3), so as to avoid interference from endogenous receptors. After 24-h serum deprivation, 10% of preadipocytes and 2% of adipocytes appeared apoptotic as determined by fluorescence-activated cell sorter (FACS) analysis of cells stained with propidium iodide (PI) and Annexin V. When NT-3 was added, the two chimeras inhibited apoptosis to the same extent by 80% in preadipocytes and 50% in adipocytes. Mutation of juxtamembrane tyrosines (IR Y960F, IGFR Y950F) abrogated these anti-apoptotic effects. Qualitatively similar results were obtained by determination of viable rather than apoptotic cells. We conclude that IR and IGFR have equal potential to inhibit apoptosis in cell backgrounds, which are normally responsive to either IGF-I or insulin.

IGF-I inhibits apoptosis induced by serum withdrawal, but potentiates TNF-alpha-induced apoptosis, in 3T3-L1 preadipocytes.

We have previously shown that human preadipocytes in primary culture undergo apoptosis in response to serum deprivation and addition of tumour necrosis factor alpha (TNF-alpha), and have proposed that regulation of preadipocyte apoptosis in vivo may contribute to the overall control of adipose mass. In the present study we have investigated both pro- and anti-apoptotic factors, and the signalling pathways by which they act, in murine 3T3-L1 preadipocytes. Apoptotic indices (fraction of cells undergoing apoptosis) were determined by microscopic examination of acridine orange-stained cells, fluorescence-activated cell sorting of propidium iodide-stained cells, or phase-contrast video microscopy. Murine 3T3-L1 cells were more susceptible to apoptosis than human preadipocytes. In medium containing 10% newborn calf serum, the basal apoptotic index was very low (<2%), but the number of apoptotic cells increased significantly following serum withdrawal (10% after 24 h). Addition of TNF-alpha (6 nM) stimulated apoptosis in both serum-containing and serum-free media (apoptotic indices of 12% and 20% respectively after 24 h). IGF-I inhibited by approximately 50% the apoptosis induced by serum withdrawal, but increased by 25% the apoptosis induced by TNF-alpha in serum-free medium. It was shown by using specific inhibitors of lipid and protein kinases (LY294002, rapamycin, PD98059, SB203580) that both phosphoinositide 3-kinase and MAP kinase pathways contribute to the anti-apoptotic action of IGF-I on serum-starved cells, while phosphoinositide 3-kinase but not MAP kinase activity is required for the paradoxical pro-apoptotic action of IGF-I in the presence of TNF-alpha. We conclude that, in addition to its previously described anti-apoptotic action, IGF-I can also be pro-apoptotic in 3T3-L1 cells in the presence of TNF-alpha, and that both the anti- and pro-apoptotic effects of IGF-I require the activation of phosphoinositide 3-kinase.

Human preadipocytes display a depot-specific susceptibility to apoptosis.

Adipose tissue mass is determined by both the number and volume of adipose cells. Adipose cell number reflects the balance of cell acquisition and cell loss, whereas adipose cell volume represents the balance of lipolysis and lipogenesis. It is well recognized that insulin resistance, NIDDM, and other metabolic disorders are associated more strongly with increased omental adiposity than with subcutaneous adiposity. Depot-related differences exist in adipocyte responses to lipolytic and lipogenic stimuli, in adipocyte apoptosis, and in the potential for preadipocyte replication and differentiation. In the present study, we address the question of whether there might also be a site-specific difference in the susceptibility of human preadipocytes to apoptosis. Paired samples of human omental and subcutaneous preadipocytes from 12 individuals were cultured, and apoptosis was induced by serum deprivation or treatment with tumor necrosis factor (TNF)-alpha for 4 h. Cells were then stained with acridine orange, and apoptotic indices were calculated as the fraction of cells showing nuclear condensation. Under both conditions, in 9 of 11 subjects, apoptotic indices were substantially greater in preadipocytes from the omental depot than in those from the subcutaneous depot, and mean apoptotic indices were more than twofold higher in omental cells (serum-free medium: P < 0.05; TNF-alpha: P < 0.02; paired t test). Omental preadipocytes are therefore more susceptible to two different apoptotic stimuli than subcutaneous preadipocytes, demonstrating another intrinsic site-specific difference between human adipose cells of the two depots. These results suggest that the regulation of adipose tissue distribution in humans could involve depot-specific differences in rates of preadipocyte apoptosis.

Tumor necrosis factor-alpha induces apoptosis of human adipose cells.

Tumor necrosis factor-alpha (TNF-alpha) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-alpha mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-alpha has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-alpha in limiting increase in fat mass. The aim of the present study was to determine if TNF-alpha could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-alpha at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-alpha induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-alpha induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-alpha in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-alpha.